DNA Probe is a mixture of oligo probes that specifically targets the HER2 gene (also

VENTANA HER2 Dual ISH DNA Probe Cocktail known as ERBB2 and NEU), which is located on human Chromosome 17 (17q12). The
Chromosome 17 probe is a mixture of oligo probes that target sequences within the
800-6043 centromeric region and serves as a reference for aneusomy. Copy numbers of both
probes are enumerated in tumor nuclei and results are reported as a ratio of
08314373001 HER2/Chromosome 17 to determine HER2 amplification status (HER2/Chromosome 17
ratio ≥ 2.0 is amplified, while a ratio < 2.0 is non-amplified). The VENTANA HER2 Dual
30 ISH DNA Probe Cocktail is optimally formulated for use with VENTANA Silver ISH DNP
Detection Kit, VENTANA Red ISH DIG Detection Kit, and accessory reagents on a
BenchMark IHC/ISH instrument.
INTENDED USE The detection kit contains a primary antibody and an enzyme-labeled secondary antibody
conjugated to horseradish peroxidase (HRP) or alkaline phosphatase (AP) which is used
The VENTANA HER2 Dual ISH DNA as the chromogenic enzyme. During the Dual in situ hybridization (Dual ISH) staining
Probe Cocktail is intended to determine process, DNP and DIG labeled probes are co-hybridized to their respective specific target
HER2 gene status by enumeration of DNA sequences within the cell nuclei. Detection of the DNP-labeled HER2 probe occurs
the ratio of the HER2 gene to first, using the VENTANA Silver ISH (SISH) DNP Detection Kit, which contains the
Chromosome 17 by light microscopy. following dispensers: mouse anti-DNP primary antibody labeled with hydroxyquinoxaline
The HER2 and Chromosome 17 probes (HQ), mouse anti-HQ secondary antibody conjugated to horseradish peroxidase (HRP),
are detected using two-color Chromogen A (Silver A), Chromogen B (Silver B) and Chromogen C (Silver C). Following
chromogenic in situ hybridization (ISH) incubation with the HQ-labeled mouse anti-DNP primary antibody and then mouse anti-
in formalin-fixed, paraffin-embedded HQ HRP secondary antibody conjugate, the SISH reaction occurs. Briefly described, this
human breast and gastric carcinoma reaction is driven by the sequential addition of Chromogens A (silver acetate), B
Figure 1. Amplified HER2 status with
tissue specimens, including the
VENTANA HER2 Dual ISH DNA Probe (hydroquinone) and C (H2O2). Here, the silver ions (Ag+) are reduced by hydroquinone to
gastroesophageal junction, following
Cocktail assay, Breast Carcinoma. metallic silver atoms (Ag0). This reaction is fueled by the substrate for HRP, hydrogen
staining on BenchMark IHC/ISH
instruments. peroxide (Chromogen C). The silver precipitate is deposited in the nuclei and a single
copy of the HER2 gene is visualized as a black dot. Figure 2 illustrates the SISH reaction.
The VENTANA HER2 Dual ISH DNA Probe Cocktail is indicated as an aid in the
assessment of patients for whom Herceptin (trastuzumab) is being considered. This Following SISH detection for HER2, the DIG-labeled Chromosome 17 probe is detected
product should be interpreted by a qualified pathologist in conjunction with histological with the VENTANA Red ISH DIG Detection Kit. This kit includes the following dispensers:
examination, relevant clinical information, and proper controls. mouse anti-DIG primary antibody labeled with nitropyrazole (NP), mouse anti-NP
secondary antibody conjugated to Alkaline Phosphatase (AP), pH Enhancer, Naphthol,
This product is intended for in vitro diagnostic (IVD) use. and Fast Red. Following development of the SISH signal, the slide is incubated with the
SUMMARY AND EXPLANATION NP-labeled mouse anti-DIG primary antibody, which binds to the DIG hapten on the
Chromosome 17 probe. The anti-hapten primary antibody is detected with the mouse anti-
Human epidermal growth factor receptor 2 (HER2) is a member of the epidermal growth NP conjugated to AP enzyme. The slide is incubated with the pH Enhancer solution, which
factor subfamily of transmembrane receptor tyrosine kinases that mediate the growth, provides the proper salt components and concentrations and buffered pH for optimal AP
differentiation, and survival of cells.1,2 Approximately 15 to 30 percent of breast enzyme performance. Next, naphthol phosphate is applied, which serves as the substrate
carcinomas demonstrate overexpression of the HER2 protein, amplification of the HER2 for the AP enzyme (AP dephosphorylates naphthol). Fast Red, added to the slide next,
gene (ERBB2), or both.3,4 Knowledge of HER2 gene and/or protein status in invasive combines with the dephosphorylated naphthol to form a red precipitate, which is readily
breast carcinoma enables clinicians to make more informed decisions to improve the visualized by light microscopy. Figure 3 illustrates the Red ISH reaction. The specimen is
overall management of care for these patients.5 HER2 status is an established predictive then counterstained with Hematoxylin II for interpretation by light microscopy.
factor for response to HER2 targeted therapy in breast cancer patients.5,6,7 The staining protocol consists of numerous steps in which reagents are incubated for pre-
Trastuzumab (Herceptin) is a humanized monoclonal antibody against the extracellular determined times at specific temperatures. At the end of each incubation step, the
domain of HER2 and has been shown to benefit patients with HER2 positive breast BenchMark IHC/ISH instrument washes the sections to remove unbound material and
cancer.8-13 Demonstration of HER2 gene amplification and/or protein overexpression is applies a liquid coverslip which minimizes the evaporation of the aqueous reagents from
essential for selecting patients for trastuzumab therapy.5,14 the slide. Results are interpreted using a light microscope using 20x, 40x, and/or 60x
objectives.
Similarly, HER2 gene amplification or protein overexpression occurs in gastric and
gastroesophageal junction adenocarcinoma (collectively referred to as gastroesophageal
adenocarcinoma or GEA).15,16,17 A wide range of HER2 overexpression frequency has
been reported across published studies. However, one of the largest screening datasets
which included 3,803 patients with GEA reported that 22 percent of patients tested
positive for HER2 protein expression or gene amplification.18 The majority of studies
suggest that in the absence of HER2 directed therapy, HER2 overexpression is a negative
prognostic factor.19
The HER2 targeted therapy trastuzumab is a mainstay in the management of invasive
breast carcinoma and has therapeutic value in the management of gastric/GEA cancer
patients overexpressing the receptor.15,17 Demonstration of HER2 gene amplification
and/or protein overexpression is essential for selecting patients for trastuzumab
therapy.15,19 Clinical studies have shown that breast or gastric/GEA cancer patients with
high HER2 protein overexpression and/or gene amplification benefit most from
trastuzumab.3,15
PRINCIPLE OF THE PROCEDURE
The VENTANA HER2 Dual ISH DNA Probe Cocktail contains HER2 probes (labeled with
the hapten dinitrophenyl or DNP) and Chromosome 17 probes (labeled with the hapten
digoxigenin or DIG) formulated in a formamide-based buffer. The probes are designed to
detect amplification of the HER2 gene in invasive breast carcinoma and GEA. The HER2

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FT0700-415l
 MATERIALS REQUIRED BUT NOT PROVIDED
Black Signal Silver A Staining reagents, such as VENTANA detection kits and ancillary components, are not
Silver B provided.
Silver C Not all products listed in the method sheet may be available in all geographies. Consult
your local support representative.
The following reagents and materials required for staining are not provided:
1. VENTANA Silver ISH DNP Detection Kit (Cat. No. 760-516 / 08318883001)
HRP 2. VENTANA Red ISH DIG Detection Kit (Cat. No. 760-512 / 08318832001)
HQ-labeled Mouse anti-DNP
HQ HQ
3. HybReady Solution (Cat. No. 780-4409 / 05917557001)
Mouse anti-HQ
4. ISH Protease 3 (Cat. No. 780-4149 / 05273331001)
5. Hematoxylin II (Cat. No. 790-2208 / 05277965001)
NO2 6. Bluing Reagent (Cat. No. 760-2037 / 05266769001)
DNP 7. Reaction Buffer Concentrate (10X) (Cat. No. 950-300 / 05353955001)
N 8. SSC (10X) (Cat. No. 950-110 / 05353947001)
HER2 Target H HER2 DNA Probe
Sequence NO2 (DNP-labeled) 9. EZ Prep Concentrate (10X) (Cat. No. 950-102 / 05279771001)
Region 10. ultraView Silver Wash II (Pre-dilute) (Cat. No. 780-003 / 05446724001)
11. Cell Conditioning Solution (CC1) (Cat. No. 950-124 / 05279801001)
12. Cell Conditioning Solution (CC2) (Cat. No. 950-123 / 05279798001)
13. LCS (Predilute) (Cat. No. 650-010 / 05264839001)
Figure 2. VENTANA Silver ISH DNP Detection for HER2 14. ULTRA LCS (Pre-dilute) (Cat. No. 650-210 / 05424534001)
15. ULTRA Cell Conditioning Solution (ULTRA CC1) (Cat. No. 950-224 / 05424569001)
16. ULTRA Cell Conditioning Solution (ULTRA CC2) (Cat. No. 950-223 / 05424542001)
17. BenchMark IHC/ISH instrument
Red Signal
18. Microscope slides, positively charged (Superfrost Plus or equivalent)
pH Enhancer
19. Permanent mounting medium*
Naphthol
20. Cover slip sufficient to cover tissue
Fast Red
21. Automated coverslipper
Mouse anti-NP 22. HER2 Dual ISH 3-in-1 Xenograft Slides (Cat. No. 783-4422 / 05640300001) can be
AP used for troubleshooting activities, as needed.
* See Table 30 for compatible mounting media with this assay.
NP NP
NP-labeled Mouse anti-DIG
STORAGE AND STABILITY
Upon receipt and when not in use, store at 2-8°C. Do not freeze.
To ensure proper reagent delivery and the stability of the probe, replace the dispenser cap
after every use and immediately place the dispenser in the refrigerator in an upright
position.
Every probe dispenser is expiration dated. When properly stored, the reagent is stable to
DIG
the date indicated on the label. Do not use reagent beyond the expiration date.

