Analysis of DNA sequence copy number in archival formalin-fixed, paraffin-
embedded (FFPE) tumor samples by oligo array CGH
Dione K. BaileyAnniek De WitteOctober 9, 2007
Analysis of FFPE samples by aCGH
October 2007Page 2
Agenda
•
Oligo aCGH Review•
Problems with Formalin-Fixed Paraffin Embedded (FFPE) Samples
•
Advantages of New Labeling Protocol •
Proof of Principle Experiments
•
Examples of FFPE Samples from Different Sources and Ages
•
Review Product Structure•
Questions
Analysis of FFPE samples by aCGH
October 2007Page 3
Agenda
•
Oligo aCGH Review•
Problems with Formalin-Fixed Paraffin Embedded (FFPE) Samples
•
Advantages of New Labeling Protocol •
Proof of Principle Experiments
•
Examples of FFPE Samples from Different Sources and Ages
•
Review Product Structure•
Questions
Analysis of FFPE samples by aCGH
October 2007Page 4
Agilent Oligo aCGH Solution•
Specialized probe database supporting custom and catalog array designs
•
Simple CGH Assay Protocol
•
New FFPE Labeling Protocol
•
Data Analysis: Quality Metrics and Biological Interpretation
•
Standard technology platform enables integrated genomics (GE, CGH and miRNA)
Oligo aCGH Labeling,
Hybridization and washing Kits Genome-wide Array
Custom Array
Power of Integration – Infrastructure & Knowledge
CGH Analytics Software
Analysis of FFPE samples by aCGH
October 2007Page 5
High Density and Multi-pack Arrays
• Low Density • 8 x 15K
• Medium Density • 2 x 105K• 4 X 44K
• High Density• 244K features/Array
• 244K, 2x105K, and 4x44K Human and Mouse Catalog arrays available • 244K and 2x105K Rat Catalog arrays available• Custom arrays available for all formats
Analysis of FFPE samples by aCGH
October 2007Page 6
Agenda
•
Oligo aCGH Review•
Problems with Formalin-Fixed Paraffin Embedded (FFPE) Samples
•
Advantages of New Labeling Protocol •
Proof of Principle Experiments
•
Examples of FFPE Samples from Different Sources and Ages
•
Review Product Structure•
Questions
Analysis of FFPE samples by aCGH
October 2007Page 7
•
Cross-linking between nucleic acid strands
•
DNA adducts with histones or nucleic acid binding proteins
•
DNA strand breaks•
Acid depurination of DNA•
Damage increases with amount of time after exposure to fixative
•
Damage dependent on formalin fixing protocol used
Poor DNA Quality
~ 500
1Kb
Ladd
er
12216
Pro
meg
a F
Froz
en
FFP
E_a
Froz
en
Froz
en
1.2% agarose gel, 20 ng DNA, Sybr Gold stainFF
PE
_b
FFP
E_a
FFP
E_b
FFP
E_a
FFP
E_b
DNA from blood or frozen tissues: High Molecular Weight
DNA from FFPE tissues: Low Molecular Weight
