Leica TA9217 User Manual

English

Leica HER2 FISH System - 30 Test Instructions For Use

For use on Leica Biosystems’ BOND-MAX and BOND-III System.

TA9217 is a uorescence in situ hybridization product designed to stain 30 tests (30 slides stained with LSI HER2/CEP17 Dual Probe).

IVD

Leica Biosystems Newcastle Ltd Balliol Business Park West Benton Lane Newcastle Upon Tyne NE12 8EW United Kingdom ( +44 191 215 4242

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Leica Biosystems Leica HER2 FISH System - 30 Test Instructions for Use TA9217 EN-CE-Rev_D 08/04/2013

Contents

Intended Use ...............................................................................................................................................................................3

For in vitro diagnostic use .............................................................................................................................................................................. 3

Required training ............................................................................................................................................................................................ 3

Summary and Explanation ........................................................................................................................................................3

Background .................................................................................................................................................................................................... 3

Clinical Concordance Summary Leica BOND-MAX System .......................................................................................................................... 4

Clinical Concordance Summary Leica BOND-III System ............................................................................................................................... 4

Principle of Procedure .................................................................................................................................................................................... 4

Components Provided .................................................................................................................................................................................... 5

Directions For Use .......................................................................................................................................................................................... 5

Storage and Stability ...................................................................................................................................................................................... 5

Specimen Preparation .................................................................................................................................................................................... 5

Warnings and Precautions .............................................................................................................................................................................6

Procedure ....................................................................................................................................................................................6

A. Reagents required but not supplied ...........................................................................................................................................................6

B. Equipment required but not supplied .........................................................................................................................................................6

C. Methodology .............................................................................................................................................................................................. 7

D. Bond Enzyme Pretreatment ....................................................................................................................................................................... 7

E. Default Staining Protocol ...........................................................................................................................................................................7

F. Procedure Steps ........................................................................................................................................................................................ 7

G. Slide Storage ............................................................................................................................................................................................. 8

Signal Assessment and Enumeration ......................................................................................................................................9

Recommended Method for LSI HER2 to CEP17 Ratio Determination ......................................................................................................... 10

Leica HER2 FISH System - 30 Test Interpretation Guide ......................................................................................................11

Sample Score Sheet .................................................................................................................................................................12

Quality Control ..........................................................................................................................................................................13

Limitations ................................................................................................................................................................................13

A. General Limitations ..................................................................................................................................................................................13

B. Product Specic Limitations .....................................................................................................................................................................14

Clinical Concordance of Leica HER2 FISH System - 30 Test to Abbott Molecular PathVysion HER-2 DNA Probe Kit ...14

2x2 Concordance Results Leica BOND-MAX System ................................................................................................................................. 15

2x2 Concordance Results Leica BOND-III System ...................................................................................................................................... 16

Precision Testing – Leica BOND-MAX System ......................................................................................................................17

A. Within Run Precision Study .....................................................................................................................................................................17

B. Within Instrument Precision Study ...........................................................................................................................................................17

C. Between Run Precision Study ................................................................................................................................................................. 17

D. Between Laboratory Precision Study ....................................................................................................................................................... 17

E. Between Observer Precision Study .........................................................................................................................................................17

F. Lot-to-Lot Precision Study ....................................................................................................................................................................... 18

Precision Testing – Leica BOND-III System ...........................................................................................................................18

G. Within Run Precision Study ..................................................................................................................................................................... 18

H. Within Instrument Precision Study ........................................................................................................................................................... 18

I. Between Run Precision Study .................................................................................................................................................................18

J. Between Laboratory Precision Study .......................................................................................................................................................18

K. Between Observer Precision Study .........................................................................................................................................................19

L. Lot-to-Lot Precision Study .......................................................................................................................................................................19

Assay Robustness ...................................................................................................................................................................19

Troubleshooting .......................................................................................................................................................................21

References ................................................................................................................................................................................23

License Agreement ..................................................................................................................................................................24

English

Leica Biosystems Leica HER2 FISH System - 30 Test Instructions for Use TA9217 EN-CE-Rev_D 08/04/2013

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Intended Use

For in vitro diagnostic use

The Leica HER2 FISH System - 30 Test is designed to detect amplication of the HER2/neu

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gene via uorescence in situ hybridization (FISH) in formalin-xed, parafn-embedded human

breast cancer tissue specimens. The Leica HER2 FISH System - 30 Test is indicated as an aid in the assessment of patients for whom Herceptin (see Herceptin

package insert). The Leica HER2 FISH System - 30 Test is not intended for use

®*

(trastuzumab) treatment is being considered

to screen for or diagnose breast cancer. All other available clinical information should also be

taken into consideration, such as tumor size, number of involved lymph nodes, and steroid

receptor status. No treatment decision for breast cancer patients should be based on HER2

gene amplication status alone.