Chromosome 17 DNA SPECIMEN PREPARATION

Chromosome 17 Probe (DIG-labeled) Routinely processed, formalin-fixed, paraffin-embedded (FFPE) tissues are suitable for
Centromere Region
use with this probe when used with a BenchMark IHC/ISH instrument. The recommended
tissue fixative is 10% neutral buffered formalin (NBF) for 6 to 72 hours.20 Aside from the
VENTANA assays, studies have found that the majority of inconclusive HER2 gene results
by FISH relate to pre-analytic factors including under- and over-fixation,21 as well as
Figure 3. VENTANA Red ISH DIG Detection for Chromosome 17 delayed fixation.22 Strict implementation of fixation procedures (e.g., a dedicated
processor to ensure a minimum of 6 hours fixation) resulted in a 68.5% reduction in
inconclusive cases from 10.8% failures to 3.4%. Specimens fixed < 6 hours in formalin can
MATERIAL PROVIDED result in signal loss and nuclear over-digestion, as observed by pale/weak hematoxylin
The VENTANA HER2 Dual ISH DNA Probe Cocktail dispenser contains sufficient reagent staining. Only fixation in 10% NBF is recommended as some fixatives produce variable
for 30 tests. staining with ISH-based assays (including Bouin’s and Alcohol Formalin-Acetic Acid
(AFA)).21
One 6 mL dispenser of VENTANA HER2 Dual ISH DNA Probe Cocktail contains
approximately 14 μg/mL of the HER2 probes labeled with dinitrophenyl (DNP) and Slides should be stained immediately, as quality of nucleic acid targets in cut tissue
0.24 μg/mL of the Chromosome 17 probes labeled with digoxigenin (DIG) formulated in a sections may diminish over time. Internal studies have shown that breast and gastric cut
formamide-based hybridization buffer. Both probes are used to determine HER2 gene slides stored at 2-8 °C can be stable for 12 months. Positively charged slides may be
status (i.e., ratio of HER2/Chromosome 17). susceptible to environmental stresses resulting in inappropriate staining of any ISH assay
(for example, lack of staining or counterstain on the tissue). Ask your Roche
Refer to the appropriate VENTANA detection kit method sheets for detailed descriptions
representative for a copy of “Impact of environmental stress on various histology slide
of: Principles of the Procedure, Materials and Methods, Specimen Collection and
types” to better understand how to use these types of slides.
Preparation for Analysis, Quality Control Procedures, Troubleshooting, Interpretation of
Results, and Limitations. Each section should be cut to the appropriate thickness (4 μm) for the assay used and
placed on positively charged microscope slides (Superfrost Plus or equivalent). Slides
should be drained or dried to remove excess water between slide and tissue.

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Sections thicker than 4 μm may require stronger protease treatment than the This product contains components classified as follows in accordance with the Regulation
recommended condition and may exhibit more nuclear bubbling than thinner sections due (EC) No. 1272/2008:
to excess paraffin in the tissue. Nuclear bubbling appears as large or small bubbles or Table 1. Hazard information.
vacuoles in the nuclei. Often when nuclear bubbling occurs there is a spectrum of effects
on the SISH and Red ISH signals characterized by 1) nuclei with nuclear bubbles in which Hazard Code Statement
the SISH and Red ISH signals are generally still centrally located in the nucleus and 2) Danger H351 Suspected of causing cancer.
nuclei with nuclear bubbles that push the SISH and Red ISH signals to the periphery.
Often in both cases, if the SISH and Red ISH signals are clearly discernable, are not H360D May damage the unborn child.
otherwise distorted, and are still enumerable, the case can be scored. However,
occasionally severe nuclear bubbling may distort the SISH and Red ISH signals or make May cause damage to organs through prolonged or
H373
them indiscernible such that accurate enumeration is not possible. This occurs more often repeated exposure.
when SISH and Red ISH signals are pushed to the nuclear periphery. When this occurs
one can often find nuclei elsewhere in the sample that are enumerable and the case can P201 Obtain special instructions before use.
be scored. If nuclear bubbling is severe, to the degree that one cannot find sufficient nuclei
Do not handle until all safety precautions have been
in which SISH and Red ISH signals can be confidently enumerated, the case should not P202
read and understood.
be scored. Nuclear bubbling also may occur in the context of underfixation (1-3 hours with
formalin), which is a less discrete nuclear bubbling. This may be remedied at 3 hours P260 Do not breathe dust/ fume/ gas/ mist/ vapours/ spray.
fixation with changed cell conditioning/protease treatment, but at 1 hour is probably
beyond remedy. Wear protective gloves/ protective clothing/ eye
P280
The VENTANA HER2 Dual ISH DNA Probe Cocktail assay has been developed with protection/ face protection.
additional pre-treatment options that may aid in optimizing the assay in different
P308 + If exposed or concerned: Get medical advice/
laboratories and for subsequent troubleshooting of particular tissues / slides exhibiting
P313 attention.
sub-optimal staining. It is recommended that each laboratory perform initial runs on
representative control samples that have been prepared under the identical conditions as Dispose of contents/ container to an approved waste
the clinical samples to be tested. This will aid in optimizing the specific staining conditions P501
disposal plant.
for individual laboratories that may vary in their exact specimen preparation procedures.
Variable results may occur with different pre-analytical factors than recommended. This product contains CAS # 75-12-7, Formamide.
Specimens that are pre-analytically prepared using conditions that are not recommended
may never stain appropriately with the assay. STAINING PROCEDURE
VENTANA probes have been developed for use on a BenchMark IHC/ISH instrument in
WARNINGS AND PRECAUTIONS combination with VENTANA detection kits and accessories. The staining procedures for
1. For in vitro diagnostic (IVD) use. the BenchMark IHC/ISH instrument with the VENTANA Silver ISH DNP Detection Kit and
2. For professional use only. the VENTANA Red ISH DIG Detection Kit are listed in Table 2. The recommended
3. CAUTION: In the United States, Federal law restricts this device to sale by or on staining protocols are listed in Table 3.
the order of a physician. (Rx Only) The parameters for the automated procedures can be displayed, printed and edited
4. Do not use beyond the specified number of tests. according to the procedure in the instrument User Guide. Refer to the appropriate
5. Warning, Product Contains Formamide. Formamide is toxic by inhalation and VENTANA detection kit method sheet for more details
moderately toxic by ingestion. It is an irritant to skin, eyes, and mucous membranes For more details on the proper use of this device, refer to the inline dispenser method
and is absorbed through the skin. It may cause harm to the unborn child. Take sheet associated with P/N 800-6043.
precautions when handling reagents. Use disposable gloves and wear suitable Table 2. Use the following staining procedures to perform VENTANA HER2 Dual ISH
protective clothing when handling suspected carcinogens or toxic materials. DNA Probe Cocktail assay on BenchMark IHC/ISH instruments.
6. Materials of human or animal origin should be handled as potentially biohazardous
and disposed of with proper precautions. In the event of exposure, the health Instrument Platform Staining Procedure
directives of the responsible authorities should be followed.23,24 BenchMark GX GX VENTANA HER2 DISH DNA PRB CKT
7. Avoid contact of reagents with eyes and mucous membranes. If reagents come in
contact with sensitive areas, wash with copious amounts of water. Avoid inhalation BenchMark XT XT VENTANA HER2 DISH DNA PRB CKT
of reagents.
BenchMark ULTRA or
8. Ensure that the waste container is empty prior to starting a run on the instrument. If U VENTANA HER2 DISH DNA PRB CKT
BenchMark ULTRA PLUS
this precaution is not taken, the waste container may overflow and the user risks a
slip and fall.
9. Avoid microbial contamination of reagents as this may produce incorrect results. Table 3. Recommended staining conditions for VENTANA HER2 Dual ISH DNA Probe
10. For further information on the use of this device, refer to the BenchMark IHC/ISH Cocktail assay on BenchMark IHC/ISH instruments.
instrument User Guide, and instructions for use of all necessary components
located at dialog.roche.com. Staining Condition Breast Gastric
11. Consult local and/or state authorities to determine the recommended method of Baking Not selected Not selected
disposal.
12. Product safety labeling primarily follows EU GHS guidance. Safety data sheet Cell Conditioning 1 16 mins 16 mins
available for professional user on request. Cell Conditioning 2 24 mins 16 mins
13. To report suspected serious incidents related to this device, contact the local Roche
representative and the competent authority of the Member State or Country in which ISH Protease 3 20 mins 16 mins
the user is established.
76°C for BenchMark 76°C for BenchMark
GX/XT instruments GX/XT instruments
Stringency Wash
Temperature 74°C for BenchMark 74°C for BenchMark
ULTRA or ULTRA PLUS ULTRA or ULTRA PLUS
instruments instruments

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Due to variation in tissue fixation and processing, as well as general lab instrument and Assay Verification
environmental conditions, it may be necessary to increase or decrease the cell Prior to initial use of a probe or staining system in a diagnostic procedure, the specificity of
conditioning or protease pretreatment based on individual specimens. the probe should be verified by testing it on a series of tissues with known ISH
Starting a Run on BenchMark IHC/ISH instruments performance characteristics (refer to the probe method sheet and to the Quality Control
recommendations of the College of American Pathologists Laboratory Accreditation
1. Apply slide bar code label that corresponds to the probe protocol to be performed.
Program, Anatomic Pathology Checklist,25 or the CLSI Approved Guideline26 or both
2. Load the VENTANA HER2 Dual ISH DNA Probe Cocktail, reagents from VENTANA
Red ISH DIG and VENTANA Silver ISH DNP Detection Kits, and required accessory documents). These quality control procedures should be repeated for each new lot of
reagents into the reagent tray(s) or carousel. Place reagent tray(s) or carousel on reagents, or whenever there is a change in assay parameters.
the instrument. STAINING INTERPRETATION / EXPECTED RESULTS
3. Check bulk fluids and waste.
The cellular staining pattern for VENTANA HER2 Dual ISH DNA Probe Cocktail assay is
4. The reaction buffer bulk bottles must be full. nuclear.
5. The waste container must be empty prior to the start of the run.
A qualified pathologist experienced in the microscopic interpretation of anatomic pathology
6. Load slides onto the instrument. specimens, ISH procedures and the recognition of single and amplified HER2 and
7. Start the staining run. Chromosome 17 (Chr17) copies (which require microscopic examination using 20x, 40x,
8. At the completion of the run, remove slides from the instrument. The stained slides and/or 60x objectives) must evaluate controls before interpreting results.
will have residual buffer and liquid coverslip solution on them. Proceed with rinsing Note: Use of 100x objective is not recommended. All of the tissue slides read during
and dehydration (see below). design verification and validation testing were done using 20x, 40x, and/or 60x objectives.
Dehydration Procedure The VENTANA HER2 Dual ISH DNA Probe Cocktail must be used along with the
Note: The Fast Red chromogen is soluble in alcohol and acetone. Stained slides exposed Interpretation Guide VENTANA HER2 Dual ISH DNA Probe Cocktail [P/N 1018386] for
to alcohol and/or acetone can result in a loss of specific signal. slide evaluation.
1. To remove liquid coverslip solution, wash slides in 2 sequential solutions of a mild The following sections describe how to interpret and score slides. Table 4 illustrates how
dishwashing detergent (do not use detergent designed for automatic dishwashers). to count discrete signals.
2. Rinse slides well with distilled water, about 1 minute. Shake off excess water. Definitions
3. Place slides in an oven (45-60°C) to dry or air dry at ambient temperature. In an 1. HER2 Gene Status. HER2 Gene status is a function of the ratio of the number of
oven, drying times range from 10 minutes to one hour (drying stained slides for a copies of the HER2 gene to the number of copies of Chr17, per cell, in an invasive
longer period of time does not appear to impact staining results). Ensure slides are breast carcinoma or GEA case. HER2 gene status is classified using the following
completely dry before coverslipping, as residual water on the slides can interfere guidelines:
with the coverslipping procedure and cause bubbles to form. a. HER2/Chr17 ratio ≥ 2.0 is amplified
4. Transfer slides into xylene bath for approximately 30 seconds.
b. HER2/Chr17 ratio < 2.0 is non-amplified
5. Place mounting media on slide.
2. Slide Adequacy. A VENTANA HER2 Dual ISH DNA Probe Cocktail slide must
6. Place coverslip on slide. Note that some mounting media are not compatible with
satisfy three criteria to be deemed adequate for enumeration; if the slide does not
the assay and should not be used (See Limitations and Troubleshooting sections).
meet these criteria, then it cannot be enumerated and the result is unsatisfactory.
QUALITY CONTROL PROCEDURES a. Internal Positive Control. Normal HER2 and Chr17signals (1 to 2 copies per
Positive Control Specimen cell) act as internal positive controls and must be visible in the sample. This
nuclear staining may be located in various non-neoplastic cells including:
Normal HER2 and Chromosome 17 signals (1 to 2 copies per cell) act as internal positive stromal fibroblasts, endothelial cells, lymphocytes, and non-neoplastic
controls and must be visible in the sample using 20x, 40x, and/or 60x objectives. epithelial cells.
However, not all cells will exhibit single gene copy due to biological heterogeneity. Specific b. Neoplastic cells. Using 20x, 40x, and/or 60x objectives, the invasive aspect of
nuclear staining may be located in various cells including: stromal fibroblasts, endothelial the tumor must exhibit an enumerable field of SISH and Red ISH signals.
cells, lymphocytes, and non-neoplastic epithelial cells. If the positive controls fail to
c. Background. Any background staining resulting from either SISH or Red ISH
demonstrate positive staining, this may indicate a reagent or instrument problem. Since
detection systems will need to be evaluated to determine if it interferes with
every specimen has an internal positive control (i.e., appropriate ISH staining in normal
enumeration of the specific SISH or Red ISH signals. SISH background
cells), this acts as the true “positive control”.
typically appears as SISH “dust” that is distinguishable from the specific
A laboratory-specific positive specimen control may be used with every staining procedure signal. Red background may appear as red haze or rarely nonspecific signals
performed. Control specimens can be specimens prepared in a manner identical to patient that are fainter in intensity compared to the specific signal.
specimens. Such controls are useful to monitor all steps of the procedure, from specimen 3. Target Areas for Signal Enumeration. An acceptable target area within the invasive
preparation through staining. Use of a specimen prepared differently from the test carcinoma exhibits an enumerable field of SISH and Red ISH signals. Signal
specimens will provide a control for the reagents, instrument and procedures but not for enumeration should not be performed in areas that contain weak SISH or Red ISH
fixation and specimen processing. Results with the test specimens should be analyzed on signal, compressed or overlapping nuclei, or necrosis. If one target area is deemed
the same run. Such controls should not replace the proper evaluation of the internal inadequate for enumeration, it often is possible to find other target areas on the
controls in each patient specimen. same slide that are adequate. This can be determined by the presence of normal
Xenograft Specimen cells exhibiting appropriate SISH and Red ISH staining in or adjacent to the target
Xenograft slides may be useful for a preliminary validation of the method used for staining area.
slides with VENTANA HER2 Dual ISH DNA Probe Cocktail assay. They also are Additional Observations for HER2 and Chromosome 17
recommended as aids for troubleshooting, when used in runs containing clinical samples. Other observations may be noted as comments on the pathologist’s report.
For more information, see the appropriate xenograft slide method sheet.
1. Heterogeneity: In some cases, the tissue may contain areas of carcinoma that are
Unexplained Discrepancies genetically heterogeneous for HER2 copy number (i.e., there may be a mixture of
Unexplained discrepancies in controls should be referred to your local support unamplified and amplified nuclei or a mixture of nuclei containing various copies of
representative immediately. If quality control results do not meet specifications, patient HER2). This may be observed among carcinoma cells within the target area itself, or
results are invalid. See the Troubleshooting section of this method sheet. Identify and between two different target areas.
correct the problem, then repeat the patient samples. 2. Aneusomy is any condition in which an organism has additional or fewer specific
chromosomes than normal, i.e., the number of a particular chromosome (in this
case, Chromosome 17) is not diploid. In polysomy, there may be three or more
copies of the chromosome rather than the expected two copies. In monosomy, the

