Agilent Pathology Solutions

Dako

IQFISH on Dako OmnisPanel for Lung Cancer

P R O D U C T I N F O R M AT I O N

ALK, ROS1, RET and MET IQFISHDako Omnis

FAST RESULTS

Integrated into your IHC workflow on Dako OmnisFast, high-quality FISH

Dako Omnis, a fully automated, walk-away solution for advanced staining, provides a fully automated, FISH solution with high efficiency and flexibility. Automated slide throughput depends on length of protocol, slide capacity and reagent positions. With the short FISH protocol and high slide and reagent capacity, Dako Omnis processes FISH slides in an IHC-like turnaround time – fast and with high quality.

IQFISH panel for lung cancer automated on Dako OmnisThe IQFISH panel for lung cancer enables the detection of rearrangements involving the ALK, ROS1 and RET genes, and the detection of MET gene amplifications by fluorescence in situ hybridization (FISH). The probes are designed using the groundbreaking, oliogonucleotide-based SureFISH technology and utilize the formamide-free IQFISH fast hybridization buffer. The probes are for use on lung paraffin-embedded (FFPE) tissue sections.

FFPE sample stained with ALK IQFISH dual color, break-apart probe

Cells show ALK gene rearrangement.

ALK IQFISH Break-Apart Probe (Dako Omnis)

ROS1 IQFISH Break-Apart Probe (Dako Omnis)

FFPE sample stained with ROS1 IQFISH dual color, break-apart probe

Cells show ROS1 gene rearrangement.

RET IQFISH Break-Apart Probe (Dako Omnis)

FFPE sample stained with RET IQFISH dual color, break-apart probe

Cells show RET gene rearrangement.

MET IQFISH Probe with CEP7 (Dako Omnis)

FFPE sample stained with MET/ CEP7 IQFISH amplification probe

Cells show MET gene amplification.

“ It is compelling to move to the Dako Omnis platform from our current because it is automated, and we today do it manually. The result is as good as the one we currently have [from another vendor], but requires less hands-on time.” Aleksandra Kolaric, University Örebro, Sweden

High efficiency

Run FISH simultaneously with IHC Unlike other systems, Dako Omnis is designed for simultaneous FISH and IHC runs. FISH slides can be run with minimal impact to IHC throughput on Dako Omnis, as shown in the chart to the right. The short FISH protocol enables a capacity of up to 45 FISH slides per day with an overnight run.

The protocols developed for use with the ALK, ROS1, RET, and MET probes on Dako Omnis share ISH Accessories reagents and allow the probes to be run together in the same rack (up to five slides are processed per rack). The ability to batch in groups of five reduces the reagents and time needed to process the different assays. Furthermore, these probes can be run alongside HER2 IQFISH pharmDx (Dako Omnis), maximizing FISH utilization on the instrument.

Figure 1. The IQFISH fast hybridization buffer reduces hybridization time to 75 minutes compared to overnight hybridization with traditional formamide-based buffer.

Figure 2. Selection of Dako Omnis slide throughput scenario.

The short FISH protocol gives you a FISH solution with high efficiency and flexibility.

IHC-like turnaround time The high-throughput Dako Omnis combined with the IQFISH fast hybridization buffer gives labs an IHC-like turnaround time for FISH. IQFISH fast hybridization buffer reduces hybridization to 75 minutes, making the overall turnaround time for FISH just 4 hours.

High flexibility

Workflow alignment across FISH applications

Deparaffinization

75 minutes

Overnight hybridization

IQFISH hybridization

buffer with 75 minutes

hybridization

Hours0 2 4 6 8 10 12 14 16 18

Traditional formamide-

based buffer

Digestion Hybridization Mounting

Heat pre-treatment Denaturation Stringent wash

IQ–Instant Quality FISH IHC-like TAT for FISH

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5FISH

105IHC 95

IHC 85IHC

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OR OR

Scenario 2 Scenario 3

Same day* FISH Same day* IHC

10FISH

*8-hour workday

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10FISH

*8-hour workday

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10FISH

*8-hour workday

FISH protocol

IHC protocol

0 1 2 3 4Time (Hours)

High signal-to-noise ratio by repeat and blocker-free designIQFISH are designed in silico and chemically synthesized using Agilent’s high-fidelity, oliogonucleotide library synthesis (OLS) technology.