Note: All of the patients in the Herceptin clinical trials were selected using an investigational immunocytochemical Clinical Trial Assay (CTA). None of the patients in those trials were selected using the Leica HER2 FISH System - 30 Test. The Leica HER2 FISH System - 30 Test has been compared to the Abbott Molecular PathVysion

®*

HER-2 DNA Probe Kit assay on an

independent set of samples and found to provide acceptably concordant results, as indicated

in the Clinical Concordance Summary. The actual correlation of the results of the Leica HER2 FISH System - 30 Test to clinical outcome has not been established.

* Herceptin® is a trademark of Genentech, Inc. and F. Hoffmann-La Roche Ltd. PathVysion® is a trademark of Abbott Molecular

Required training

Leica Biosystems will provide training in specimen preparation, assay procedure, and

interpretation of FISH testing of the HER2 gene for all users.

Summary and Explanation

Background

The HER2 gene, alternatively known as neu or c-erbB2, is located on the long arm of

chromosome 17 at position 17q11-12 (1). Both the HER2 gene and its 185 kD encoded protein have been shown to play a major role in malignant transformation and tumor progression of breast cancer (2).

HER2 functions as a prognostic marker, with gene amplication and protein over expression

being linked to an increased rate of disease recurrence and higher mortality. HER2 also functions as a predictive marker for selected systemic chemotherapy and targeted

treatments (3). Specically, amplication of the HER2 gene has been shown to be an indicator of poor prognosis in node-positive breast cancer (4-8). Furthermore, one study indicates

the prognostic value of HER2 to be stronger among patients treated with chemotherapy (7).

However, in predicting disease-free and overall survival in individual patients, other established prognostic factors such as tumor size, number of positive lymph nodes and steroid receptor

status must also be taken into consideration.

Overexpression of the HER2 oncoprotein, as a result of gene amplication found in breast cancer cells, suggests HER2 as a target for an antibody-based therapy (3). Herceptin (trastuzumab), a humanized monoclonal antibody (9) that binds with high afnity to the HER2 oncoprotein has

been shown to inhibit the proliferation of human tumor cells that overexpress HER2 oncoprotein both in vitro and in vivo (10–12). Since the development of Herceptin, the detection of both

the HER2 gene and protein have become essential tools in the assessment of breast tumors, directing both therapy selection and subsequent patient management (13,14).

In both interphase and metaphase cells derived from human breast carcinoma cell lines, FISH has been used to show HER2 gene amplication (15-18). For quantication of HER2 gene amplication, FISH assesses the level of HER2 gene amplication directly in the tumor cells.

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Leica Biosystems Leica HER2 FISH System - 30 Test Instructions for Use TA9217 EN-CE-Rev_D 08/04/2013

The characteristic morphology of the tissue and the spatial distribution of oncogene copies in individual uncultured primary breast carcinomas are retained. Aberrations in chromosome 17 copy number (aneusomy) are also commonly found in breast tumors. These may present as chromosome deletions or gains (polysomy). This chromosomal variation has critical impact on

the interpretation and reporting of HER2 gene amplication status. Therefore, measurement of

chromosome 17 copy number in conjunction with HER2 is critically important (4).

The Leica HER2 FISH System - 30 Test contains the LSI HER2 DNA probe, a 226 Kb

SpectrumOrange

locus (17q11.2-q12) and the CEP17 DNA probe, a 5.4 Kb SpectrumGreen

™

directly labeled uorescent DNA probe specic for the HER2 gene

™

directly labeled

uorescent DNA probe specic for the alpha satellite DNA sequence at the centromeric region

of chromosome 17 (17p11.1-q11.1). The probe solution has been specially formulated and

validated for use on the Leica BOND-MAX and BOND-III System and should not be modied

or used in a manual setting.

Clinical Concordance Summary Leica BOND-MAX System

The Leica HER2 FISH System - 30 Test was developed to provide a fully automated alternative

to current methodologies used to determine HER2 gene amplication status. The performance

of the Leica HER2 FISH System - 30 Test on the Leica BOND-MAX System was evaluated in an independent study comparing the results of the Leica HER2 FISH System - 30 Test to the Abbott Molecular PathVysion

HER-2 DNA Probe Kit Assay on 300 breast tumor specimens. None of

these tumor specimens were obtained from patients in the Herceptin clinical studies. The results

indicated a 99.33% concordance in a 2x2 analysis (95% condence intervals of 97.61–99.92%).