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 tumor cells may exhibit only one copy of Chromosome 17. Apparent “amplification,” Table 4. Signal Visualization.
clusters, or polysomy of Chromosome 17 (with or without HER2 SISH clusters) have
been reported.27 In cases with clusters of HER2 and Chromosome 17, care must be
taken not to consider them with a ratio of ~1.0. The reader should refer to
immunohistochemistry (IHC) results for HER2 protein overexpression analyses in Do not count if nuclei overlap.
these cases, as the majority tend to be 3+.
3. Monoallelic Deletion: The deletion of the HER2 gene from Chromosome 17 in the
tumor cells results in a HER2/Chr17 ratio < 1.0.
Signal Visualization
SISH and Red ISH signals are visualized as: Do not count if no signal is present.
1. Single Copy. A discrete black dot (SISH) is counted as a single copy of HER2.
Discrete single dots visualized in the internal, control (non-neoplastic) nuclei
represent the size of a single copy in invasive carcinoma cells for the SISH (black)
signal. For Red ISH signals, each discrete signal is counted as one copy. It should Do not count if only signal of one color is present.
be noted that the Red ISH signal from the Chr17 may appear larger than the SISH
signals, and sometimes elongated in shape. Pink haze may occur and should not be
mistaken for signal. Red signals that are very light in color compared to the signal in
internal positive control nuclei and overall pattern of staining should not be
enumerated, as they may be non-specific. Specific red signals have discrete edges, Do not count if signals are outside the nuclei.
as shown in Table 4.
2. Multiple Copies. Discrete single SISH signals visualized in the internal positive
control nuclei represent the size of a single copy HER2 in invasive carcinoma cells.
The size of the single SISH signals is used as a reference to determine the relative
number of amplified copies in the cancer nuclei. For Red ISH signals, each discrete Count as 1 black (HER2) and 1 red (Chr17) signal.
signal is counted as one copy.
3. Clusters. Presence of multiple overlapping signals in the nuclei that cannot be
enumerated. A cluster is defined as numerous overlapping SISH signals in the
nuclei that cannot be individually discerned. Clusters of HER2 can only be estimated Count as 2 black (HER2) and 2 red (Chr17) signals.
by the reader. For example, a large cluster of multiple SISH signals could be
estimated as 12 copies, while smaller clusters may be estimated as 6 copies. The
estimation is made by using the single SISH copies present in the internal positive Count as 1 black (HER2) and 2 red (Chr17) signals. The
control cells as a reference. The presence of HER2 clusters is noted on the score black signal is a “doublet”. Count two adjacent signals of
sheet. same color only if the distance between the signals is equal
4. Overlapping nuclei, nuclei with only one color present, and specimens with non- to or greater than the diameter of a single signal.
specific staining should not be enumerated. Any nuclei with overlapping Red ISH
and SISH signals that cannot be discerned should be visualized at higher Small SISH clusters can only be estimated by using the size
magnifications to discern the two signals or should not be counted. Nuclei that of a single signal as reference. Use stromal cells to estimate
appear bubbled should not be counted. signal size (smaller cell). For instance, this cluster could be
Enumeration of the SISH and Red ISH signals to determine HER2 gene status estimated as 6 SISH signals - adding the other 2 single
Examine the H&E stained slide to locate areas containing invasive breast or signals yields a total count of 8. Count as 2 red signals.
gastroesophageal carcinoma. Examine the HER2 Dual ISH stained slide corresponding to Note on scoring sheet that clusters are present for HER2.
the H&E, and identify an invasive breast or gastroesophageal carcinoma target area.
Before enumerating HER2 and Chromosome 17 signals to determine HER2 gene status, it Estimate the large cluster. Here, the cluster can be
is critical to determine whether the invasive target area (the lesional tissue) is adequately estimated as 12 black signals - adding the other 4 single
stained and satisfies the criteria described for slide adequacy (see the Definitions section signals yields a total count of 16. Count red signals as 2
above, 2. Slide Adequacy). copies of Chr17. Note on scoring sheet that clusters are
The scoring algorithm developed for the assay maximizes precision and efficiency in present for HER2.
counting. Twenty nuclei, each containing red (Red ISH) and black (SISH) signals, should
be enumerated. A red signal close to a black signal should be counted as
Cell Selection Criteria one red signal and one black signal. This may require
enumeration at 60x objective to discern. Therefore, count as
Count only nuclei with diameters that are representative of the average population of
4 black (HER2) and 2 red (Chr17) signals. If overlapping
invasive carcinoma nuclei in the target area. Do not count signals in nuclei that are:
signals cannot be distinguished, do not count that nucleus.
1. Much larger in diameter than the average size of carcinoma nuclei
2. Much smaller in diameter than the average size of carcinoma nuclei Cluster of black dots obscuring red signal(s). Higher
Count only nuclei that are representative of the population of invasive carcinoma nuclei magnification (60x) may be utilized in attempts to confirm
with the highest average number of signals (both SISH and Red ISH). presence or absence of red signal(s); otherwise do not
In target areas that are genetically heterogeneous for HER2 copy number, count only count: always count nuclei with clear red signals. Note the
nuclei that are representative of the population of invasive carcinoma nuclei with the presence of SISH clusters on the score sheet. Nuclei with
highest average number of signals (both SISH and Red ISH). Note that heterogeneity is visible and higher numbers of red signal should be scored in
present on the score sheet. nuclei with SISH clusters.

If background SISH “dust” occurs in the nuclei, only count if

specific SISH signals are clearly distinguishable from
background.

