Advances in Understanding

Structural Aberrations in Cancer -

Utilization of CGH

Not approved for use in diagnostic

procedures
1
Structural Aberrations in Cancer

Chromosomal aberrations are one

of the hallmarks of cancer

Extensive catalogs of Cancer

cytogenetics aberrations available:
Mitelman Database
cGAP
Sanger Institute
COSMIC
Etc.

Not approved for use in diagnostic

procedures
2
Technology Advancements: Smaller Aberrations

3-5Mb 50-100kb 5-10Mb 0.5Mb >60bp 1bp

Not approved for use in diagnostic

procedures
3
Needs/Challenges of Cancer Research Community

What is aCGH and how does it work?

Can aCGH technology address cancer challenges

(mosaicism, complex rearrangements, LOH, FFPE, etc)?

How do I handle copy number variants (CNVs) and

differentiate between benign/pathogenic changes?
How can I target probe content to interrogate genomic
regions of interest to my lab?

Not approved for use in diagnostic

procedures
4
Needs/Challenges of Cancer Research Community

What is aCGH and how does it work?

Can aCGH technology address cancer challenges

(mosaicism, complex rearrangements, LOH, FFPE, etc)?

How do I handle copy number variants (CNVs) and

differentiate between benign/pathogenic changes?
How can I target probe content to interrogate genomic
regions of interest to my lab?

Not approved for use in diagnostic

procedures
5
CGH Workflow

Control
gDNA

Sample
gDNA

Not approved for use in diagnostic

procedures
6
CGH Data Interpretation
Genet Med. 2008 Apr;10(4):278-89.

Trisomy 21

Not approved for use in diagnostic

procedures
7
aCGH Detects Net Copy Changes
 Technology Advancements: CGH vs. Karyotype

10.9Mb Deletion 7.2Mb Duplication

60 genes
Not approved for use in diagnostic
procedures
9
Technology Advancements: CGH vs. Karyotype

Miller el. al. Amer J. Human Genet 86, 749764, May 14, 2010 749

Not approved for use in diagnostic

procedures
10
Analysis of a Cytogenetic Sample

NA14117 46,XY,del(5)(qter>p14:).ishdel(5)
(D5S23-)

Chromosome view Gene View

Higher Density Higher Resolution

Genomics. 2007 May;89(5):647-53. Epub 2007 Feb 2.

Not approved for use in diagnostic

procedures
12
Needs/Challenges of Cancer Research Community

What is aCGH and how does it work?

Can aCGH technology address cancer challenges

(mosaicism, complex rearrangements, LOH, FFPE, etc)?

How do I handle copy number variants (CNVs) and

differentiate between benign/pathogenic changes?
How can I target probe content to interrogate genomic
regions of interest to my lab?

Not approved for use in diagnostic

procedures
13
Range of Challenges for aCGH

Adapted from Nat Genet. 2005 Jun; 37 Suppl:S11-7

Not approved for use in diagnostic

procedures
14
Needs/Challenges of Cancer Research Community

What is aCGH and how does it work?

Can aCGH technology address cancer challenges

(mosaicism, complex rearrangements, LOH, FFPE, etc)?

How do I handle copy number variants (CNVs) and

differentiate between benign/pathogenic changes?
How can I target probe content to interrogate genomic
regions of interest to my lab?

Not approved for use in diagnostic

procedures
15
Challenges: Low-level Mosaicism

Aim:
To identify the 10-15% missing alterations
in EXT mutation-negative Multiple osteo-
chondroma (MO) cases.

Method:
Custom 4x44k CGH array, tiling EXT1,
EXT2 and 68 candidate genes
Screen 17 patients with previously
undetected mutations to identify other
causative aberrations

Results:
Somatic mosaicism in EXT genes in 3
previously negative cases.
Aberrations detected at 10-15%
mosaicism.

Hum. Mutat., 32: E2036E2049. doi: 10.1002/humu.21423

Not approved for use in diagnostic

procedures
16
Challenges: Low-level Mosaicism

30% tumor vs. pooled REF

Affymetrix
Illumina
Agilent

Agilent
Nimblegen
30% purity

Curtis et al. BMC Genomics Dec 09

Not approved for use in diagnostic

procedures
17
Needs/Challenges of Cancer Research Community

What is aCGH and how does it work?

Can aCGH technology address cancer challenges

(mosaicism, complex rearrangements, LOH, FFPE, etc)?

How do I handle copy number variants (CNVs) and

differentiate between benign/pathogenic changes?
How can I target probe content to interrogate genomic
regions of interest to my lab?

Not approved for use in diagnostic

procedures
18
Challenges: Complex Rearrangements

Complex rearrangements can be

detected by high resolution custom array
that spans the MECP2 locus.

The duplications and triplications are

detected by arrays while inversion were
detected by sequencing the break point.

Duplication Triplication Inversion-Dup

Not approved for use in diagnostic
procedures
19
Needs/Challenges of Cancer Research Community

What is aCGH and how does it work?

Can aCGH technology address cancer challenges

(mosaicism, complex rearrangements, LOH, FFPE, etc)?

How do I handle copy number variants (CNVs) and

differentiate between benign/pathogenic changes?
How can I target probe content to interrogate genomic
regions of interest to my lab?

Not approved for use in diagnostic

procedures
20
Challenges: Copy-Neutral Loss of Heterozygosity

# SNPs Genome 17,000,000

Overlap Alu/Rsa 800,000
Polymorphic by HapMap 124,000
MAF >5% 65,000

Resolution 5-10Mb

Not approved for use in diagnostic

procedures
22
Challenges: Copy-Neutral Loss of Heterozygosity

Clonal Fraction
is reported out in Copy Number LOH Copy Number LOH
the software

Not approved for use in diagnostic

procedures
23
Challenges: Copy-Neutral Loss of Heterozygosity

~85% major clone fraction (AML case)

3
2
1
0

Mix of Major
Genotypes Genotypes Genotypes A , B
and Minor
AAA, AAB, ABB, BBB AA, AB, BB
clone

Not approved for use in diagnostic

procedures
24
Needs/Challenges of Cancer Research Community

What is aCGH and how does it work?