Analysis of FFPE samples by aCGH
October 2007Page 8
Specific activity (pmol/μg)
0
5
10
15
20
25
30
35
40
45
50
Frozen
_1 C
y-5
FFPE_1 C
y-5
Frozen
_2 C
y-3
FFPE_2 C
y-3
Frozen
_3 C
y-5
FFPE_3 C
y-5
Frozen
_4 C
y-3
FFPE_4 C
y-3
Frozen
_5 C
y-5
FFPE_5 C
y-5
Frozen
_6 C
y-3
FFPE_6 C
y-3
Frozen
_7 C
y-5
FFPE_7 C
y-5
Frozen
_8 C
y-3
FFPE_8 C
y-3
Frozen
_9 C
y-5
FFPE_9 C
y-5
Frozen
_10 C
y-3
FFPE_10 C
y-3
Frozen
_11 C
y-5
FFPE_11 C
y-5
Frozen
_12 C
y-3
FFPE_12 C
y-3Low Specific Activity Compared to Frozen Samples with Klenow Labeling
Frozen Cy-5
FFPE Cy-5
Frozen Cy-3
FFPE Cy-3
Analysis of FFPE samples by aCGH
October 2007Page 9
Noisy Array DataDLRSD = Derivative Log2
Ratio SpreaD = probe-to-probe log ratio noise
Cell line DNA (colon cancer)
DLRSD 0.16
FFPE DNA (breast cancer)
DLRSD 0.55
Analysis of FFPE samples by aCGH
October 2007Page 10
Agenda
•
Oligo aCGH Review•
Problems with Formalin-Fixed Paraffin Embedded (FFPE) Samples
•
Advantages of New Labeling Protocol•
Proof of Principle Experiments
•
Examples of FFPE Samples from Different Sources and Ages
•
Review Product Structure•
Questions
Analysis of FFPE samples by aCGH
October 2007Page 11
New Oligo aCGH Labeling Kit for FFPE Samples •
New protocol based on a non-enzymatic labeling method:–
Avoids enzyme processivity
problems due to crosslinking/complexing–
Independent of fragment length•
Universal Linkage System (ULS™) is a platinum-based labeling technology that allows for the direct coupling of Cy-3 or Cy-5 labels to DNA
Source www.kreatech.com
Analysis of FFPE samples by aCGH
October 2007Page 12
De-paraffinization
(~2 20µm slices)
De-crosslinking + proteinaseK
DNA purification (Qiagen)
2 hr Restriction Digestion
Heat Fragmentation (from 0 to 10’)
Random primers + 3’
Denaturation
2 hr Klenow Labeling
30 min ULS™
Labeling
Microcon clean-up
KreaPURE clean-up
40 hr Hybridization
Wash
Scan
Comparison of Enzymatic and New ULSTM-based Protocols
ENZYMATIC ULS™
+ KreaBLOCK
Analysis of FFPE samples by aCGH
October 2007Page 13
Oligo aCGH Labeling Kit for FFPE Samples Protocol
1.
De-paraffinization10 minutes at 90°C, spin, ice, remove wax disc
2.
DNA isolationBased on the protocol described by van Beers et al. (Br J Cancer. 2006 Jan 30;
94(2): 333-7)•
NaSCN
treatment (1 night)•
Proteinase
K (2 nights)•
Qiagen
DNeasy
Blood & Tissue kit (replace wash 2 with 80% EtOH, elute in water)
Analysis of FFPE samples by aCGH
October 2007Page 14
3.
Heat fragmentation
4.
Labeling(500 ng for 4-packs, 1 µg for 2-packs, 2 µg for 1-packs)
30 minutes at 85°C
5.