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 Controls
Pink haze may be observed and should not be mistaken for
signal. Small, faint Red ISH signals may be seen and could Normal cells within, or adjacent to, the target area serve as internal controls of the
represent nonspecific binding of the Chr17 probe to other staining. At least 50% of the normal cell nuclei should contain at least one SISH signal and
chromosomes. The image shows 2 discrete red (Chr17) at least 50% should contain at least one Red ISH signal (the SISH and Red ISH signals do
signals and 2 black (HER2) signals. not have to be in the same cells) for the target area to be deemed adequate. Failure to
detect adequate signal in normal cells on any slide on the run indicates that the particular
slide is inadequate for enumeration. Using positive control samples or xenograft slides will
HER2 Gene Status: Scoring Algorithm for the VENTANA HER2 Dual ISH DNA Probe aid in troubleshooting potential instrument and/or reagent problems.
Cocktail
LIMITATIONS
Twenty nuclei (each containing red (Chr17) and black (HER2) signals) should be
enumerated. The final results for the HER2 status are reported based on the ratio formed General Limitations
by dividing the sum of HER2 signals for all 20 nuclei divided by the sum of Chromosome 1. ISH is a multiple step methodology that requires specialized training in the selection
17 signals for all 20 nuclei. The amplification status is defined as Amplified if the of the appropriate reagents, specimen preparation, processing, preparation of the
HER2/Chr17 ratio ≥ 2.0 and as Non-Amplified if the HER2/Chr17 ratio < 2.0. If the ISH slide, and interpretation of the results.
HER2/Chr17 ratio falls between 1.8 to 2.2, an additional 20 nuclei should be enumerated. 2. Tissue staining is dependent on the handling and processing of the tissue prior to
A new ratio should then be formed on the basis of all 40 nuclei, and the amplification staining. Improper fixation, freezing, thawing, washing, drying, heating, sectioning,
status reported as already described. or contamination with other tissues or fluids may produce artifacts, reagent trapping,
false negative, or false positive results. Inconsistent results may be a consequence
of variations in fixation and embedding methods, or inherent irregularities within the
Start
HER2/Chr17 tissue.
Stained Slide
3. Excessive or incomplete counterstaining may compromise proper interpretation of
results.
4. The clinical interpretation of staining must be evaluated within the context of clinical
Inadequate.
Slide history, morphology and other histopathological criteria. It is the responsibility of a
Repeat staining with
Adequate? No new slide qualified pathologist to be familiar with the reagents and methods used to produce
the stained preparation. Staining must be performed in a certified, licensed
Yes
laboratory under the supervision of a pathologist who is responsible for the review of
Identify and Select Target Area the stained slides and ensuring the adequacy of controls.
5. VENTANA reagents are provided at optimal dilution for use when the provided
instructions are followed. Any deviation from recommended test procedures may
Count HER2 and Chromosome 17 Signals in 20 nuclei invalidate expected results. Users who deviate from recommended test procedures
must accept responsibility for interpretation of patient results.
6. Due to variations in specimen processing it may be necessary to either increase or
Calculate HER2/Chr17 ratio by dividing the total
decrease the ISH protease treatment time. Additionally, increasing or decreasing
number of HER2 signals from Target Area 1 by the cell conditioning will affect staining results. Such changes must be validated by
total number of Chr17 signals from Target Area 1 the user. Users who deviate from recommended test procedures are responsible for
interpretation of patient results under these circumstances.
7. Reagents may demonstrate unexpected reactions in previously untested tissues.
Is The possibility of unexpected reactions even in tested tissue groups cannot be
1.8 ≤ HER2/Chr17 ≤ 2.2 Count additional 20 nuclei completely eliminated because of biological variability of tissues. Contact your local
? Yes support representative with documented unexpected reactions.
No SPECIFIC LIMITATIONS
Report Results 1. Not all fixatives are compatible with the assay. The recommended fixative is 10%
Non-Amplified
Calculate HER2/Chr17 ratio by dividing the NBF for 6 to 72 hours.
total number of HER2 signals from Target
HER2/Chr17 < 2.0
Areas 1 and 2 by the total number of Chr17 2. The VENTANA HER2 Dual ISH DNA Probe Cocktail assay was developed to stain
Amplified signals from Target Areas 1 and 2 tissue sections that are cut at ~4 μm in thickness.28 Sections thicker than 4 μm may
HER2/Chr17 ≥ 2.0
experience tissue loss.
3. All assays might not be registered on every instrument. Please contact your local
support representative for more information.
4. Oxidation, fading, and/or disappearance of the SISH signal may be due to certain
brands of mounting media. See Table 30 for compatibility of mounting media.
5. To prevent the Red ISH signal from dissolving, stained slides must not be
submerged in alcohol or acetone baths for dehydration. Air drying or drying in an
oven is recommended. The stained slides must be completely dry before
coverslipping.
6. As with any test, a negative result means that the specific target was not detected,
not that the specific target was absent in the cells or tissue assayed.
7. This probe has been optimized for use with VENTANA reagents on BenchMark
IHC/ISH instruments. Users who deviate from recommended test procedures are
responsible for interpretation of patient results under these circumstances.

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PERFORMANCE CHARACTERISTICS table below along with positive percent and negative percent agreement rates where
The performance of the VENTANA HER2 Dual ISH DNA Probe Cocktail was evaluated PathVysion HER-2 FISH is the reference assay. Acceptance criteria for demonstrating
through analytical and clinical studies. All staining was performed using the VENTANA equivalent performance of these two assay methods when using the BenchMark ULTRA
HER2 Dual ISH DNA Probe Cocktail protocol as noted in Table 3 on BenchMark IHC/ISH instrument required the two-sided 95% score confidence interval lower bounds be 85% or
instruments unless otherwise specified. higher when pooling data from all three sites. These acceptance criteria were met
(Table 7). Additionally, positive and negative agreement rates by site were all greater
Table 5 and Table 6 summarize the performance data across these studies: Concordance
than 85% (Table 8).
Studies, Repeatability and Precision, Within Reader and Between Reader Precision, Lot to
Lot Precision, Instrument Inter-Laboratory Precision, Analytical Sensitivity and Specificity, Table 7. Agreement between VENTANA HER2 Dual ISH DNA Probe Cocktail and
Assay Characterization, and Stability. A subset of these studies are described in greater Abbott/Vysis PathVysion HER-2 DNA Probe Kit in a cohort of human breast carcinoma
detail in the following sections. specimens.
Table 5. Summary of performance results for breast across analytical and clinical PathVysion HER-2 FISH Result
studies.
VENTANA HER2
Dual ISH DNA Probe
Failure Modes
Cocktail Result Amplified Non-Amplified Total
Amplified 270 12 282
Pass Fail Total Weak/no
HER2/Chr17 Background Non-Amplified 32 291 323
No tissue Other
(internal control Failures
Total 302 303 605
or target cells)

2893 127 3020 113 (3.74%) 5 (0.17%) 6 (0.20%) 3 (0.10%) n/N % (95% Score CI)
Positive Percent
270/302 89.4 (85.4, 92.4)
Agreement
Table 6. Summary of performance results for gastric across analytical and clinical
studies. Negative Percent
291/303 96.0 (93.2, 97.7)
Agreement
Failure Modes
Table 8. Summary of negative, positive, and overall agreement rates for VENTANA
Weak/no HER2 Dual ISH DNA Probe Cocktail and Abbott/Vysis PathVysion HER-2 DNA Probe Kit
Pass Fail Total
HER2/Chr17 Background on human breast carcinoma specimens, presented by site.
No tissue Other
(internal control Failures VENTANA HER2
or target cells) Dual ISH DNA
Probe Cocktail vs Positive
1340 17 1357 17 (1.25%) 0 (0%) 0 (0%) 0 (0%) PathVysion Percent Negative Percent Overall Percent
HER-2 FISH Agreement Agreement Agreement
Site A: n/N (%) 92/100 (92.0%) 92/93 (98.9%) 184/193 (95.3%)
(95% CI) (85.0, 95.9) (94.2, 99.8) (91.4, 97.5)
CLINICAL PERFORMANCE
Concordance Study with PathVysion Assay: VENTANA HER2 Dual ISH DNA Probe Site B: n/N (%) 93/103 (90.3%) 108/119 (90.8%) 201/222 (90.5%)
Cocktail on BenchMark ULTRA Instrument vs. Abbott/Vysis PathVysion HER-2 DNA (95% CI) (83.0, 94.6) (84.2, 94.8) (86.0, 93.7)
Probe Kit
Site C: n/N (%) 85/99 (85.9%) 91/91 (100.0%) 176/190 (92.6%)
To evaluate the concordance of the VENTANA HER2 Dual ISH DNA Probe Cocktail assay
(95% CI) (77.7, 91.4) (95.9, 100.0) (88.0, 95.6)
to the comparator device, the Abbott/Vysis PathVysion HER-2 FISH Kit, in determination
of HER2 gene status in invasive breast carcinoma, a multi-site concordance study was
performed. Three central laboratories participated for the VENTANA HER2 Dual ISH DNA These data indicate excellent agreement between the VENTANA HER2 Dual ISH DNA
Probe Cocktail assay testing. Six hundred thirty-six cases of human invasive breast Probe Cocktail assay and PathVysion HER-2 FISH Kit in determining HER2 gene status in
carcinoma were provided from three clinical enrollment sites for potential inclusion in the human breast carcinoma specimens.
study based on HER2 protein expression obtained previously with IHC. The study sponsor
supplemented 133 cases. The central laboratories conducting the VENTANA HER2 Dual Secondary Results
ISH DNA Probe Cocktail assay and the PathVysion HER-2 FISH assay were blinded to Overall percent agreement between VENTANA HER2 Dual ISH DNA Probe Cocktail and
IHC status and original case identifier to prevent bias in evaluation of the specimens. One PathVysion HER-2 FISH Kit and its two-sided 95% score CI, pooling data from all clinical
central laboratory performed IHC staining on all samples using PATHWAY anti-HER-2/neu sites, was 92.7% (90.4, 94.5).
(4B5) Rabbit Monoclonal Primary Antibody (PATHWAY anti-HER2 (4B5) antibody) for the Secondary Results: IHC vs. ISH for HER2 status
additional analyses. The FISH and VENTANA HER2 Dual ISH DNA Probe Cocktail assay The concordance study comparing VENTANA HER2 Dual ISH DNA Probe Cocktail and
staining results were enumerated by counting at least 20 nuclei in each specimen. The PathVysion FISH was designed to also evaluate cases based on their IHC scores for
results were reported as: HER2/Chr 17 ratio ≥ 2.0 as amplified; HER2/Chr 17 < 2.0 as HER2 protein levels (see PATHWAY anti-HER2 (4B5) antibody method sheet [ P/N
non-amplified. Of the 678 cases that were stained by both the FISH and VENTANA HER2 14427EN], for IHC scoring). This enabled a secondary analysis to compare agreement
Dual ISH DNA Probe Cocktail assays, 605 specimens were enumerable by both assays rates between PATHWAY anti-HER2 (4B5) antibody and the VENTANA HER2 Dual ISH
and therefore included in the analysis of agreement rates. DNA Probe Cocktail assay, and between PATHWAY anti-HER2 (4B5) antibody and the
Primary Results PathVysion FISH assay. In this study IHC scores of 2+/3+ were considered positive for
The primary analysis compared positive and negative percent agreement rates to assess HER2 overexpression. Agreement data for the PathVysion HER-2 FISH assay and
concordance between the VENTANA HER2 Dual ISH DNA Probe Cocktail and PATHWAY HER2/neu (4B5) antibody are shown in Table 9. Agreement data for the
PathVysion HER-2 FISH assays in breast carcinoma. Data for amplified and non-amplified VENTANA HER2 Dual ISH DNA Probe Cocktail assay and PATHWAY HER2/neu (4B5)
clinical assessments for each assay, pooling data across all sites, are presented in a 2x2 antibody are shown in Table 10.