The concordance data also indicates that a positive result with the Leica HER2 FISH System 30 Test is highly likely to correspond with a positive result on the Abbott Molecular PathVysion HER-2 DNA Probe Kit assay. The Leica HER2 FISH System - 30 Test is interpreted as negative

for HER2 gene amplication when the HER2:CEP17 gene ratio is less than 2.0 and positive when the HER2:CEP17 gene ratio is greater than or equal to 2.0. Equivocal (borderline) results, where the HER2:CEP17 gene ratio is between or equal to 1.8-2.2, should be interpreted with

caution. Count an additional 20 nuclei and recalculate the ratio.

Clinical Concordance Summary Leica BOND-III System

The Leica HER2 FISH System - 30 Test was developed to provide a fully automated alternative

to current methodologies used to determine HER2 gene amplication status. The performance

of the Leica HER2 FISH System - 30 Test on the Leica BOND-III System was evaluated in an independent study comparing the results of the Leica HER2 FISH System - 30 Test to the Abbott Molecular PathVysion HER-2 DNA Probe Kit Assay on 300 breast tumor specimens. None of these tumor specimens were obtained from patients in the Herceptin clinical studies. The results

indicated a 99.67% concordance in a 2x2 analysis (95% condence intervals of 98.16–99.99%).

caution. Count an additional 20 nuclei and recalculate the ratio.

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Principle of Procedure

The Leica HER2 FISH System - 30 Test contains components required to complete a uorescence in situ hybridization based staining procedure for formalin-xed, parafn-embedded tissues. Following appropriate pretreatment, incubation with the ready-to-use LSI HER2/CEP17 Dual Probe and appropriate stringency washing, tissue sections are then dehydrated and mounted with DAPI. Results are interpreted by uorescence microscopy using the recommended lters

at the appropriate wavelengths.

Leica Biosystems Leica HER2 FISH System - 30 Test Instructions for Use TA9217 EN-CE-Rev_D 08/04/2013

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The Leica HER2 FISH System - 30 Test is for use only on the Leica BOND-MAX and BOND-III System.

Components Provided

The materials listed below (Table 1) are sufcient to stain 30 tests (30 slides stained with LSI

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HER2/CEP17 Dual Probe).

LSI HER2/CEP17 Probe

6.6 mL

Post Hybridization Wash 2

9 mL

Leica BOND Enzyme Concentrate 2

Contains ready-to-use LSI HER2/CEP17 Dual Probe. Contains <60% (v/v) formamide.

Contains ready-to-use post hybridization wash solution. Contains <50% (v/v) formamide.

Contains Proteinase K solution at 1.7 mg/mL.

1 mL

Leica BOND Enzyme Diluent

Contains Enzyme Diluent.

65 mL

Leica BOND Open Container

BOND Open Container used for Enzyme 5.

3 x 7 mL

Table 1: Leica HER2 FISH System - 30 Test Components

Refer to individual MSDS for further product safety information, available from www.LeicaBiosystems.com/TA9217-IFU

Directions For Use

All reagents supplied are formulated specically for use with this assay and lot numbers are specic for each Leica HER2 FISH System - 30 Test. For the assay to be valid, no substitutions

should be made.

Storage and Stability

Store at 2–8 °C. Do not freeze. Return to 2–8 °C immediately after use. Any deviation from these conditions will invalidate the assay. Ensure the Leica HER2 FISH System - 30 Test is used within its designated expiry date. The signs indicating contamination and/or instability of the Leica HER2 FISH System - 30 Test are turbidity of the solutions (except for the probe solution) and

odor development. The user must verify storage conditions other than those specied above.

Specimen Preparation

Standard methods of tissue processing should be used for all specimens (19). It is recommended

that tissues are prepared in formalin-based xatives and are routinely processed and parafnembedded. For example, specimens should be sampled at a thickness of 3–4 mm and xed

for 18–24 hours in 10% neutral-buffered formalin. The tissues should then be dehydrated in a

series of alcohols and cleared through xylene, followed by impregnation with molten parafn wax, held at no more than 60 °C. Tissue specimens should be sectioned between 4–6 µm.