Free dye removal with Agilent- KREApureTM
columns
Protocol (Continued)
1018
1636
12218
~500
1 Kb LadderIntact gDNA10’ at 95°CAfter labeling
Cold labeling
Average Molecular
WeightSample Type Recommended
Fragmentation Time
> 10 KB Intact gDNA 10 minutes at 95 oC> 7 KB FFPE sample 5 minutes at at 95 oC< 7 KB FFPE sample No fragmentation
Analysis of FFPE samples by aCGH
October 2007Page 15
Agenda
•
Oligo aCGH Review•
Problems with Formalin-Fixed Paraffin Embedded (FFPE) Samples
•
Advantages of New Labeling Protocol•
Proof of Principle Experiments
•
Examples of FFPE Samples from Different Sources and Ages
•
Review Product Structure•
Questions
Analysis of FFPE samples by aCGH
October 2007Page 16
Enzymatic vs. ULS™ Labeling: FFPE Lung Chromosome 16Enzymatic ULS™
DLRSD 0.46 DLRSD 0.29
Analysis of FFPE samples by aCGH
October 2007Page 17
Normal FFPE Tissues Hybridized Against Sex Mismatched Reference DNA
Autosomal probes
X-chromosome probes
Log2
ratio
Num
ber o
f pro
bes
Legend:
Promega
M –
Promega F
FFPE normal tissue (2 Colon, 1 Lung, 1 Lymph Node) –
Promega sex mismatched
Autosomal probes ratios:
1 => log2
ratio = 0
X-chromosome probes ratios:
0.5 => log2
ratio = -1
Analysis of FFPE samples by aCGH
October 2007Page 19
Agenda
•
Oligo aCGH Review•
Problems with Formalin-Fixed Paraffin Embedded (FFPE) Samples
•
Advantages of New Labeling Protocol•
Proof of Principle Experiments
•
Examples of FFPE Samples from Different Sources and Ages
•
Review Product Structure•
Questions
Analysis of FFPE samples by aCGH
October 2007Page 20
HER-2 Gene Amplification in 1-, 4-, and 10-Year Old FFPE Breast Cancer Samples on 4x44K arrays
Source: ICR Norway
Analysis of FFPE samples by aCGH
October 2007Page 21
Point to point
All probes shown
High resolution breakpoint mapping
~10 Kb
1Mb moving average
FFPE Breast Cancer Sample on Agilent 1x244K Arrays
Point to point
Analysis of FFPE samples by aCGH
October 2007Page 22
Summary of Technical Review
•DNA extracted from FFPE tissues is often degraded. Using the new oligo aCGH Labeling kit for FFPE samples, we obtained good quality data for more than 90% of the samples tested in-house and at different test sites which consisted of different ages and tissue types (~125 samples processed to date).•
When DNA isolated from normal FFPE tissue was hybridized against sex-
mismatched pooled reference DNA, the log2 ratios were close to the expected values.•
The same amplifications and deletions could be detected in DNA extracted from FFPE and fresh frozen tissue for the same tumor.•
Previously known aberrations associated with breast cancer, such as HER-2 (human epithelial receptor 2) could be detected in DNA isolated from breast tumor FFPE tissue.•
This new labeling method can be applied to a wide range of challenging sample types, including solid tumor tissues, heterogeneous samples, tissue biopsies, and archived formalin-fixed paraffin embedded (FFPE) samples.
Analysis of FFPE samples by aCGH
October 2007Page 23
Agenda
•
Oligo aCGH Review•
Problems with Formalin-Fixed Paraffin Embedded (FFPE) Samples
•
Advantages of New Labeling Protocol •
Proof of Principle Experiments
•
Examples of FFPE Samples from Different Sources and Ages
•
Review Product Structure•
Questions
Analysis of FFPE samples by aCGH
October 2007Page 24
Product Structure
Part Number Product Name5190-0419* Oligo aCGH Labeling Kit for FFPE samples
5190-0418 Genomic DNA Purification Module
*This kit can be used to label 5 slides:5 -
244k arrays; 10 -
105k arrays; 20 -
44K arrays; 40 -
15k arraysIt also contains 12 purification columns.
Additional purification columns should be ordered for multi-pack arrays (Genomic DNA Purification Module -
5190-0418).
Analysis of FFPE samples by aCGH
October 2007Page 25
Introductory Promotion: 25% Discount on FFPE Kit + Catalog or Custom aCGH ArraysEffective August 15 –
December 31, 2007
Catalog Arrays:•
Up to 5 catalog aCGH kits plus FFPE labeling kit
Custom Arrays:•
Up to 5 custom aCGH slides plus FFPE labeling kit
Analysis of FFPE samples by aCGH
October 2007Page 26
Agenda
•
Oligo aCGH Review•
Problems with Formalin-Fixed Paraffin Embedded (FFPE) Samples
•
Advantages of New Labeling Protocol •
Proof of Principle Experiments
•
Examples of FFPE Samples from Different Sources and Ages
•
Review Product Structure•
Questions
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