FISH probes from other vendors are purified from bacterial library clones (BAC) harboring human genomic DNA fragments. They therefore include repetitive sequences that can bind nonspecifically throughout the nuclei, producing a hazy background. Consequently, BAC-based probes usually come premixed with Cot-1 DNA, which blocks the background signals from repeated sequences, and also suppresses the overall hybridization signal.

Agilent IQFISH probes address this by targeting only the unique sequences. During probe design, all repetitive elements are removed. This increases signal specificity and decreases hybridization background. It also eliminates the need for blocking agent and associated signal suppression. The combined effect is a high signal-to-noise ratio and easy visualization.

Unique micro-gap designAgilent’s oliogonucleotide-based SureFISH technology permits probe placement with base-pair-level precision. This allows a unique micro-gap design for ALK and RET IQFISH probes. Whereas the spacing between child probes with BAC-based probes is typically 100-300 kb, the spacing between Agilent’s child probes is only about 0.4 kb.

This design methodology is beneficial because the ALK-EML4 fusion is the result of an inversion between genes that are separated by approximately 12 Mb. Similarly, RET fusions with KIF5B are the result of an 11 Mb inversion. Detecting such intra-chromosomal inversions can be challenging due to the limited signal separation compared to a different chromosome.

Agilent’s micro-gap design provides tighter co-localization of orange-red and green signals in nuclei without the inversion, so cases with the inversion are easier and faster to analyze.

Easy, accurate and fast scoring Spend less time at the microscope thanks to high signal-to-noise ratio and micro-gap probe design

Figure 3. Oliogonucleotide-based FISH (oligo FISH) probe design strategy. Repetitive elements are identified and removed during probe design process.

Figure 4. An inversion event.

Figure 5. The IQFISH ALK probe (top left) shows tighter co-localized signals than the ALK BAC probe from vendor (bottom left). Right: Inversion positive nuclei easy to distinguish from negative nuclei with IQFISH ALK probe.

Genomic DNA

Oligo FISH probe

Standard FISH probe

Repetitive & non-unique sequences

Binds across genome yielding “hazy” background

Figure 6. Probe map and typical signal pattern for ALK, ROS1, RET and MET IQFISH probes.

Probe maps

ALK IQFISHprobe

Chr. 2

SHGC

-144

795

SHGC

-852

75

CentromereTelomereALK

0.4 kbALK 3’ BA 300 kb ALK 5’ BA 599 kb

3' 5'

Chr. 6

SHGC

-102

962

RH93

170

Centromere TelomereROS1

137 kbROS1 3’ BA 1099 kb ROS1 5’ BA 1153 kb

5'3'

MET IQFISHprobe

Chr. 7

STSG

5979

83

STSG

5982

93

Centromere TelomereMET

Chr7 CEP 767kb

7q31.2 MET 400 kb

3'5'

Chr. 10

SHGC

-889

4

SHGC

-836

59

Centromere TelomereRET

0.3 kb RET 3’ BA 602 kbRET 5’ BA 668 kb

3'5'

A typical signal pattern for negative specimens

A typical signal pattern for positive specimens

ROS1 IQFISHprobe

RET IQFISHprobe

The same high-quality IQFISH on Dako Omnis as manualPerforming FISH with the ALK, ROS1, RET, and MET IQFISH (Dako Omnis) probes together with all of the ISH accessory reagents and devices results in equivalent performance compared to the manual procedure. Nine NSCLC samples were processed both manually and on Dako Omnis. Probe signal intensity was evaluated on a 0-3 scale with: 0= no signal, 1= weak signal 2= moderate signal, 3= strong signal. For all samples evaluated, the staining intensity was moderate to strong with Dako Omnis processed samples giving slightly higher scores.

Furthermore, processing of the ALK, ROS1, RET, and MET IQFISH (Dako Omnis) probes is both repeatable and reproducible across multiple instruments.

The optimized probes and protocols for use on Dako Omnis provide high-intensity staining, work well across multiple tissue types, and provide reproducible results. Together these attributes can lead to reduced analysis time and fewer assay repeats.