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Leica Biosystems Leica HER2 FISH System - 30 Test Instructions for Use TA9217 EN-CE-Rev_D 08/04/2013

Tissue sections mounted on charged slides (Leica BOND Plus Slides S21.2113) can be held for

up to 12 months at 2–8 °C before staining. Following sectioning, it is recommended that slides

are incubated at 60 °C for one hour. Stained sections should be stored at -20 °C to preserve

uorescent signal and prevent fading. Allow stored slides to reach room temperature prior to

reading.

Warnings and Precautions

For professional users only.

One or more components in the product are hazardous and may cause harm to the unborn child.

As a rule, persons under 18 years of age are not allowed to work with this product. Users must be carefully instructed in the proper work procedure, the hazardous properties of the product

and the necessary safety instructions.

Specimens, before and after xation, and all materials exposed to them, should be handled as

if capable of transmitting infection and disposed of with proper precautions. Never pipette reagents by mouth and avoid contacting the skin and mucous membranes with

reagents and specimens. If reagents or specimens come into contact with sensitive areas, wash with copious amounts of water. Seek medical advice. Consult federal, state or local regulations

for disposal of any potentially toxic components.

Minimize microbial contamination of reagents or an increase in nonspecic staining may occur.

Procedure

A. Reagents required but not supplied

• Leica BOND Dewax Solution (AR9222)

• Leica BOND Epitope Retrieval Solution 1 (AR9961)

• Leica BOND Wash Solution x10 Concentrate (AR9590)

• Standard solvents used in uorescence in situ hybridization based assays (eg ethanol,

absolute and graded)

• Distilled or de-ionized water

• DAPI Counterstain

• Leica HER2 FISH Control Slides (TA9123)

• Leica BOND Aspirating Probe Cleaning System (CS9100)

B. Equipment required but not supplied

• Pipettes (capable of measuring 1-20 µL and 100 – 1000 µL volumes)

• Charged slides (Leica BOND Plus Slides – S21.2113)

• Leica BOND-MAX (21.0051) or Leica BOND-III (21.2201)

• Leica BOND Universal Covertiles

• Leica BOND Mixing Stations (S21.1971)

• Leica BOND Slide Label & Print Ribbon (S21.4564)

• Coverslips

• Drying oven (capable of maintaining 60 °C)

• Fluorescence Microscope (60–100x objective) with appropriate light source. Record the

number of hours that the bulb has been used and replace the bulb before it exceeds the rated time. Ensure that the lamp is properly aligned.

• Appropriate Fluorescence Filter Set (SpectrumOrange Emission Peak at 588nm, SpectrumGreen Peak at 524nm and DAPI – Excitation Peak at 367nm, Emission Peak at 452nm). Multi-

™

(S21.2001)

™

– Excitation Peak at 497nm, Emission

– Excitation Peak at 559nm,

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Leica Biosystems Leica HER2 FISH System - 30 Test Instructions for Use TA9217 EN-CE-Rev_D 08/04/2013

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bandpass uorescence microscope lter sets optimized for use with the Leica HER2 FISH System - 30 Test are available for most microscope models. The recommended lter sets for the Leica HER2 FISH System - 30 Test are the DAPI/9-Orange dual bandpass, DAPI/ Green dual bandpass, Green/Orange(V.2) dual bandpass and the DAPI/Green/Orange

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(V.2) triple bandpass.

C. Methodology

• Prior to undertaking this methodology, users are required to be suitably trained in the automated in situ uorescence technique.

• Each test section stained with the LSI HER2/CEP17 Dual Probe enables same cell

analysis of both HER2 and centromeric chromosome 17 signals. A subsequent ratio of HER2 to chromosome 17 signals will enable a quantitative value to be assigned to the

sample, indicating a negative (non-amplied) or positive (amplied) result. Equivocal

(borderline) results (1.8-2.2) should be interpreted with caution. Count an additional 20 nuclei and recalculate the ratio.

D. BOND Enzyme Pretreatment

Prior to staining dilute supplied Leica BOND Enzyme Concentrate 2 at a 1:300 dilution using supplied Leica BOND Enzyme Diluent in one of the Leica BOND Open Containers

provided. For example, to stain 10 slides prepare 3 mL of working enzyme solution by diluting 10 µL of Leica BOND Enzyme Concentrate 2 in 2990 µL of Leica BOND Enzyme

Diluent. It is recommended that the enzyme is freshly prepared before each staining run

and that a minimum volume of 900 µL be used per run.

E. Default Staining Protocol

It is recommended that the Leica HER2 FISH System - 30 Test is used with the recommended default staining protocol shown in Table 2 below.

