Analysis of DNA sequence copy number in archival formalin-fixed, paraffinembedded. Dione K. Bailey Anniek De Witte October 9, 2007

Transcription

1 Analysis of DNA sequence copy number in archival formalin-fixed, paraffinembedded (FFPE) tumor samples by oligo array CGH Dione K. Bailey Anniek De Witte October 9, 2007

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 Page 2

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 Page 3

4 Agilent Oligo acgh Solution Oligo acgh Labeling, Hybridization and washing Kits Genome-wide Array Custom Array Specialized probe database supporting custom and catalog array designs Simple CGH Assay Protocol Power of Integration Infrastructure & Knowledge New FFPE Labeling Protocol Data Analysis: Quality Metrics and Biological Interpretation CGH Analytics Software Standard technology platform enables integrated genomics (GE, CGH and mirna) Page 4

5 High Density and Multi-pack Arrays High Density 244K features/array Medium Density 2 x 105K 4 X 44K Low Density 8 x 15K 244K, 2x105K, and 4x44K Human and Mouse Catalog arrays available 244K and 2x105K Rat Catalog arrays available Custom arrays available for all formats Page 5

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 Page 6

7 Poor DNA Quality 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 Kb Ladder Promega F Frozen FFPE_a FFPE_b Frozen FFPE_a FFPE_b Frozen FFPE_a FFPE_b DNA from blood or frozen tissues: High Molecular Weight ~ 500 DNA from FFPE tissues: Low Molecular Weight 1.2% agarose gel, 20 ng DNA, Sybr Gold stain Page 7

8 Low Specific Activity Compared to Frozen Samples with Klenow Labeling Frozen Cy-5 Specific activity (pmol/μg) FFPE Cy-5 Frozen Cy-3 FFPE Cy Frozen_1 Cy-5 FFPE_1 Cy-5 Frozen_2 Cy-3 FFPE_2 Cy-3 Frozen_3 Cy-5 FFPE_3 Cy-5 Frozen_4 Cy-3 FFPE_4 Cy-3 Frozen_5 Cy-5 FFPE_5 Cy-5 Frozen_6 Cy-3 FFPE_6 Cy-3 Frozen_7 Cy-5 FFPE_7 Cy-5 Frozen_8 Cy-3 FFPE_8 Cy-3 Frozen_9 Cy-5 FFPE_9 Cy-5 Frozen_10 Cy-3 FFPE_10 Cy-3 Frozen_11 Cy-5 FFPE_11 Cy-5 Frozen_12 Cy-3 FFPE_12 Cy-3 Page 8

9 Noisy Array Data DLRSD = Derivative Log 2 Ratio SpreaD = probe-to-probe log ratio noise FFPE DNA (breast cancer) DLRSD 0.55 Cell line DNA (colon cancer) DLRSD 0.16 Page 9

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 Page 10

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 Page 11

12 Comparison of Enzymatic and New ULS TM -based Protocols 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 + KreaBLOCK ENZYMATIC Scan ULS Page 12

13 Oligo acgh Labeling Kit for FFPE Samples Protocol 1. De-paraffinization 10 minutes at 90 C, spin, ice, remove wax disc 2. DNA isolation Based on the protocol described by van Beers et al. (Br J Cancer 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) Page 13

14 Protocol (Continued) 10 at 95 C Intact gdna 1 Kb Ladder Cold labeling After labeling 3. Heat fragmentation Average Molecular Weight Sample Type Recommended Fragmentation Time > 10 KB Intact gdna 10 minutes at 95 o C > 7 KB FFPE sample 5 minutes at at 95 o C < 7 KB FFPE sample No fragmentation 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- KREApure TM columns ~500 Page 14

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 Page 15

16 Enzymatic vs. ULS Labeling: FFPE Lung Chromosome 16 Enzymatic ULS DLRSD 0.46 DLRSD 0.29 Page 16

17 Normal FFPE Tissues Hybridized Against Sex Mismatched Reference DNA Autosomal probes Autosomal probes ratios: 1 => log 2 ratio = 0 Number of probes X-chromosome probes X-chromosome probes ratios: 0.5 => log 2 ratio = -1 Legend: Promega M Promega F FFPE normal tissue (2 Colon, 1 Lung, 1 Lymph Node) Promega sex mismatched Log 2 ratio Page 17

18 Frozen vs. FFPE From the Same Tumor Page 18

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 Page 19

20 HER-2 Gene Amplification in 1-, 4-, and 10-Year Old FFPE Breast Cancer Samples on 4x44K arrays Source: ICR Norway Page 20

21 FFPE Breast Cancer Sample on Agilent 1x244K Arrays All probes shown ~10 Kb High resolution breakpoint mapping Point to point Point to point 1Mb moving average Page 21

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 sexmismatched pooled reference DNA, the log 2 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. Page 22

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 Page 23

24 Product Structure Part Number Product Name * Oligo acgh Labeling Kit for FFPE samples Genomic DNA Purification Module *This kit can be used to label 5 slides: 5-244k arrays; k arrays; 20-44K arrays; 40-15k arrays It also contains 12 purification columns. Additional purification columns should be ordered for multi-pack arrays (Genomic DNA Purification Module ). Page 24

25 Introductory Promotion: 25% Discount on FFPE Kit + Catalog or Custom acgh Arrays Effective 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 Page 25

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 Page 26