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Table 9. IHC on the BenchMark ULTRA instrument vs. FISH Comparison: Pooled Data Table 12. Summary of negative, positive, and overall agreement rates for VENTANA
from All Sites. HER2 Dual ISH DNA Probe Cocktail and Dako HER2 IQFISH pharmDx™ on human GEA
specimens.
PathVysion HER-2 FISH Result
Negative Positive Overall
Non-
Agreement Rate Agreement Rate Agreement Rate
Amplified Amplified Total
Raw Percent Raw Percent Raw Percent
PATHWAY Positive 277 63 340
Data / (95% Score Data / (95% Score Data / (95% Score
HER2 (4B5) (2+/3+ cases)
# of CI) # of CI) # of CI)
antibody
Negative 27 238 265 Cases Cases Cases
Results
(0/1+)
VENTANA
Total 304 301 605 HER2 Dual 91.3 90.7 91.1
ISH DNA 84/92 49/54 133/146
Probe (83.8 – 95.5) (80.1 – 96.0) (85.4 – 94.7)
n/N % (95% Score CI) Cocktail
Positive Percent Agreement 277/304 91.1 (87.4, 93.8)
ANALYTICAL PERFORMANCE
Table 10. IHC on the BenchMark ULTRA instrument vs. VENTANA HER2 Dual ISH DNA BenchMark IHC/ISH instrument Repeatability and Precision with Breast Carcinoma
Probe Cocktail assay Comparison: Pooled Data from All Sites.
The repeatability and precision of VENTANA HER2 Dual ISH DNA Probe Cocktail were
VENTANA HER2 Dual ISH DNA Probe Cocktail evaluated on BenchMark IHC/ISH instruments in combination with the VENTANA Silver
Result ISH DNP Detection Kit and VENTANA Red ISH DIG Detection Kit.
Non- Within-Run Repeatability was evaluated using 28 breast carcinoma specimens. Two
Amplified Amplified Total replicate slides from each specimen were stained with VENTANA HER2 Dual ISH DNA
Probe Cocktail on a single BenchMark ULTRA, BenchMark XT, or BenchMark GX
PATHWAY Positive 248 78 326 instrument. For the analysis of the BenchMark XT and GX instrument data, the cases with
HER2 (4B5) (2+/3+ cases) ratios between 1.5 to 2.5 were weighted to their prevalence.
antibody Between-Day Intermediate Precision was also evaluated using breast carcinoma
Negative 18 253 271
Results specimens. Replicate slides from each of the 28 specimens were stained with VENTANA
(0/1+)
HER2 Dual ISH DNA Probe Cocktail on BenchMark IHC/ISH instruments on 5 non-
Total 266 331 597 consecutive days. For the analysis of the BenchMark XT and BenchMark GX instrument
data, the cases with ratios between 1.5 to 2.5 were weighted to their prevalence.
Within-Run Repeatability was determined with average positive agreement (APA),
n/N % (95% Score CI) average negative agreement (ANA), and overall percent agreement (OPA). The Between-
Positive Percent Agreement 248/266 93.2 (89.6, 95.7) Day Intermediate Precision was determined with positive percent agreement (PPA),
negative percent agreement (NPA), and overall percentage agreement (OPA) across all
the observations from the evaluable population. A summary of the results of both studies
Concordance Study: VENTANA HER2 Dual ISH DNA Probe Cocktail assay on can be found in Table 13.
BenchMark ULTRA instrument vs. Dako HER2 IQFISH pharmDx™ Kit assay Table 13. BenchMark IHC/ISH Instrument Within-Run Repeatability and Between-Day
A concordance study was performed to evaluate the VENTANA HER2 Dual ISH DNA Intermediate Precision.
Probe Cocktail assay compared to Dako HER2 IQFISH pharmDx™ Kit for fluorescent in
situ hybridization (FISH) in determination of HER2 gene status in GEA. Comparability of Repeatability Clinical Agreement
the assay on GEA specimens was determined by comparing the staining results from the Platform
/ Precision Status
two assays (Table 11). One hundred thirty-four (134) human GEA specimens (a mix of Type n/N % 95% CI
amplified and non-amplified cases) were stained using VENTANA HER2 Dual ISH DNA
Probe Cocktail. The same cohort was stained using the Dako HER2 IQFISH pharmDx™ Amplified APA 194/194 100 (98.1, 100)
assay. The results detailing negative, positive and overall agreement rates for the 146 Within-Run Non-
samples of this cohort that were enumerable with both the Dako HER2 IQFISH ULTRA ANA 186/186 100 (98.0, 100)
Repeatability Amplified
pharmDx™ assay and the VENTANA HER2 Dual ISH DNA Probe Cocktail assay are
shown in Table 11 and Table 12. Total OPA 190/190 100 (98.0, 100)
Table 11. Agreement between VENTANA HER2 Dual ISH DNA Probe Cocktail and the Amplified PPA 139/139 100 (97.3, 100)
Dako HER2 IQFISH pharmDx™ assay in a cohort of human GEA specimens. Between-
Day Non-
Dako HER2 IQFISH pharmDx™ assay ULTRA NPA 135/135 100 (97.2, 100)
VENTANA HER2 Dual Intermediate Amplified
ISH DNA Probe Cocktail Amplification Status Precision
Total OPA 274/274 100 (98.6, 100)
Amplification Status Amp Non-Amp
128.8/
49 8 Amplified APA 100 (97.1, 100)
Amp 128.8

Non-Amp 5 84 Within-Run Non- 151.2/

XT ANA 100 (97.5, 100)
Repeatability Amplified 151.2
140.0/
Total OPA 100 (97.3, 100)
140.0
Between- 128.8/
XT Amplified PPA 100 (97.1, 100)
Day 128.8

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 Table 14. BenchMark IHC/ISH Between-Instrument Intermediate Precision.
Repeatability Clinical Agreement
Platform
/ Precision Status Clinical Agreement
Type n/N % 95% CI Platform Precision
Status
Intermediate Non- 151.2/ Type n/N % 95% CI
Precision NPA 100 (97.5, 100)
Amplified 151.2 Amplified PPA 84/84 100 (95.6, 100)
Between-
280.0/ Instrument Non-
Total OPA 100 (98.6, 100) ULTRA NPA 84/84 100 (95.6, 100)
280.0 Intermediate Amplified
128.8/ Precision
Amplified APA 100 (97.1, 100) Total OPA 168/168 100 (97.8, 100)
128.8
77.3/
Within-Run Non- 151.2/ Amplified PPA 100 (95.3, 100)
GX ANA 100 (97.5, 100) 77.3
Repeatability Amplified 151.2 Between-
Instrument Non- 90.7/
140.0/ XT NPA 100 (95.9, 100)
Total OPA 100 (97.3, 100) Intermediate Amplified 90.7
140.0 Precision
168.0/
128.8/ Total OPA 100 (97.8, 100)
Amplified PPA 100 (97.1, 100) 168.0
128.8
Between- 76.2/
Day Non- 151.2/ Amplified PPA 100 (95.2, 100)
GX NPA 100 (97.5, 100) 76.2
Intermediate Amplified 151.2 Between-
Precision Instrument Non- 90.7/
280.0/ GX NPA 100 (95.9, 100)
Total OPA 100 (98.6, 100) Intermediate Amplified 90.7
280.0 Precision
166.9/
Note: 95% CIs were calculated using the percentile bootstrap method; in instances Total OPA 100 (97.8, 100)
166.9
where the point estimate was 100%, Wilson Score method was used. . Four cases
with ISH ratios between 1.5 to 2.5, inclusive, were included in the study. Note: 95% CIs were calculated using the percentile bootstrap method; in instances
where the point estimate was 100%, Wilson Score method was used. Four cases
with ISH ratios between 1.5 to 2.5, inclusive, were included in the study.
Between-Instrument Intermediate Precision with Breast Carcinoma
BenchMark IHC/ISH instrument between-instrument intermediate precision of the
VENTANA HER2 Dual ISH DNA Probe Cocktail was determined by staining replicate Within-Reader and Between-Reader Precision with Breast Carcinoma
slides of 28 breast carcinoma specimens on 3 BenchMark IHC/ISH instruments with the BenchMark IHC/ISH instrument within-reader and between-reader precision of the
VENTANA HER2 Dual ISH DNA Probe Cocktail using the VENTANA Silver ISH DNP VENTANA HER2 Dual ISH DNA Probe Cocktail was determined by having three readers
Detection Kit and VENTANA Red ISH DIG Detection Kit. The between-instrument evaluate 60 breast carcinoma specimens stained with the VENTANA HER2 Dual ISH DNA
intermediate precision was determined with PPA, NPA, and OPA across all the Probe Cocktail using the VENTANA Silver ISH DNP Detection Kit and VENTANA Red ISH
observations from the evaluable population. The cases with ratios between 1.5 to 2.5 DIG Detection Kit on BenchMark ULTRA instrument. For within-reader precision, the same
were weighted to their prevalence (BenchMark XT / BenchMark GX instruments). A set of slides were read twice after a minimum of two weeks between reads. The within-
summary of the results of this study can be found in Table 14. reader and between-reader precision was determined with APA, ANA, and OPA across all
the observations from the evaluable population. A summary of the results of this study can
be found in Table 15.
Table 15. BenchMark ULTRA Instrument Within-Reader and Between-Reader Precision.

Clinical Agreement
Precision
Status
Type n/N % 95% CI
Amplified APA 178/181 98.3 (96.3, 100)
Non-
Within-Reader ANA 174/177 98.3 (96.1, 100)
Amplified
Total OPA 176/179 98.3 (96.1, 100)
Amplified APA 350/362 96.7 (93.2, 99.4)
Between
Reader Non-
ANA 342/354 96.6 (92.8, 99.4)
Amplified
Total OPA 346/358 96.6 (92.8, 99.4)
Note: 95% CIs were calculated using the percentile bootstrap method. Six cases
with ISH ratios between 1.5 to 2.5, inclusive, were included in the study.

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Between Platform Precision with Breast Carcinoma Table 17. BenchMark IHC/ISH Instrument: Within-Run Repeatability and Between-Day
BenchMark IHC/ISH instrument between platform precision of the VENTANA HER2 Dual Intermediate Precision.
ISH DNA Probe Cocktail was determined by evaluating 28 breast carcinoma specimens
stained with the VENTANA HER2 Dual ISH DNA Probe Cocktail using the VENTANA Repeatability Clinical Agreement
Silver ISH DNP Detection Kit and VENTANA Red ISH DIG Detection Kit on BenchMark Platform
/ Precision Status
IHC/ISH instruments. The between platform precision was determined with PPA, NPA, Type n/N % 95% CI
and OPA across all the observations from the evaluable population. The cases with ratios
70.0/
between 1.5 to 2.5 were weighted to their prevalence. A summary of the results of this Amplified APA 100 (94.8, 100)
70.0
study can be found in Table 16.
Table 16. BenchMark IHC/ISH Instrument Between Platform Precision. Within-Run Non- 70.0/
ULTRA ANA 100 (94.8, 100)
Repeatability Amplified 70.0
Clinical Agreement 70.0/
Precision Total OPA 100 (94.8, 100)
Status 70.0
Type n/N % 95% CI
70.0/
230.8/ Amplified PPA 100 (94.8, 100)
Amplified PPA 100 (98.4, 100) 70.0
230.8 Between-
Between Day Non- 70.0/
Non- 271.0/ ULTRA NPA 100 (94.8, 100)
Platform NPA 99.6 (98.3, 100) Intermediate Amplified 70.0
Amplified 272.2 Precision
Precision 140.0/
501.8/ Total OPA 100 (97.3, 100)
Total OPA 99.8 (99.2, 100) 140.0
502.9
70.0/
Note: 95% CIs were calculated using the percentile bootstrap method; in instances Amplified APA 100 (94.8, 100)
70.0
where the point estimate was 100%, Wilson Score method was used. Four cases
with ISH ratios between 1.5 to 2.5, inclusive, were included in the study. Within-Run Non- 70.0/
XT ANA 100 (94.8, 100)
Repeatability Amplified 70.0

BenchMark IHC/ISH Instrument: Repeatability and Precision with Gastric 70.0/

Total OPA 100 (94.8, 100)
Adenocarcinoma 70.0
The repeatability and precision of VENTANA HER2 Dual ISH DNA Probe Cocktail were 70.0/
Amplified PPA 100 (94.8, 100)
evaluated on the BenchMark IHC/ISH instruments in combination with the VENTANA 70.0
Between-
Silver ISH DNP Detection Kit and VENTANA Red ISH DIG Detection Kit.
Day Non- 70.0/
Within-Run Repeatability was evaluated using fourteen gastric adenocarcinoma XT NPA 100 (94.8, 100)
Intermediate Amplified 70.0
specimens. Two replicate slides from each of the gastric adenocarcinoma specimens were Precision
stained with VENTANA HER2 Dual ISH DNA Probe Cocktail on a single BenchMark 140.0/
Total OPA 100 (97.3, 100)
IHC/ISH instrument. The cases with ratios between 1.5 to 2.5 were weighted to their 140.0
prevalence. 64.6/
Between-Day Intermediate Precision was also evaluated using gastric adenocarcinoma Amplified APA 99.1 (95.9, 100)
65.1
specimens. Replicate slides from each of the fourteen specimens were stained with
VENTANA HER2 Dual ISH DNA Probe Cocktail on BenchMark IHC/ISH instruments on 5 Within-Run Non- 70.0/
GX ANA 99.2 (95.2, 100)
non-consecutive days. The cases with ratios between 1.5 to 2.5 were weighted to their Repeatability Amplified 70.6
prevalence. 67.3/
Total OPA 99.2 (96.9, 100)
Within-Run Repeatability was determined with average positive agreement (APA), 67.9
average negative agreement (ANA), and overall percent agreement (OPA). The Between-
Day Intermediate Precision was determined with positive percent agreement (PPA), 67.3/
Amplified PPA 99.2 (96.5, 100)
negative percent agreement (NPA), and overall percentage agreement (OPA) across all 67.9
Between-
the observations from the evaluable population. A summary of the results of both studies Day Non- 70.0/
can be found in Table 17. GX NPA 100 (94.8, 100)
Intermediate Amplified 70.0
Precision
137.3/
Total OPA 99.6 (98.5, 100)
137.9
Note: 95% CIs were calculated using the percentile bootstrap method; in instances
where the point estimate was 100%, Wilson Score method was used. Two cases with
ISH ratios between 1.5 to 2.5, inclusive, were included in the study.