Protocol Type Protocol Name

Staining *FISH Protocol A

Preparation *Dewax

HIER *HIER 25 min with ER1 (97)

Enzyme *Enzyme 5 for 25 min

Denaturation *D10

Hybridization *ISH Hybridization (12Hr)

Table 2: Default Leica HER2 FISH System - 30 Test Staining Protocol

F. Procedure Steps

These instructions should be read in conjunction with the Leica BOND-MAX and BOND-III System user manual. A new Leica BOND Universal Covertile should be used with each slide.

The use of Leica BOND Universal Covertiles, which have previously been utilized for either

immunohistochemical or in situ hybridization staining have not been validated with this test.

1. On the Leica BOND-MAX and BOND-III System, ensure the bulk and hazardous waste

containers have enough capacity to perform the required staining runs.

2. Ensure there is adequate alcohol, distilled or de-ionized water, Leica BOND Dewax

Solution, Leica BOND Epitope Retrieval Solution 1 and Leica BOND Wash Solution

in the bulk reagent containers to perform the required staining runs.

3. Ensure that a clean Leica BOND Mixing Station is installed.

4. Turn on the Leica BOND-MAX and BOND-III System.

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Leica Biosystems Leica HER2 FISH System - 30 Test Instructions for Use TA9217 EN-CE-Rev_D 08/04/2013

5. Turn on the PC attached to the Leica BOND-MAX and BOND-III System.

6. Open the Leica BOND software.

7. For a new Leica HER2 FISH System - 30 Test kit, scan the reagent tray barcode with

the handheld scanner to enter the system into the Leica BOND reagent inventory (Single barcode only).

8. Prepare Leica BOND Enzyme 5 in the supplied Leica BOND Open Container at a dilution

of 1:300. For example, for 10 slides add 10µL of Leica BOND Enzyme Concentrate 2 to 2990 µL of Leica BOND Enzyme Diluent.

9. Scan in supplied Leica BOND Open Container and register as Bond Enzyme 5.

10. Go to the Slide setup screen and click Add case.

11. Enter details for the rst case. Ensure the dispense volume is set to 150 µL and the

preparation protocol is *Dewax. Click OK.

12. With the case highlighted in the Slide setup screen click Add slide.

13. First, add patient test slides. Ensure tissue type is set to Test tissue.

14. Select staining mode Single.

15. Select process ISH.

16. Select *LSI HER2/CEP17 Dual Probe – 30 Test from the probe list. The Protocols tab

defaults to the correct staining protocol (*FISH Protocol A), HIER protocol (*HIER 25 min with ER1 (97)), EIER protocol (*Enzyme 5 for 25 min), denaturation (*D10) and hybridization (*ISH Hybridization (12Hr)).

17. Repeat steps 10 to 16 until patient test slides and controls (Leica HER2 FISH control

slides and/or in-house controls) have been created. Print slide labels.

18. Label slides appropriately.

19. Open the lids of all Leica HER2 FISH System - 30 Test containers and load the reagent

tray onto the Leica BOND-MAX and BOND-III System.

20. Apply new Covertiles to each slide.

21. Load the slide tray onto the Leica BOND-MAX and BOND-III System and press the

Load/Unload button.

22. Conrm that the slides have been scanned and click the Run (Play) button on the System

status screen to commence the run immediately (for the Leica HER2 FISH System 30 Test it is recommended that this assay is run overnight utilizing the delayed start functionality).

23. Ensure that the tray indicator eld displays Proc (OK) and batch number and nish time

are displayed.

24. When the run is completed press the Load/Unload button and remove the slide tray from

the Leica BOND-MAX and BOND-III System.

25. Remove Covertiles and rinse the slides in de-ionized water.

26. Dehydrate rapidly in two changes of alcohol, air dry.

27. Dispense 20µL of DAPI directly onto the sample.

28. Apply coverslip and allow the solution to spread to its full extent, taking care to remove

any air bubbles.

29. Seal edge of coverslip with nail varnish, or similar sealant.

30. Place slides in dark to facilitate signal development before viewing under uorescence

microscope.

31. To preserve signal intensity store stained slides at -20 °C.

G. Slide Storage

Store stained slides at -20 °C in the dark. Allow slides to reach room temperature prior to viewing following removal from -20 °C.

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Leica Biosystems Leica HER2 FISH System - 30 Test Instructions for Use TA9217 EN-CE-Rev_D 08/04/2013

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