# Presentation: 714571

## Technology Development:
Detecting gene rearrangement 
associated with intragenic CNA

Cameron Brennan
Department of Neurosurgery
Memorial Sloan-Kettering Cancer Center

- Detecting gene rearrangement
- associated with intragenic CNA
- Cameron Brennan
- Department of Neurosurgery
- Memorial Sloan-Kettering Cancer Center

## Candidate gene fusions among tyrosine kinases in GBM

**Tumors (n=40)**

**Cell Lines (n=30)**

## Sequence-verified, in-frame, expressed

**Tumors**

**Cell Lines**

- Sequence-verified, in-frame, expressed

## Functional validation

**Tumors**

**Cell Lines**

**?**

## Search systematically for intragenic CNA:  Cancer Genome Atlas

_**Strategy 1**_**:**  Statistical approach to identifying iCNA in whole-genome copy-number data
-    _**iCNA analysis, GTS package (R language), **_( cbio.mskcc.org/~brennan )
_**Strategy 2**_**:** Integration of exon expression to identify clearly expressed transcripts with exon imbalance
-    _**run on iCNA significant candidates**_
_**Strategy 3**_**:** Custom high density CGH array: confirm intragenic CNA and map at exon-level resolution

**Notes:**
KP fusion has been found only once but:
One other constitutively active deletion mutant PDGFRA has been reported in GBM
like EGFRvIII, PDGFRA deletion is associated with amplification
PDGFRA regional amplification is seen in XX% of GBM
2/5 tumors with PDGFRA regional amplification show deletion mutation of PDGFRA
Jak3-SPTBN4 in glioma cell line and HCK truncation mutant in glioblastoma tumor also discovered 
In GBM, TK mutation by rearrangement (EGFRvIII) exceeds point mutation in frequency
Multiple TK mutations in the same tumor may point to a more general phenomenon of multiple and redundant mechanisms of signaling activation.

## iCNA: estimating boundary error

**       ****Gene                                  **

## Permuting raw data on either side:boundary may shift

**       ****Gene                                  **

**permuted**

**permuted**

**New change point**

## Confidence intervals

**       ****Gene                                  **

## iCNA algorithm summary

- Determine iCNA boundaries with confidence interval
- Count recurrence across tumors
- Estimate expected rate for each gene as a function of gene size and rate of breaks per sample
- Account for non-independence of breaks
- Estimate p-value based on (observed – expected)/SE
- run on 175 Prostate CA profiles  40,000 breaks(William Gerald, MSKCC)
- ~250 genes significantly altered (many are CNVs)
- ERG is top gene, >30 samples  (TMPRSS-ERG)

## iCNA in GBM samples

- 224 samples
- 37,464 autosomal segments
- 11,470 intragenic boundaries (31%)
- 9,172 Δlog2 >0.25
- 3,728 unique genes
- 1,010 genes significant above background (p<0.05)
- 571 are in at least 2 samples
- 450 are in at least 1 at high confidence
- 303 genes show no iCNA in matched normals
- 262 do not match known CNV site*
- 150 genes are altered in >=3 samples
- EGFR, PDGFRA, KIT, PTEN, NF1, NF2, etc...
- Most are CNVs

## iCNA vs. point mutation

**EGFR**

- Histogram of intragenic breaks

*450 genes with only one iCNA event*

*115 genes with iCNA in two samples*

- Number per sample

- Number of genes

- Number per sample

- Boris Reva, John Major

- Histogram of point mutations

- Number of genes

*750 genes with only one mutation event*

## Search systematically for intragenic CNA:  Cancer Genome Atlas

_**Strategy 1**_**:**  Statistical approach to identifying iCNA in whole-genome copy-number data
_**Strategy 2**_**:** **Integration of exon expression to identify clearly expressed transcripts with exon imbalance**
-    _**run on iCNA-significant candidates**_

**Notes:**
KP fusion has been found only once but:
One other constitutively active deletion mutant PDGFRA has been reported in GBM
like EGFRvIII, PDGFRA deletion is associated with amplification
PDGFRA regional amplification is seen in XX% of GBM
2/5 tumors with PDGFRA regional amplification show deletion mutation of PDGFRA
Jak3-SPTBN4 in glioma cell line and HCK truncation mutant in glioblastoma tumor also discovered 
In GBM, TK mutation by rearrangement (EGFRvIII) exceeds point mutation in frequency
Multiple TK mutations in the same tumor may point to a more general phenomenon of multiple and redundant mechanisms of signaling activation.

## NUP107: recurrent iCNA (n=7)

**(mapped)**

**Exon-expression**
**(spread out)**

**Nuclear pore complex protein, located at 12q15**
**Related *****NUP98***** and *****NUP214*****, involved in many fusion oncogenes**

**Array-CGH**

## MET translocation candidate

- Promoter substitution ?
- Exon 1 non-coding
- High MET expression
- Proneural expression cluster

## NELL2 (-): recurrent iCNA

_**N**_**eural **_**E**_**GF-**_**l**_**ike **_**l**_**ike 2; located at 12q13**
**Function unclear, interacts with PKC-beta**
**Same change point in 2 cases, **
**would remove signal sequence + laminin G domain, leave EGF-like repeats**

## iCNA in ovarian samples

- 39 ovarian tumor samples (45 normals)
- 9,932 autosomal segments
- 3,414 intragenic boundaries (34%)
- 3,242 Δlog2 >0.25
- 2,085 unique genes
- 810 genes significant above background (p<0.05)
- 274 are in at least 2 samples
- 197 are in at least 1 at high confidence
- 120 genes show no iCNA in matched normals
- 74 do not match known CNV site*

## GBE1, 3p12, recurrent iCNA

- GBE1:  glucan (1,4-alpha) branching enzyme 1
- glycogen storage disease type IV
- iCNA in 3 samples, all high-confidence (95% CI)

**GBE1: one of 13 differentially-expressed genes on chr3 vs. normal ovary (cDNA array)**

## NF1 is focus of iCNA, deletion

- NF1:  neurofibromatosis 1
- iCNA in 3 samples, 2 high-confidence (95% CI)

## NF1 deletion mutations in GBMassociation with altered transcript

## NF1 loss recently reported in serous ovarian carcinoma

**~22% of tumors have NF1 deletion or splice mutation leading to low expression **

## Search systematically for intragenic CNA:  Cancer Genome Atlas

_**Strategy 1**_**:**  Statistical approach to identifying iCNA in whole-genome copy-number data
_**Strategy 2**_**:** Integration of exon expression to identify clearly expressed transcripts with exon imbalance
_**Strategy 3**_**:** **Custom high density CGH array: confirm intragenic CNA and map at exon-level resolution**

**Notes:**
KP fusion has been found only once but:
One other constitutively active deletion mutant PDGFRA has been reported in GBM
like EGFRvIII, PDGFRA deletion is associated with amplification
PDGFRA regional amplification is seen in XX% of GBM
2/5 tumors with PDGFRA regional amplification show deletion mutation of PDGFRA
Jak3-SPTBN4 in glioma cell line and HCK truncation mutant in glioblastoma tumor also discovered 
In GBM, TK mutation by rearrangement (EGFRvIII) exceeds point mutation in frequency
Multiple TK mutations in the same tumor may point to a more general phenomenon of multiple and redundant mechanisms of signaling activation.

## TCGA high-density CGH array

- 244K platform (initially)
- Stable discovery probeset
- 6449 candidate fusion genes: 120K probes, ~**2,400bp**
- Tyrosine and other kinases:  80K, **~700bp**
- Flexible (adaptive) probesets
- “zoom in” on sites of recurrent iCNA
- Custom array designed per-batch
- Adaptive analysis 40K  **~700bp**

## whole genome vs custom array

**WG**

**CA**

## High-density custom array forintragenic deletions, translocations

- High-density custom array forintragenic deletions, translocations

## High-density custom array forintragenic deletions, translocations

_**Custom array:  580bp**_**  **resolution vs**  **_**whole genome 10,000bp**_

- High-density custom array forintragenic deletions, translocations

## Custom array coverage, 6400 select genes

**Mean probe interval (log 10 scale)**

**~700bp**

**~2.4kb**

**Custom array + 244k**

## Custom array vs. 244K array, 6400 select genes

**~9.0kb**

**~700bp**

**~2.4kb**

**Mean probe interval (log 10 scale)**

**Custom array + 244k**
**244k alone**

## Custom array vs. 1M array

**~700bp**

**~9.0kb**

**~1.5kb**

**Mean probe interval (log 10 scale)**

**Custom array + 244k**
**244k array alone**
**1M array alone**

## Summary

- Intragenic CNA can mark unbalanced translocations and fusion genes--  have validated this approach
- Statistical approach to intragenic CNA is important to define driver events from background noise: ~200 genes from GBM
- Integration of exon expression analysis identifies 82 genes in which CNA breakpoint defines a truncated transcript
- These are lead candidates RACE  or other sequencing strategies to identify translocation/fusion partners.
- Fine-mapping of breakpoints: custom adaptive array

## Acknowledgements

_**Memorial Sloan-Kettering**_
**Alicia Pedraza,Tatsuya Ozawa**
**Marc Ladanyi**
**Eric Holland**

_***The Cancer Genome Atlas Network***_
_***Glioblastoma Sample Source Sites***_