Between-Instrument Intermediate Precision with Gastric Adenocarcinoma

BenchMark IHC/ISH instrument between-instrument intermediate precision of the
VENTANA HER2 Dual ISH DNA Probe Cocktail was determined by staining replicate
slides of fourteen gastric adenocarcinoma specimens on 3 BenchMark IHC/ISH
instruments with the VENTANA HER2 Dual ISH DNA Probe Cocktail using the VENTANA
Silver ISH DNP Detection Kit and VENTANA Red ISH DIG Detection Kit. The between-
instrument intermediate precision was determined with PPA, NPA, and OPA across all the
observations from the evaluable population. The cases with ratios between 1.5 to 2.5 were
weighted to their prevalence. A summary of the results of this study can be found in
Table 18.

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Table 18. BenchMark IHC/ISH Between-Instrument Intermediate Precision. specimens stained with the VENTANA HER2 Dual ISH DNA Probe Cocktail using the
VENTANA Silver ISH DNP Detection Kit and VENTANA Red ISH DIG Detection Kit on
Clinical Agreement BenchMark IHC/ISH instruments. The between platform precision was determined with
Platform Precision PPA, NPA, and OPA across all the observations from the evaluable population. The cases
Status
Type n/N % 95% CI with ratios between 1.5 to 2.5 were weighted to their prevalence. A summary of the results
of this study can be found in Table 20.
42.0/
Amplified PPA 100 (91.6, 100) Table 20. BenchMark IHC/ISH Instrument: Between Platform Precision.
42.0
Between-
Instrument Non- 42.0/ Clinical Agreement
ULTRA NPA 100 (91.6, 100) Precision
Intermediate Amplified 42.0 Status
Precision Type n/N % 95% CI
84.0/
Total OPA 100 (95.6, 100)
84.0 123.3/12
Amplified PPA 99.5 (98.1, 100)
40.4/ 3.9
Amplified PPA 98.6 (94.1, 100) Between
40.9 Non- 124.9/12
Between- Platform NPA 100 (97.0, 100)
Instrument Non- 40.9/ Amplified 4.9
XT NPA 100 (91.4, 100) Precision
Intermediate Amplified 40.9 248.2/24
Precision Total OPA 99.8 (99.2, 100)
81.3/ 8.8
Total OPA 99.3 (97.5, 100)
81.9 Note: 95% CIs were calculated using the percentile bootstrap method; in instances
40.9/ where the point estimate was 100%, Wilson Score method was used. Two cases with
Amplified PPA 100 (91.4, 100) ISH ratios between 1.5 to 2.5, inclusive, were included in the study.
40.9
Between-
Instrument Non- 42.0/
GX NPA 100 (91.6, 100) Lot-to-Lot Precision with Breast Carcinoma
Intermediate Amplified 42.0
Precision Lot-to-Lot Precision was determined by testing 3 production lots of the VENTANA HER2
82.9/ Dual ISH DNA Probe Cocktail, VENTANA Silver ISH DNP Detection Kit, and VENTANA
Total OPA 100 (95.6, 100)
82.9 Red ISH DIG Detection Kit on BenchMark ULTRA instruments. Twenty-eight breast
Note: 95% CIs were calculated using the percentile bootstrap method; in instances carcinoma cases were stained with each probe and detection kit. A summary of the results
where the point estimate was 100%, Wilson Score method was used. Two cases for Lot-to-Lot Precision of the assay is shown in Table 21.
with ISH ratios between 1.5 to 2.5, inclusive, were included in the study. Table 21. Lot-to-Lot Precision.

Clinical Agreement
Within-Reader and Between-Reader Precision with Gastric Adenocarcinoma Precision
Status
BenchMark IHC/ISH instrument within-reader and between-reader precision of the Type n/N % 95% CI
VENTANA HER2 Dual ISH DNA Probe Cocktail was determined by having three readers
evaluate 28 gastric adenocarcinoma specimens stained with the VENTANA HER2 Dual Amplified PPA 121/121 100 (96.9, 100)
ISH DNA Probe Cocktail using the VENTANA Silver ISH DNP Detection Kit and
Non-
VENTANA Red ISH DIG Detection Kit on BenchMark ULTRA instrument. All slides were Lot-to-Lot NPA 123/123 100 (97.0, 100)
Amplified
randomized and masked to the case diagnosis. For within-reader precision, the same set
of slides were read twice after a minimum of two weeks between reads. The within-reader Total OPA 244/244 100 (98.5, 100)
precision and between-reader precision was determined with APA, ANA, and OPA across
all the observations from the evaluable population. A summary of the results of this study Note: 95% CIs were calculated using the percentile bootstrap method; in instances
can be found in Table 19. where the point estimate was 100%, Wilson Score method was used. Four cases with
Table 19. BenchMark ULTRA Instrument Within-Reader and Between-Reader Precision. ISH ratios between 1.5 to 2.5, inclusive, were included in the study.

Clinical Agreement BenchMark ULTRA Instrument Inter-laboratory Reproducibility Study with Breast
Precision Carcinoma and Gastric Adenocarcinoma
Status
Type n/N % 95% CI An inter-laboratory reproducibility (ILR) study was conducted to evaluate the
Amplified APA 80/84 95.2 (90.5, 100) reproducibility of VENTANA HER2 Dual ISH DNA Probe Cocktail to determine HER2 gene
status in breast carcinoma and gastric adenocarcinoma tissue stained on the BenchMark
Between Non- 80/84 95.2 (90.5, 100) ULTRA instrument in combination with the VENTANA Silver ISH DNP Detection Kit and
ANA
Reader Amplified VENTANA Red ISH DIG Detection Kit.
Total OPA 80/84 95.2 (90.5, 100) Twenty-eight FFPE breast and gastric adenocarcinoma tissue specimens were used and
approximately half of these cases were amplified for HER2 expression status and half
Amplified APA 82/84 97.6 (95.2, 100) were non-amplified for HER2 status.
Non- 82/84 97.6 (95.2, 100) Multiple tissue sections were cut from each specimen and provided to 3 external study
Within-Reader ANA
Amplified sites. Each site stained 28 breast and 28 gastric cases on each of 5 non-consecutive days
over a minimum of 20 days. Following staining on the BenchMark ULTRA instrument a
Total OPA 82/84 97.6 (95.2, 100) reader evaluated each slide to assign HER2 gene status.
Note: 95% CIs were calculated using the percentile bootstrap method. Two cases The results of the study are summarized in Table 22 and Table 23, below. The data was
with ISH ratios between 1.5 to 2.5, inclusive, were included in the study. analyzed for PPA and NPA across all observations. For each case, all evaluable
observations (amplified vs non-amplified) were compared against the modal result for
each case. The cases with ratios between 1.5 to 2.5 were weighted to their prevalence.
Between Platform Precision with Gastric Adenocarcinoma
These comparisons were pooled across sites and days, and then results were aggregated
BenchMark IHC/ISH instrument between platform precision of the VENTANA HER2 Dual across cases.
ISH DNA Probe Cocktail was determined by evaluating fourteen gastric adenocarcinoma