Outstanding performance

Figure 7. Performing FISH on Dako Omnis gives green and orange-red signals that are equivalent to the manual protocol.

Figure 8. Images of IQFISH probes hybridized to negative (left column) and positive (right column) samples using Dako Omnis.

ALK RET

ROS1 MET

Negative specimen Positive specimen Negative specimen Positive specimen

Dako OmnisManualDako OmnisManual

Two IQFISH protocols are available for processing on Dako Omnis: The IQFISH and IQFISH (Extra Wash). The ALK IQFISH protocol provides a slightly faster run-time and allows for the processing of HER2 IQFISH pharmDx (Dako Omnis) slides in the same Dako Omnis Slide Rack. Using the IQFISH (Extra Wash) protocol may result in lower background on challenging samples. (Note that the two protocols cannot be run in the same Slide Rack.)

Filter recommendationsFilters are individually designed for specific fluorochromes and for each microscope. For the interpretation of IQFISH staining assays, the following combination of filters should be used:

• Specific DAPI filter• High-quality Cy3/FITC double filter (alternatively specific

Cy3 and FITC single filters)

Table 1. Simplified overview of the automated staining protocols for ALK IQFISH performed onboard Dako Omnis.

Two IQFISH protocols

Fluorochrome Excitation Wavelength Emission Wavelength

FITC 495 nm 517 nmCy3 547 nm 565 nm

For identifying the relevant tissue/regions for scoring, an oil-immersion 20x objective with a minimum numerical aperture (N.A.) of 0.75 is recommended.

For scoring signal patterns, an oil-immersion 60x or 100x objective with a numerical aperture (N.A.) of 1.4 is recommended.

Microscope objective recommendation

Table 1 Simplified overview of the automated staining protocols for ALK IQFISH performed onboard Dako Omnis

Step Reagent Time and Temperature

C°83 ,setunim 01)tnega gniraelc( yfiraelCgnixaweD

Target retrieval ISH Pre-Treatment Solution (Dako Omnis) 15 minutes, 97°C

Wash ISH Ethanol Solution, 96% (Dako Omnis) 2 × 3 minutes, 32°C

Digestion ISH Pepsin (Dako Omnis) 30 minutes

C°54 ,setunim 51—gniyrD

C°66 ,setunim 01—noitarutaneD

Hybridization ALK IQFISH Break-Apart Probe (Dako Omnis) 75 minutes, 45°C

Stringent wash ISH Stringent Wash Buffer (Dako Omnis) 10 minutes, 61°C

Cool down washALK IQFISH protocol: ISH Stringent Wash Buffer (Dako Omnis) Brief rinse, 1 cycle

ALK IQFISH (Extra Wash) protocol: Wash Buffer (Dako Omnis) Brief rinse, 2 cycles

2915

0 20

16JU

N01

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Agilent Pathology Solutions

Dako

Ordering information

Companion devices

Product Code Vol. & tests per unit

ALK IQFISH BA Probe (Dako Omnis) G111800-2 2 mL, 20 tests

ROS1 IQFISH BA Probe (Dako Omnis) G111801-2 2 mL, 20 tests

RET IQFISH BA Probe (Dako Omnis) G111802-2 2 mL, 20 tests

MET IQFISH Probe with CEP7 (Dako Omnis) G111803-2 2 mL, 20 tests

ISH Pepsin (Dako Omnis) GM302 7 mL, RTU, 20 tests

ISH Ethanol Solution, 96 (Dako Omnis) GM300 14 mL, RTU, 20 tests

ISH Pre-Treatment Solution (20x) (Dako Omnis) GM301 175 mL for 3.5 L bulk, 5-25 tests

FISH Stringent Wash Buffer (20x) (Dako Omnis) GM303 175 mL for 3.5 L bulk, 5-25 tests

Fluorescence Mounting Medium (Dako Omnis) GM304 0.8 mL, 20 tests

ISH Cleaning Solution (Dako Omnis) GC207 10 mL, 100 tests

Dako Omnis FISH Lid GC102 Box of 5, 25 tests

Dako Omnis Mixing Device GC116 1 pcs

Dako Omnis Mixing Device Dako Omnis FISH Lids Cy3/FITC double filter