Can aCGH technology address cancer challenges

(mosaicism, complex rearrangements, LOH, FFPE, etc)?

How do I handle copy number variants (CNVs) and

differentiate between benign/pathogenic changes?
How can I target probe content to interrogate genomic
regions of interest to my lab?

Not approved for use in diagnostic

procedures
25
FFPE Samples using ULS Labeling

Not approved for use in diagnostic

procedures
26
FFPE Samples using ULS Labeling

Chromosome 17 of two different FFPE breast cancer (infiltrating ductal carcinoma) samples. DNA Analytics settings: 0.2 Mb moving
average, ADM-2, threshold 5, filter: 2 probes, 0.25 log2 ratio.

Not approved for use in diagnostic

procedures
27
FFPE Samples using ULS Labeling

Agilent 180K

Nimblegen 135K

Affymetrix OncoScan
330,000 MIP probes

Krijgsman et al. Genes Chromosomes & Nasri et al. Cancer Genet Cytogenet Apr
Cancer Dec 2011 2010

Not approved for use in diagnostic

procedures
28
Needs/Challenges of Cancer Research Community

What is aCGH and how does it work?

Can aCGH technology address cancer challenges

(mosaicism, LOH, FFPE, limited DNA, etc)?
How do I handle copy number variants (CNVs) and
differentiate between benign/pathogenic changes?

How can I target probe content to interrogate genomic

regions of interest to my lab?

Not approved for use in diagnostic

procedures
29
Normal Copy Number Variants (CNVs)
Unpublished data

Nature Genetics 2004; 36 [9] Amer J Hum Genet 2008 (82) 685695

Confidentiality Label
30 July 18, 2013
Normal Copy Number Variants (CNVs)
CNVs account for greater genomic variation than SNPs
CNVs associated with diverse diseases (Autism, Schizophrenia, Crohns disease)

Nature 2011 (470) 59-65.

Confidentiality Label
31 July 18, 2013
CNV Consideration for Targeted Designs

Microarray Content (to include CNV or not)

Reference DNA (Single vs. Multiple)

Confirmation (DGV, FISH, Parental, MLPA, etc)

International Databases/ Consortia

Confidentiality Label
32 July 18, 2013
 CNV Consideration for Targeted Designs

MLL CCD5 CBL2

CGH probes
SNP probes

Backbone Backbone

Not approved for use in diagnostic

procedures
33
 CNV Consideration for Targeted Designs

Upload
custom
tracks

Link out from genes associated with Link out to and compare
aberrations to external Databases: DGV, aberrations in DGV and Preloaded tracks:
OMIM, Entrez UCSC genome browser DGV-CNV

Not approved for use in diagnostic

Slide 11 out of 15 procedures
Needs/Challenges of Cancer Research Community

What is aCGH and how does it work?

Can aCGH technology address cancer challenges

(mosaicism, LOH, FFPE, limited DNA, etc)?
How do I handle copy number variants (CNVs) and
differentiate between benign/pathogenic changes?

How can I target probe content to interrogate

genomic regions of interest to my lab?

Not approved for use in diagnostic

procedures
35
Custom Designs: Where to Begin?

Discovery (gene-centric or random tiling) vs. Targeted Design?

Format # Probes

Not approved for use in diagnostic

procedures
36
Free Design Iteration: eArray

Genomic Tiling
Probes

Repeats

HD Probe Search
Probes

Repeats

Based on Tm, GC content, hairpin G, sequence complexity, homology with the rest of the reference
genome. Followed by pair-wise reduction based on the user-selected average HD probe spacing per
interval or the total number of HD probes

Not approved for use in diagnostic

procedures
37
Free Design Iteration: eArray

Empirical
eArray Validation eArray

Not approved for use in diagnostic

procedures
38
Free Design Iteration: CLL and GLI3

Mapping of 13q14 deletions in 41 CLL

GLI3 Targeted CGH Array
cases that had detectable deletions

Journal Mol.Diagnostics, Vol. 11(1): January 2009 J Med Genet 2007 44 (1), e59

Not approved for use in diagnostic

procedures
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Free Design Iteration: Ch18 Array

Heard P. et al. Am J Med Genet A. 2009 July; 149A(7): 14311437.

Not approved for use in diagnostic

procedures
40
Free Design Iteration: Dystrophin gene

del Gaudio D et al. Human Mutation 29(9),1100-1107,2008

Not approved for use in diagnostic

procedures
41
Single Cell Analysis <24 hrs

Not approved for use in diagnostic

procedures
42
Needs/Challenges of Cancer Research Community

What is aCGH and how does it work?

Can aCGH technology address cancer challenges

(mosaicism, LOH, FFPE, limited DNA, etc)?
How do I handle copy number variants (CNVs) and
differentiate between benign/pathogenic changes?

How can I target probe content to interrogate genomic

regions of interest to my lab?

Not approved for use in diagnostic

procedures
43
Microarray Workflow
Conclusions

aCGH is well-suited for challenging cancer samples (LOH,

FFPE, limited input, etc).
It is simple/free to iterate probe content and to design targeted
aCGH microarrays for high-resolution coverage.
Agilent offers a complete workflow solution including
automation, software, customization, instrumentation, reagents.

Not approved for use in diagnostic

procedures
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