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Table 22. ILR: Agreement Rates on the BenchMark ULTRA instrument for Breast Performance of VENTANA HER2 Dual ISH DNA Probe Cocktail on the BenchMark
Carcinoma. ULTRA PLUS Instrument
Concordance Between BenchMark ULTRA PLUS and BenchMark ULTRA
Inter-Laboratory Agreement Instruments for Breast Carcinoma
Reproducibility Three laboratories, from separate institutions in the United States, participated in a
Type n/N % 95% CI concordance study between the BenchMark ULTRA PLUS instrument versus the
PPA 208.9/208.9 100 (98.2, 100.0) BenchMark ULTRA instrument. There were 193 unique FFPE invasive breast carcinoma
cases which represented the staining range of the VENTANA HER2 Dual ISH Cocktail
Between-Site
NPA 198.1/200.3 98.9 (96.8, 100.0) assay, with approximately equal distribution between HER2-amplified and HER2–non-
(3 sites)
amplified cases. Tissue slides from all cases were stained with H&E as well as VENTANA
OPA 407.0/409.3 99.5 (98.4, 100.0) HER2 Dual ISH Cocktail by Roche on a BenchMark ULTRA instrument using the
(92.3, 100.0) recommended staining protocol. Unstained tissue slides from all cases were randomized
PPA 72/74 97.3
and equally distributed (64-65 cases/per site) across study sites for staining on a
Site A NPA 63/63 100 (94.3, 100.0) BenchMark ULTRA PLUS instrument using the recommended VENTANA HER2 Dual ISH
Cocktail staining protocol. Pathologists, blinded to case status, evaluated the slides
OPA 135/137 98.5 (95.6, 100.0) stained on one BenchMark IHC/ISH instrument and determined the HER2 gene status.
Between- After a two week period, pathologists evaluated the slides stained on the second
PPA 70/70 100 (94.8, 100.0)
Day BenchMark IHC/ISH instrument. HER2 gene status was determined using the ratio of
(5 non- Site B NPA 63/64 98.4 (95.8, 100.0) HER2 gene signals to chromosome 17 (Chr17) signals (i.e., the HER2:Chr17 ratio) in the
consecutive tumor cell nuclei. If the ratio was 2.0 or greater, the case was considered HER2-amplified;
days) OPA 133/134 99.3 (97.8, 100.0) if it was less than 2.0, it was considered HER2–non-amplified. The results were analyzed
by Roche. The OPA, PPA and NPA rates were 97.1% (535/551), 97.3% (248/255), and
PPA 70/70 100 (94.8, 100.0)
97.0% (287/296), respectively. The results are summarized in Table 24.
Site C NPA 69/69 100 (94.7, 100.0) Table 24. Pooled Agreement of HER2 Gene Status for Breast Carcinoma Cases Stained
with VENTANA HER2 Dual ISH Cocktail on the BenchMark ULTRA PLUS vs BenchMark
OPA 139/139 100 (97.3, 100.0) ULTRA Instrument
Note: 95% CIs were calculated using the percentile bootstrap method; in instances BenchMark ULTRA HER2 Gene
where the point estimate was 100%, Wilson Score method was used. Four cases with Status
BenchMark ULTRA PLUS
ISH ratios between 1.5 to 2.5, inclusive, were included in the study.
HER2 Gene Status Amplified Non-Amplified Total
Amplified 248 9 257
Table 23. ILR: Agreement Rates on the BenchMark ULTRA instrument for Gastric
Adenocarcinoma. Non-Amplified 7 287 294
Total 255 296 551
Inter-Laboratory Agreement
Reproducibility n/N % (95% CI)
Type n/N % 95% CI PPA 248/255 97.3 (95.0, 99.2)
PPA 206.8/206.8 100 (98.2, 100.0) NPA 287/296 97.0 (94.8, 99.0)
Between-Site
NPA 208.4/208.9 99.7 (99.2, 100.0) OPA 535/551 97.1 (95.5, 98.6)
(3 sites)
OPA 415.1/415.7 99.9 (99.6, 100.0) Note: Two-sided 95% CIs were calculated using the percentile bootstrap method with
2000 replicates selected with stratification on the 4 diagnostic score bins used during
PPA 70/70 100 (94.8, 100.0) case selection [amplified (not borderline), non-amplified (not borderline), borderline
(96.0, 100.0) amplified, borderline non-amplified]
Site A NPA 69/70 98.6
OPA 139/140 99.3 (97.9, 100.0)
BenchMark ULTRA PLUS Instrument Inter-laboratory Reproducibility Study with
Between- PPA 67/67 100 (94.6, 100.0) Breast Carcinoma
Day An inter-laboratory reproducibility (ILR) study was conducted to evaluate the
(5 non- Site B NPA 69/69 100 (94.7, 100.0) reproducibility of VENTANA HER2 Dual ISH DNA Probe Cocktail to determine HER2 gene
consecutive status in breast carcinoma tissue stained on the BenchMark ULTRA PLUS instrument in
days) OPA 136/136 100 (97.3, 100.0)
combination with the VENTANA Silver ISH DNP Detection Kit and VENTANA Red ISH
PPA 70/70 100 (94.8, 100.0) DIG Detection Kit.
(94.8, 100.0) Twenty-eight unique FFPE invasive breast carcinoma cases, which represented the
Site C NPA 70/70 100
staining range of the VENTANA HER2 Dual ISH Cocktail assay, were used with an
OPA 140/140 100 (97.3, 100.0) approximate equal distribution between HER2-amplified and HER2–non-amplified cases.
Multiple tissue sections were cut from each specimen and provided to 3 external study
Note: 95% CIs were calculated using the percentile bootstrap method; in instances sites. All 28 cases were stained on a BenchMark ULTRA PLUS instrument on each of 5
where the point estimate was 100%, Wilson Score method was used. Four cases with non-consecutive days over a minimum of 20 days at each site. Readers evaluated the
ISH ratios between 1.5 to 2.5, inclusive, were included in the study. slides and determined the HER2 gene status.
Results are summarized in Table 25 and Table 26. The data was analyzed for PPA, NPA
and OPA in Table 25 and APA, ANA, OPA in Table 26 across all observations. For each
case, all evaluable observations (amplified vs non-amplified) were compared against the
modal result for each case. These comparisons were pooled across sites and days, and
then results were aggregated across cases.

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Table 25. ILR: Agreement Rates with Modal Status on the BenchMark ULTRA PLUS All agreement rates were calculated using a two-sided 95% confidence intervals using the
instrument for Breast Carcinoma. percentile bootstrap method.
Inter-Laboratory Agreement For Within-Run Repeatability, five slides per case were stained on the BenchMark ULTRA
Reproducibility PLUS instrument. The overall percent agreement for VENTANA HER2 Dual ISH Cocktail
Type n/N % 95% CI staining, based on HER2 gene status (HER2-amplified, HER2-non-amplified), was 91.7%
(95% CI: 81.7, 100.0).
Overall PPA 372/381 97.6 (95.3, 100.0) For Between-Day Intermediate Precision, two slides per case were stained on the
BenchMark ULTRA PLUS instrument in five staining runs conducted over a five, non-
NPA 421/440 95.7 (91.1, 99.3) consecutive day period. The overall percent agreement for VENTANA HER2 Dual ISH
Cocktail staining based on HER2 gene status (HER2-amplified, HER2-non-amplified) was
OPA 793/821 96.6 (94.3, 98.5)
90.8% (95% CI: 80.8, 100.0)
Between-Site PPA 380/389 97.7 (95.3, 100.0) For Between-Instrument Intermediate Precision, two slides per case were stained on each
(3 sites) of three BenchMark ULTRA PLUS instruments. The overall percent agreement for
NPA 421/432 97.5 (95.3, 99.3) VENTANA HER2 Dual ISH Cocktail staining based on HER2 gene status (HER2-
amplified, HER2-non-amplified) was 92.6% (95% CI: 84.5, 100.0).
OPA 801/821 97.6 (96.3, 98.7)
Sensitivity and Specificity
Between-Reader PPA 383/389 98.5 (97.1, 99.5) Analytical specificity (hybridization efficacy) of the VENTANA HER2 Dual ISH DNA Probe
Cocktail assay was determined by staining normal human metaphase spreads on a
NPA 424/432 98.1 (97.1, 99.0) BenchMark ULTRA instrument. Of 100 metaphase spreads analyzed, 100% exhibited
specific co-localization of both HER2 and Chromosome 17 probes.
OPA 807/821 98.3 (97.5, 99.0)
Analytical sensitivity measures the ability of the probe to detect its specific target, while
Note: Two-sided 95% CIs were calculated using the percentile bootstrap method. specificity is its ability to distinguish the target from other sequences in the specimen. The
assay has an analytical sensitivity and specificity control built into each human tissue.
Normal human cells (including: stromal fibroblasts, endothelial cells, lymphocytes, and
Table 26. ILR: Pairwise Agreement Rates on the BenchMark ULTRA PLUS instrument for non-neoplastic breast epithelial cells) should contain 1-2 copies of HER2 and Chr17.
Breast Carcinoma Therefore, 1-2 copies for HER2 and Chr17 in normal human cells indicates that the probes
are detecting their specific target (a measure of sensitivity). One to two copies for HER2
Inter-Laboratory Agreement
and Chr17 in normal cells also indicates that the probe is detecting only its specific targets
Reproducibility
Type n/N % 95% CI (a measure of specificity). The first pass rate for the VENTANA HER2 Dual ISH DNA
Probe Cocktail assay on 40 breast samples fixed within the ASCO CAP guidelines (10%
Between-Site (3 sites) APA 7204/7652 94.1 (91.1, 96.9) NBF for 6 to 72 hours) was 97.5% (87.1– 99.6) on BenchMark ULTRA instruments, 100%
(91.2 – 100) on BenchMark XT instruments, and 97.5% (87.1 – 99.6) on BenchMark GX
ANA 7968/8416 94.7 (91.5, 97.4) instruments. Specificity on the same 40 breast samples with no probe control was 100%
(91.2 – 100) on BenchMark ULTRA instruments.
OPA 7586/8034 94.4 (91.5, 97.1)
The first pass rate for the VENTANA HER2 Dual ISH DNA Probe Cocktail assay on 39
Between-Reader APA 370/390 94.9 (92.5, 97.1) gastric samples fixed within the ASCO CAP guidelines (10% NBF for 6 to 72 hours) was
97.4% (86.8 – 99.5) on BenchMark ULTRA instruments, 97.4% (86.8 – 99.5) on
ANA 408/428 95.3 (92.7, 97.5) BenchMark XT instruments, and 100% (91 – 100) on BenchMark GX instruments.
Analytical sensitivity and specificity was also assessed by staining multiple cases of
OPA 389/409 95.1 (92.7, 97.3)
normal and neoplastic human tissues with VENTANA HER2 Dual ISH DNA Probe Cocktail
Between-Day APA 1472/1519 96.9 (95.5, 98.2) assay, VENTANA Silver ISH DNP Detection Kit, and VENTANA Red ISH DIG Detection
(5 non-consecutive Kit. The results are listed in Table 27 and Table 28. No unexpected staining was observed
days) ANA 1642/1689 97.2 (95.8, 98.5) with VENTANA HER2 Dual ISH DNA Probe Cocktail assay on the normal and neoplastic
tissues.
OPA 1557/1604 97.1 (95.7, 98.3) Table 27. Sensitivity/Specificity of VENTANA HER2 Dual ISH DNA Probe Cocktail assay
Note: Two-sided 95% CIs were calculated using the percentile bootstrap method. was determined by testing FFPE normal tissues.
# acceptable / # acceptable /
Tissue total cases Tissue total cases
Concordance Between BenchMark ULTRA PLUS and BenchMark ULTRA
Instruments for Gastric Carcinoma Adrenal gland 3/3 Lung 3/3
There were 109 unique FFPE invasive gastric carcinoma cases which represented the
staining range of the VENTANA HER2 Dual ISH Cocktail assay, with approximately equal Bladder 3/3 Lymph node 3/3
distribution between HER2-amplified and HER2–non-amplified cases. Tissue slides were 3/3 3/3
Bone marrow Mesothelium
stained on a BenchMark ULTRA PLUS instrument and BenchMark ULTRA instrument
using the recommended staining protocol. Stained slides were scored by a pathologist. Ovary 3/3 Pancreas 3/3
The overall percent agreement for VENTANA HER2 Dual ISH Cocktail staining based on
HER2 gene status (HER2-amplified, HER2-non-amplified) was 92.4%. Two-sided 95% Breast 3/3 Parathyroid gland 3/3
confidence intervals, which is 84.4% to 96.5%, were calculated using the Wilson score
method. Background and morphology acceptability rates for all cases were 100% for the Cerebellum 3/3 Peripheral nerve 3/3
BenchMark ULTRA PLUS instrument. 3/3 3/3
Cerebrum Prostate
BenchMark ULTRA PLUS Instrument Precision Studies for Gastric Carcinoma
Twelve gastric carcinoma tissue cases, which represented the staining range of the Cervix 3/3 Skeletal Muscle 3/3
VENTANA HER2 Dual ISH Cocktail assay, were tested on the BenchMark ULTRA PLUS
Colon 3/3 Skin 3/3
instrument. The cases had approximately equal distribution between HER2-amplified and
HER2–non-amplified HER2 gene status. Stained slides were evaluated by a pathologist.

