Analysis of DNA sequence copy number in archival Anniek De ... · Problems with Formalin-Fixed...

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 2007

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


October 2007

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


October 2007

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


October 2007

Agenda

•



•



•


•


Questions


October 2007

•

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


October 2007

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


October 2007

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


October 2007

Agenda

•



•

Advantages of New Labeling Protocol•


•


•


Questions


October 2007

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

http://www.kreatech.com/


October 2007

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


October 2007

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)


October 2007

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


October 2007

Agenda

•



•



•


•


Questions


October 2007

Enzymatic vs. ULS™ Labeling: FFPE Lung Chromosome 16Enzymatic ULS™

DLRSD 0.46 DLRSD 0.29


October 2007

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


October 2007

Frozen vs. FFPE From the Same Tumor


October 2007

Agenda

•



•



•


•


Questions


October 2007

HER-2 Gene Amplification in 1-, 4-, and 10-Year Old FFPE Breast Cancer Samples on 4x44K arrays

Source: ICR Norway


October 2007

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


October 2007

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.


October 2007

Agenda

•



•



•


•


Questions


October 2007

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).


October 2007

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


October 2007

Agenda

•



•



•


•


Questions

Analysis of DNA sequence copy number in archival Anniek De ... · Problems with Formalin-Fixed...

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