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FT0700-415l
 # acceptable / # acceptable / # acceptable /
Tissue total cases Tissue total cases Pathology total cases
Endometrium 3/3 Spleen 3/3 Hepatocellular carcinoma (Liver) 1/1

Esophagus 3/3 Stomach 3/3 Clear cell carcinoma (Kidney) 1/1

Heart 3/3 Testis 3/3 Adenocarcinoma (Prostate) 2/2

Hypophysis (Pituitary) 3/3 Thymus 3/3 Leiomyoma (Uterus) 1/1

Intestine 3/3 Thyroid 3/3 Endometrioid adenocarcinoma (Uterus) 1/1

Kidney 3/3 Tongue/Salivary gland 3/3 Clear cell carcinoma (Uterus) 1/1

Liver 3/3 Tonsil 3/3 Squamous cell carcinoma (Cervix) 2/2

Embryonal rhabdomyosarcoma (Striated muscle) 1/1

Table 28. Sensitivity/Specificity of VENTANA HER2 Dual ISH DNA Probe Cocktail assay Squamous cell carcinoma (Skin) 1/1
was determined by testing a variety of FFPE neoplastic tissues.
Basal cell carcinoma (Skin) 1/1
# acceptable /
Pathology total cases Neurofibroma (Lumbar) 1/1
Glioblastoma (Cerebrum) 3/3 Neuroblastoma (Retroperitoneum) 1/1

Meningioma (Cerebrum) 1/1 Mesothelioma (Peritoneum) 1/1

Oligodendroglioma (Cerebrum) 1/1 B-cell lymphoma; NOS (Lymph node) 2/2

Endometrioid Carcinoma (Ovary) 1/1 Hodgkin lymphoma (Lymph node) 3/3

Adenocarcinoma (Ovary) 1/1 Anaplastic large cell lymphoma (Lymph node) 1/1

Pancreatic neuroendocrine neoplasm (Pancreas) 1/1 Leiomyosarcoma (Bladder) 1/1

Adenocarcinoma (Pancreas) 1/1 Urothelial carcinoma (Bladder) 1/1

Seminoma (Testis) 1/1 Osteosarcoma (Bone) 1/1

Embryonal carcinoma (Testis) 1/1 Mesothelioma (Peritoneum) 1/1

Medullary carcinoma (Thyroid) 1/1 Leiomyosarcoma (Smooth muscle) 1/1

Papillary carcinoma (Thyroid) 1/1

Ductal carcinoma in situ (Breast) 1/1 TROUBLESHOOTING

Table 29. Troubleshooting Solutions.
Invasive ductal carcinoma (Breast) 2/2
Issue Solution
B-cell lymphoma; NOS (Spleen) 1/1
Absent or 1. Ensure reagent dispensers are functioning properly (i.e., not
Small cell carcinoma (Lung) 1/1 clogged or empty) and bulk solutions are filled. Check the
Weak SISH
1/1 Staining reagent dispenser priming chamber or meniscus for foreign
Squamous cell carcinoma (Lung)
materials or particulates, such as fibers or precipitates. If the
Adenocarcinoma (Esophagus) 1/1 dispenser is blocked, do not use the dispenser and contact
your local support representative. Otherwise, re-prime the
Squamous cell carcinoma (Esophagus) 1/1 dispenser by aiming the dispenser over a waste container,
removing the nozzle cap, and pressing down on the top of
Adenocarcinoma (Stomach) 1/1 the dispenser. If staining is still weak or absent, proceed to 2
1/1 below.
Adenocarcinoma (Gastroesophageal junction)
2. Ensure fixation type, time and section thickness is
Adenocarcinoma (Small intestine) 1/1 appropriate for ISH-based assays.
3. Ensure use of SISH compatible mounting media (see
Gastrointestinal stromal tumor (GIST) (Small intestine) 1/1 Table 30) to preserve SISH signal. If staining is still weak or
Gastrointestinal stromal tumor (GIST) (Colon) 1/1 absent, proceed to 4 below.
4. Increase CC1 time to > 16 min.
Adenocarcinoma (Colon) 1/1
5. Increase CC2 time to > 16 min for gastric carcinoma/GEA or
Adenocarcinoma (Rectum) 1/1 > 24 min for breast carcinoma.
6. Increase ISH Protease 3 time to > 16 min for gastric
Gastrointestinal stromal tumor (GIST) (Rectum) 1/1 carcinoma or > 20 min for breast carcinoma if nuclear
morphology is intact.
Hepatoblastoma (Liver) 1/1

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 Issue Solution Type
(Xylene, Compatibility
Absent or 1. Ensure reagent dispensers are functioning properly (i.e., not Mounting Media Manufacturer
clogged or empty) and bulk solutions are filled. If staining is alcohol, with SISH
Weak Red aqueous)
ISH Staining still weak or absent, proceed to 2 below.
2. Ensure alcohol baths and extended xylene washes are not BioMount 2 BBInternational Xylene Yes
used to dehydrate stained slides, as this will degrade Red
ISH signals. If staining is still weak or absent, proceed to 3 Cytoseal 60 Richard Allan Scientific Xylene Yes
below.
Cytoseal XYL Richard Allan Scientific Xylene Yes
3. Ensure fixation type, time and section thickness is
appropriate for ISH-based assays. Diamount Diapath Xylene Yes
4. Increase CC1 time to > 16 min.
5. Increase CC2 time to > 16 min for gastric carcinoma or > 24 DPX BDH: Raymond Lamb Xylene Yes
min for breast carcinoma.
FloTexx Lerner Labs Xylene Yes
6. Increase ISH Protease 3 time to > 16 min for gastric
carcinoma or > 20 min for breast carcinoma if nuclear Gel Mount Biomeda Aqueous Yes
morphology is intact.
1. Ensure that positively charged slides are used and specimen Histomount Raymond Lamb Xylene Yes
Nonspecific
Red ISH is fixed and sectioned appropriately for ISH-based assays.
MicroMount SurgiPath Xylene Yes
Background 2. If Red ISH background is discernible from specific Red ISH
signal, enumerate the slide but do not count non-specific Red MM24 SurgiPath Xylene Yes
ISH signals.
3. If Red ISH background in the nucleus interferes with Mountex Histolab Xylene Yes
enumeration, repeat the staining using 76°C or 78°C
MountQuick Daido Sangyo Co. Aqueous Yes
stringency wash temperature. Decreasing protease or cell
conditioning time also mitigates red background. Paramount Protaqs Quartett: Dako Xylene Yes
Nonspecific 1. Ensure that positively charged slides are used and specimen
is fixed and sectioned appropriately for ISH-based assays. Permount Fisher Xylene Yes
SISH
Background 2. If SISH background is discernible from specific SISH signal, Pertex Cell Path Xylene Yes
enumerate the slide but do not count non-specific signals.
3. If SISH background in the nucleus interferes with Shandon Consul mount Thermo Scientific Xylene Yes
enumeration, repeat the staining with lower protease
treatment or lower cell conditioning time. Softmount WAKO Lemasol A Yes

Precipitation 1. If precipitation artifact interferes with enumeration, repeat the SureMount Triangle Biomedical Xylene Yes
staining. If SISH background is discernible from specific SISH Sciences
signal, enumerate the slide but do not count non-specific
signals. Thermo EZ Mount Thermo Scientific Xylene Yes
2. Ensure that barcode slide labels are centered and applied to
the glass slide with no label overhang. Do not double label or Ultramount Dako Xylene Yes
reapply barcode labels.
Bubbling If bubbling interferes with enumeration, ensure pre analytical REFERENCES
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 metastatic breast cancer. A phase III, multinational, randomized controlled trial. Symbols
N Engl J Med. 2001;344:783-792. Ventana uses the following symbols and signs in addition to those listed in the ISO
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epidermal growth factor receptor 2-positive metastatic breast cancer administered Global Trade Item Number
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Trastuzumab in combination with chemotherapy versus chemotherapy alone for Specimen Preparation, Warnings and Precautions, Staining Procedure,
treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer Quality Control Procedures, Staining Interpretation/Expected Results,
(ToGA): A phase 3, open-label, randomised controlled trial. Lancet 2010;376: 687- Specific Limitations, Performance Characteristics, Clinical Performance,
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5):v38-v49 VENTANA, BENCHMARK, HYBREADY, PATHWAY, ultraView, and the VENTANA logo
18. Van Cutsem E, Bang YJ, Feng-Yi F, et al. Her2 Screening Data from Toga: are trademarks of Roche. All other trademarks are the property of their respective owners.
Targeting Her2 in Gastric and Gastroesophageal Junction Cancer. Gastric Cancer. © 2022 Ventana Medical Systems, Inc.
2015;18(3):476-484.
CONTACT INFORMATION
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NOTE: A point (period/stop) is always used in this document as the decimal separator to
mark the border between the integral and the fractional parts of a decimal numeral.
Separators for thousands are not used.
The summary of safety and performance can be found here:
https://ec.europa.eu/tools/eudamed

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FT0700-415l
 Appendix A: VENTANA HER2 Dual ISH DNA Probe Cocktail Scoring Form

1. Case ID / Patient ID: 2. Is this case enumerable? 2a. Yes-Proceed to #3 3. Is tumor heterogeneity present? 3a. Yes, Skip #4. Proceed to #5.
2b. No-Skip. #3. Proceed to #4 3b. No, Skip #4. Proceed to #5.
4. This case is not enumerable 4a. There was no tissue left on 4b. There was no invasive 4c. Nuclear Morphology is 4d. Background unacceptable,
because (mark ALL that apply): the ISH stained slide carcinoma in tissue on the ISH unacceptable; unable to distinguish tissue interferes with scoring ISH stained slide
stained slide structural elements of normal cells from
those of target carcinoma cells. HER2 Chr 17
4e. Internal Positive Control 4f. Weak/absent ISH staining 4g. Other (specify):
signal not detectable in target cells, unable to score
HER2 Chr 17 HER2 Chr 17
5. Enumerate Target Area 1: Count HER2 signal and Chr17 signal in each of 20 nuclei. Add the HER2 signal counts. Add the Chr 17 counts. Create the gene status ratio by dividing the TOTAL
HER2 signal count by the TOTAL Chr 17 signal count. Round to .1 decimal place. Document whether clusters of signals were counted.
SIGNAL COUNT TARGET AREA 1 – 20 nuclei
5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m 5n 5o 5p 5q 5r 5s 5t 5u 5v 5w 5x Comments
01 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 17 18 19 20 TOTAL RATIO Clusters Present?
HER2 Yes No

Chr17 Yes No

6. Result from 20 nuclei: 6a. Non- amplified: HER2/Chr17 < 2.0 or 6b. Amplified: HER2/Chr17 ≥ 2.0
If the HER2/Chr17 ratio falls between 1.8 and 2.2 then 20 additional nuclei should be enumerated.
7. Enumerate Target Area 2: Count HER2 signal and Chr17 signal in each of 20 nuclei. Add the HER2 signal counts. Add the Chr 17 counts. Document whether clusters of signals were counted.
. SIGNAL COUNT TARGET AREA 2 – 2nd 20 nuclei (if target area 1 ratio is 1.8 – 2.2)
7a 7b 7c 7d 7e 7f 7g 7h 7i 7j 7k 7l 7m 7n 7o 7p 7q 7r 7s 7t 7u 8w 8x Comments
01 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 17 18 19 20 TOTAL Clusters
Present?
HER2 Yes No

Chr17 Yes No

8. Result from all 40 nuclei:

8a. Target Area 1 HER2 Total __________ + Target Area 2 HER2 Total __________ = Total HER2 count _________ 8c. Ratio: Total HER2 / Total Chr17
(from box 5u) (from box 7u)

8b. Target Area 1 Chr17 Total __________ + Target Area 2 Chr17 Total ___________ = Total Chr17 count _________ ______________
(from box 5u) (from box 7u)
8d. Final Result from 40 nuclei: Non- amplified: HER2/Chr17 < 2.0 or Amplified: HER2/Chr ≥ 2.0

Scored by: Date: