possibility of human error and inherent variability resulting from individual reagent dilution,
manual pipetting, and manual reagent application.
CLINICAL SIGNIFICANCE
PATHWAY速 anti-HER-2/neu (4B5) Rabbit Monoclonal Breast cancer is the most common carcinoma occurring in women, and the second leading
Primary Antibody cause of cancer related death. In North America, a womans chance of contracting breast
cancer is one in eight.9 Early detection and appropriate treatment therapies can significantly
Catalog number 790-2991 (50 tests) affect overall survival.10 Small tissue samples may be easily used in routine
immunohistochemistry (IHC), making this technique, in combination with antibodies that
INDICATIONS AND USE
detect antigens important for carcinoma interpretation, an effective tool for the pathologist in
Intended Use
their diagnosis and prognosis of disease. One important marker in breast cancer today is
This antibody is intended for in vitro diagnostic use.
c-erbB-2 oncoprotein (HER2).
Ventana Medical Systems, Inc.'s (Ventana) PATHWAY anti-HER-2/neu (4B5) Rabbit
HER2 is an intracellular membrane protein detected in the cellular membrane.11 It is closely
Monoclonal Primary Antibody (PATHWAY HER2 (4B5)) is a rabbit monoclonal antibody
related to EGFR and, like EGFR, has tyrosine kinase activity.1 Gene amplification and the
intended for laboratory use for the semi-quantitative detection of HER2 antigen in sections
corresponding overexpression of c-erbB-2 has been found in a variety of tumors, including
of formalin-fixed, paraffin-embedded normal and neoplastic tissue on a Ventana automated
breast carcinomas.11,12
immunohistochemistry slide staining device. It is indicated as an aid in the assessment of
The therapeutic drug Herceptin has been shown to benefit some breast carcinoma patients
breast cancer patients for whom Herceptin速 treatment is considered.
by arresting, and in some cases reversing the growth of their cancer.8 The drug is a
Note: All of the patients in the Herceptin clinical trials were selected using a clinical trial
humanized monoclonal antibody that binds to HER2 protein on cancer cells. Thus only
assay. None of the patients in those trials were selected using PATHWAY anti-HER-2/neu
patients with HER-2/neu positive breast carcinomas should benefit from treatment with
(4B5). PATHWAY anti-HER-2/neu (4B5) was compared to PATHWAY HER-2 (clone
Herceptin. In vitro diagnostics for the determination of HER2 status in breast carcinomas
CB11) Primary Antibody on an independent sample set and found to provide acceptably
are important to aid the clinician in determination of therapy with Herceptin.
concordant results. The actual correlation of PATHWAY anti-HER-2/neu (4B5) to clinical
Interpretation of the results of any detection system for HER2 must take into consideration
outcome has not been established.
the fact that HER2 is expressed in both breast cancer tumors and healthy tissue, albeit at
differing levels and with different patterns of expression.13 Histological tissue preparations
The Ventana Image Analysis System (VIAS?) is an adjunctive optional computer-
have the advantage of intact tissue morphology to aid in the interpretation of the HER2
assisted image analysis system functionally connected to an interactive microscope. It is
positivity of the sample. All histological tests should be interpreted by a specialist in breast
intended for use as an aid to the pathologist in the detection, classification and counting of
cancer morphology, and/or pathology, and the results should be complemented by
cells of interest based on marker intensity, size and shape using appropriate controls to
morphological studies and proper controls and used in conjunction with other clinical and
assure the validity of the VIAS scores.
laboratory data.
Prescription use only.
Principles and Procedures
PATHWAY HER2 (4B5) is a rabbit monoclonal antibody, which binds to HER2 in paraffin-
Summary and Explanation
embedded tissue sections. The specific antibody can be localized by either a biotin
PATHWAY anti-HER-2/neu is a rabbit monoclonal antibody (clone 4B5) directed against the
conjugated secondary antibody formulation that recognizes rabbit immunoglobulins
internal domain of the c-erbB-2 oncoprotein (HER2). c-erbB-2 oncoprotein was cloned and
followed by the addition of a streptavidin-horseradish peroxidase (HRP) conjugate (iVIEW
characterized by Akiyama et al in 1986.1 It is an approximately 185 kD transmembrane
DAB detection kit) or a secondary antibody-HRP conjugate (ultraView Universal DAB
glycoprotein which is structurally similar to epidermal growth factor receptor (EGFR). The
detection kit). The specific antibody-enzyme complex is then visualized with a precipitating
protein is associated with tyrosine kinase activity similar to that of several growth factor
enzyme reaction product. Each step is incubated for a precise time and temperature. At the
receptors, and to that of the transforming proteins of the src family. The coding sequence is
end of each incubation step, the Ventana automated slide stainer washes the sections to
consistent with an extracellular binding domain and an intracellular kinase domain. This
stop the reaction and to remove unbound material that would hinder the desired reaction in
suggests that HER2 may be involved in signal transduction and stimulation of mitogenic
subsequent steps. It also applies Liquid Coverslip?, which minimizes evaporation of the
activity.1
aqueous reagents from the specimen slide.
Clone 4B5 has been shown to react with a 185 kD protein from SK-BR-3 cell lysates via Clinical cases should be evaluated within the context of the performance of appropriate
Western blotting. SK-BR-3 is a breast carcinoma cell line which has a 128-fold over controls. Ventana recommends the inclusion of a positive tissue control fixed and
expression of HER2 mRNA.2 The size of the band identified correlates well with that processed in the same manner as the patient specimen (for example, a weakly positive
reported by Akiyama et al for HER2 protein (185 kD).1 Immunohistochemistry has been breast carcinoma or uterus). In addition to staining with PATHWAY HER2 (4B5), a second
used to detect specific antigens in cells or tissue since 1950.3 The use of enzymes and slide should be stained with Ventana CONFIRM? Negative Control Rabbit Ig. For the test
peroxidase as markers for immunohistochemistry was reported by Nakane and Pierce in to be considered valid, the positive control tissue should exhibit membrane staining of the
1967.4 The increased sensitivity of the avidin-biotin-peroxidase detection system over the tumor cells. These components should be negative when stained with CONFIRM Negative
enzyme labeled antibody method was documented by Hsu et al in 1981.5 Control Rabbit Ig. In addition, it is recommended that a negative tissue control slide (for
The HER2 protein is expressed at a level detectable by immunohistochemistry in up to 20 example, a HER-2/neu negative breast carcinoma) be included for every batch of samples
percent of adenocarcinomas from various sites. Between 15 and 30 percent of invasive processed and run on the Ventana automated slide stainer. This negative tissue control
ductal cancers are positive for HER2.6 Almost all cases of Pagets disease of breast7 and should be stained with PATHWAY HER2 (4B5) to ensure that the antigen enhancement
up to 90 percent of cases of ductal carcinoma in situ of comedo type are positive.6 The and other pretreatment procedures did not create false positive staining.
immunohistochemical detection of HER2 protein overexpression is also used as an aid in
The VIAS is an interactive histology imaging device that performs image processing using a
determination of patients for whom Herceptin therapy is indicated.8
microscope, digital color video camera, computer, and image analysis software to acquire
Staining results in normal tissues, neoplastic tissues, and 322 cases of breast carcinoma
and analyze user-selected images on the PATHWAY HER-2/neu (4B5) stained slides.
with PATHWAY HER2 (4B5) were evaluated by Ventana. In the normal tissues tested,
expression was consistent with the published literature in that there was no unexpected
The device is intended to aid the pathologist in their analysis by providing semi-quantitative
specific cytoplasmic/membrane staining, with the following exceptions: two cases of tonsil
input to supplement the pathologists qualitative interpretation of PATHWAY HER-2/neu
showing with epithelial cell membrane staining, one case of parathyroid, and one case of
(4B5) slides. The pathologist performs the usual manual read of the HER-2/neu slides to
esophageal epithelium. Of the neoplastic tissues tested, cytoplasmic/membrane staining
assess the HER-2/neu expression as scored on a scale (0,1+,2+,3+) for the slide using the
was seen in cancer cells of the breast, colon and ovary. Three hundred twenty-two (322)
VIAS microscope. The pathologist then has the opportunity to select multiple fields of view
breast carcinomas were evaluated with Ventana PATHWAY HER2 (4B5) in a method
using the VIAS microscope and computer for semi-quantitative analysis. The VIAS device
comparison study with PATHWAY HER-2 (CB11). There is a significant correlation of
processes the user-selected color images to assess the HER-2/neu expression using a
staining between these two tests. See Summary of Expected Results section for further
software algorithm to aid the pathologists qualitative read.
information.
Ventana PATHWAY HER2 (4B5) in combination with Ventana iVIEW DAB Detection Kit,
The pathologist makes the final call based on both the semi-quantitative and qualitative
utilizes biotinylated secondary antibodies to locate the bound PATHWAY HER2 (4B5)
information. It is recommended that in this application of the Ventana Image Analysis
primary antibody (produced by using a synthetic peptide corresponding to a site on the
System the user follow the appropriate instructions in the Ventana PATHWAY HER-2/neu
internal domain of the HER2 protein). This is followed by the binding of an
(4B5) product insert and associated scoring guide.
avidin/streptavidin-enzyme conjugate to the biotin. The complex is then visualized using a
precipitating enzyme generated product. For further information refer to the Ventana Image Analysis System (VIAS) Operators
The use of Ventana pre-diluted PATHWAY HER2 (4B5) and ready-to-use iVIEW DAB Manual.
detection kits, in combination with a Ventana automated slide stainer, reduces the
2007-02-14 14427EN Rev B
Page 1 of 8
Every antibody dispenser is expiration dated. When properly stored, the reagent is stable to
MATERIALS AND METHODS
the date indicated on the label. Do not use reagent beyond the expiration date for the
Reagents Provided
prescribed storage method. The product has been designed to have 18 months dating after
Catalog #790-2991: PATHWAY anti-HER-2/neu (4B5) Primary Antibody contains sufficient
the date of manufacture.
reagent for 50 tests.
There are no definitive signs to indicate instability of this product; therefore, positive and
1 ? 5 ml dispenser PATHWAY anti-HER-2/neu (4B5) Primary Antibody contains
negative controls should be run simultaneously with unknown specimens. Your local
approximately 30 袖g of a rabbit monoclonal antibody directed against human c-erbB-2
Ventana office should be contacted immediately if there is an indication of reagent
antigen.
instability.
The antibody is diluted in 0.05 M Tris buffered saline, 0.01 M EDTA, 0.05% Brij-35 with
0.3 % carrier protein and 0.05 % sodium azide, a preservative. There is trace fetal calf Specimen Collection and Preparation for Analysis
Formalin fixed, paraffin embedded tissues which have been antigen enhanced are suitable
serum, approximately 0.25 %, present from the stock solution.
for use with PATHWAY HER2 (4B5) when used with Ventana detection kits and a Ventana
automated slide stainer (see Materials and Reagents Needed But Not Provided).
Total protein concentration of the reagent is approximately 16 mg/ml. Specific antibody
concentration is approximately 6 袖g/ml. PATHWAY anti-HER-2/neu (4B5) Primary Antibody The recommended fixative is 10% neutral buffered formalin. The amount used is 15 to 20
times the volume of tissue. No fixative will penetrate more than 2 to 3 mm of solid tissue or
is a rabbit IgG diluted from tissue culture supernatants. There is no known irrelevant
5 mm of porous tissue in a 24 hour period. A 3 mm or smaller section of tissue should be
antibody reactivity observed in this product.
fixed no less than 4 hours and no more than 8 hours. Fixation can be performed at room
temperature (15-25属C).14
Reconstitution, Mixing, Dilution, Titration
This antibody is optimized for use on a Ventana automated slide stainer in combination with Osseous tissues should be decalcified prior to tissue processing to facilitate tissue cutting
and prevent damage to microtome blades.14
Ventana iVIEW DAB and compatible with ultraView Universal DAB detection kits. No
Properly fixed and embedded tissues expressing the antigen will remain stable for at least 2
reconstitution, mixing, dilution, or titration is required.
years if stored in a cool location (15-25属C). The Clinical Laboratory Improvement Act (CLIA)
Further dilution may result in loss of antigen staining. The user must validate any such
of 1988, 42CFR493.1259(b) requires that The laboratory must retain stained slides at least
changes. Differences in tissue processing and technical procedures in the laboratory may
ten years from the date of examination and retain specimen blocks at least two years from
produce significant variability in results and require regular use of controls (see Quality
the date of examination?.
Control Procedures section).
Approximately 5 袖m thick sections should be cut and picked up on glass slides. The slides
should be Superfrost Plus or equivalent. Tissue should be air dried by placing the slides at
Materials and Reagents Needed But Not Provided
ambient temperature overnight.14 Studies at Ventana indicate that air dried cut tissue and
The following reagents and materials may be required for staining but are not provided:
Ventana CONFIRM Negative Control Rabbit Ig (Cat. No. 760-1029) (negative reagent cell line sections stored at 2-8属C are stable for at least 6 months. Each laboratory should
1.
validate the cut slide stability for their own procedures and environmental storage
control)
conditions.
2. Microscope slides, Superfrost? Plus [VWR Cat. No. 48311-703 or equivalent]
3. Positive and negative tissue controls (invasive breast carcinoma and normal breast
tissue) Manual Deparaffinization Procedure
Bar code labels (appropriate for negative reagent control and primary antibody being Required when using the NexES IHC automated slide stainers or if deparaffinization is not
4.
selected on a BenchMark Series automated slide stainer:
tested)
1. For instructions on when to label slides with bar code label, refer to the Instructions
5. Staining jars or baths
for Use section of the specific automated slide stainer.
Tissue-Tek速 staining dishes
6.
2. Immerse the slides sequentially in 3 xylene baths for 5賊1 minute each.
7. Timer (capable of 2-10 minute intervals)
3. Transfer the slides to 100 % ethanol and immerse sequentially in 2 baths for 3賊1
8. Xylene (histological grade)
minute each.
9. Ethanol or reagent alcohol (histological grade)
4. Transfer the slides to 95 % ethanol and immerse them in a bath of this solution for
-100% solution: undiluted ethanol or reagent alcohol
3賊1 minute.
-95% solution: mix 95 parts ethanol or reagent alcohol with 5 parts of deionized water
5. Transfer the slides to 80 % ethanol and immerse them in this solution for 3賊1 minute.
-80% solution: mix 80 parts ethanol or reagent alcohol with 20 parts deionized water
6. Transfer the slides to a bath of deionized or distilled water and dip a minimum of 10
10. Deionized or distilled water
times.
11. Biocare Medicals Decloaking Chamber (Cat. No. DC2002) (NexES速 IHC automated
7. Transfer slides to APK Wash (1X) solution or buffer solution as appropriate. For APK
slide stainers)
Wash solution, the slides should remain until you are ready to perform the staining
12. NexES IHC, BenchMark速 Series automated slide stainers
run. For buffer solution, the slides should remain until you are ready to perform the
13. Ventana iVIEW DAB (Cat. No. 760-091) or ultraView Universal DAB (Cat. No. 760-
antigen unmasking procedure. Do not allow the slides to dry.
500) detection kits
Slides stained on the BenchMark Series automated slide stainers can be deparaffinized on
14. Ventana Endogenous Biotin Blocking Kit (Cat. No. 760-050)
15. Ventana APK Wash (10X)* (Cat. No. 250-042) (NexES IHC automated slide stainers) the instrument. If this option is selected, barcode slides and place them on the instrument. If
the option is not selected follow the Manual Deparaffinization Procedure above.
16. Ventana Liquid Coverslip? (Low Temperature) (Cat. No. 250-009) (NexES IHC
automated slide stainers)
WARNINGS AND PRECAUTIONS
Ventana EZ Prep? (10X)* (Cat. No. 950-102) (BenchMark Series automated slide
17.
1. This antibody is intended for in vitro diagnostic use.
stainers)
2. Take reasonable precautions when handling reagents. Use disposable gloves when
18. Ventana Reaction Buffer (10X)* (Cat. No. 950-300) (BenchMark Series automated
handling suspected carcinogens or toxic materials (example: xylene or
slide stainers)
formaldehyde). Do not use near open flame.
19. Ventana Liquid Coverslip (High Temperature) (Cat. No. 650-010) (BenchMark Series
3. Do not smoke, eat or drink in areas where specimens or reagents are being handled.
automated slide stainers)
4. Avoid contact of reagents with eyes and mucous membranes. If reagents come in
20. Ventana Cell Conditioning 1 (Pre-dilute) (CC1) (Cat. No. 950-124)
contact with sensitive areas, wash with copious amounts of water.
21. Ventana Hematoxylin II counterstain (Cat. No. 790-2208)
5. Patient specimens and all materials contacting them should be handled as
22. Ventana Bluing Reagent (Cat. No. 760-2037)
biohazardous materials and disposed of with proper precautions. Never pipette by
23. Permanent Mounting Medium (Permount速, Fisher Cat. No. SP15-500 or equivalent)
mouth.
24. Cover glass (sufficient to cover tissue such as VWR Cat. No. 48393-60 or equivalent)
6. Avoid microbial contamination of reagents, as this could produce incorrect results.
Automated coverslipper (such as Tissue-Tek速 SCA automated coverslipper).
25. 7. Incubation times and temperatures other than those specified may give erroneous
26. Absorbent wipes (If performing manual antigen unmasking) results. The user must validate any such change.
27. Light microscope (20-80X) or Ventana Image Analysis System (VIAS)* 8. The reagents have been optimally diluted, and further dilution may result in loss of
* As needed for specific applications. antigen staining. The user must validate any such change.
9. When used according to instructions, this product is not classified as a hazardous
Storage and Handling substance. The preservative in the reagent is sodium azide. Symptoms of
Store at 2-8属C. Do not freeze. The user must validate any storage conditions other than overexposure to sodium azide include skin and eye irritation, and irritation of mucous
those specified in the package insert. membranes and upper respiratory tract. The concentration of sodium azide in this
PATHWAY HER2 (4B5) should be allowed to stand at least 30 minutes at room product is 0.05% and does not meet the OSHA criteria for a hazardous substance.
temperature prior to use. To ensure proper reagent delivery and stability of the antibody Build up of NaN3 may react with lead and copper plumbing to form highly explosive
after every run, the cap must be replaced and the dispenser must be immediately placed in metal azides. Upon disposal, flush with large volumes of water to prevent azide
the refrigerator in an upright position. accumulation in plumbing.21 Systemic allergic reactions are possible in sensitive
individuals.
2007-02-14 14427EN Rev B
Page 2 of 8
10. Consult local or state authorities with regard to recommended method of disposal. 5. Place each Tissue-Tek staining dish, filled with 250 ml of Cell Conditioning 1 Solution
and the appropriate slides, on the heat shield that is placed in the center of the pan.
6. Put the Decloaking Chamber lid on and secure. (Align the open arrow with the white
INSTRUCTIONS FOR USE
dot on the pan handle. Grip the lid handle, and rotate clockwise to the close position.
Step by Step Procedure
When the lid is locked in the proper position, the Vent Lever will lower the weight on
Ventana primary antibodies have been developed for use on Ventana automated slide
the vent nozzle.)
stainers in combination with Ventana detection kits and accessories. Recommended
7. Turn the rheostat to 10 and lock into place (approximately 120属C).
staining protocols for the automated slide stainers are listed below in Table 1 and Table 2.
8. Turn on the Decloaking Chamber and monitor until the pressure reaches 17-25 psi
The parameters for the automated procedures can be displayed, printed and edited
and the temperature is 120属-125属C. Once the Decloaking Chamber reaches the
according to the procedure in the Operator's Manual. Other operating parameters for the
desired temperature, time for 2 minutes using a calibrated manual timer, as the
automated slide stainers have been preset at the factory.
Decloaking Chamber timer is not real time? consistent.
9. Once the cell conditioning procedure is completed, turn off the Decloaking Chamber.
Table 1. Recommended Staining Protocols for PATHWAY anti-HER-2/neu (4B5) with
NOTE: Technician must monitor temperature and pressure conditions to confirm
iVIEW DAB Detection Kit
desired specifications are met. If they were not, the procedure is invalidated and the
Procedure Type Platform or Method
specimens must be re run.
NexES IHC BenchMark Series
10. The technician can monitor the declining pressure by periodically checking the
Deparaffinization Off Line (manual) Selected pressure gauge. When pressure reaches 0 psi, the Decloaking Chamber can now be
Off Line (manual), Cell opened safely. Rotate the lid counterclockwise and remove it slowly, allowing steam
Conditioning 1,
Cell Conditioning Cell Conditioning 1, to escape away from your hand.
2 minutes, Decloaking
(Antigen Unmasking) Standard 11. Remove the container of slides from the pan and gently rinse in deionized water.
Chamber, 120属C NOTE: Be very careful when opening lid as surface and liquid temperatures remain
high.
Enzyme (Protease) None required None required
12. Once rinsing is complete, place the slides in a Tissue-Tek slide rack filled with
Approximately 32 Approximately 32
Antibody (Primary) deionized water for maintaining hydration while barcode labels are applied to the
minutes, 37属C minutes, 37属C
slides. One by one, remove the slides from the slide rack; blot the frosted end dry,
A/B Block (Biotin Blocking) Required Required ensuring the tissue sections do not dry during the process. Label the slide with the
Hematoxylin II, 4 Hematoxylin II, 4 appropriate barcode label, and return it to the slide container. Repeat this process for
Counterstain (Hematoxylin)
minutes minutes all slides.
13. Once all slides have been barcoded, empty the deionized water from the slide
Post Counterstain Bluing, 4 minutes Bluing, 4 minutes
container and refill it with 1X APK Wash. Slides should remain in this solution until
ready to perform staining run. NOTE: Slides must be stained within 4 hours of being
Table 2. Recommended Staining Protocols for PATHWAY anti-HER-2/neu (4B5) with
cell conditioned. They may be left in wash solution during this time to prevent drying
ultraView Universal DAB Detection Kit
of tissue.
Procedure Type Platform or Method
NexES IHC* BenchMark Series BenchMark Series Automated Slide Stainers
1. Apply slide bar code label which corresponds to the antibody protocol to be
Deparaffinization N/A Selected
performed.
Cell Conditioning N/A Cell Conditioning 1,
2. Load the primary antibody, appropriate detection kit dispensers, and required
(Antigen Unmasking) Mild
accessory reagents onto the reagent tray and place them on the automated slide
Enzyme (Protease) N/A None required
stainer. Check bulk fluids and waste.
N/A Approximately 16 3. Load the slides onto the automated slide stainer.
Antibody (Primary)
minutes, 37属C
A/B Block (Biotin Blocking) N/A N/A For All Instruments
1. Start the staining run.
N/A Hematoxylin II, 4
Counterstain (Hematoxylin)
2. At the completion of the run, remove the slides from the automated slide stainer.
minutes
3. Wash in a mild dishwashing detergent to remove the coverslip solution; dehydrate,
Post Counterstain N/A Bluing, 4 minutes
clear, and coverslip with permanent mounting media in the usual manner.
* ultraView Universal DAB is not available for use with PATHWAY HER2 (4B5) on NexES
4. The stained slides should be read within two to three days of staining, and are stable
IHC
for at least two years if properly stored at room temperature (15 to 25属 C).
The procedures for staining on the Ventana automated slide stainers are as follows. For
Quality Control Procedures
more detailed instructions and additional protocol options, refer to your Operators Manual.
Cell Line System Controls
Ventana has available as a separate product four formalin-fixed cell line controls embedded
NexES IHC Automated Slide Stainers
in paraffin, sectioned and placed on a single charged slide (catalog # 781-2991).
Manual antigen unmasking is required when using the NexES IHC:
PATHWAY HER-2 4 in 1 Control Slides may be useful for a preliminary validation of the
1. Slides are to be deparaffinized through a series of xylene and gradient alcohols to
processing method used for staining slides with PATHWAY HER2 (4B5). These four cell
water and then to appropriate buffer. Perform manual antigen unmasking procedure
line controls are characterized by in situ hybridization for gene copy number. When
listed below and transfer slides to APK Wash (1X).
processed and stained appropriately, the cell lines should stain as described in Table 4. If
2. Load the primary antibody, appropriate detection kit dispensers and required
the indicated staining is not evident in the appropriate cores, especially the 1+ and 2+
accessory reagents onto the reagent tray and place the reagent tray on the
controls, the staining of the tissues should be repeated.
automated slide stainer. Check bulk fluids and waste.
3. Dry the painted end of the slide and then apply slide barcode label that corresponds
Table 3. Characteristics of PATHWAY HER-2 4 in 1 Control Slides
to the antibody protocol to be performed.
HER2
4. Load the deparaffinized, antigen unmasked, labeled slides from the APK Wash (1X) HER2 IHC
Cell Line Gene
onto the NexES IHC Automated Slide Stainer. Avoid tissue drying. Score
Copy #*
0 MCF-7 1.7
Manual Antigen Unmasking Procedure
Antigen enhancement (cell conditioning) procedure (for tissue slides to be stained on 1+ T47D 2.9
NexES: 2+ MDA-MB-453 5.2
1. Prepare the Decloaking Chamber for use.
3+ BT-474 18.9
2. Place the pan into the chamber.
*Average of 3 lots of PATHWAY HER-2 4 in 1 control slides determined using PathVysion速
3. Align the handles of the pot with the handles of the chamber. NOTE: Make sure that
HER2 Probe.
the outside of the pan is completely dry prior to placing it in the chamber. If the
outside of the pan is wet, the Decloaking Chamber will make a cracking noise and
Positive Tissue Control
any water in the chamber will cause a malfunction.
A positive control tissue fixed and processed in the same manner as the patient specimens
4. Fill the pan with 500 ml of deionized water. Place the heat shield (circular screen) in
must be run for each set of test conditions and with every PATHWAY HER2 staining
the center of the pot. Note: The heat shield keeps the plastic containers from
procedure performed. This tissue could contain both positive staining cell/tissue
warping.
2007-02-14 14427EN Rev B
Page 3 of 8
components and negative cell tissue components and serve as both the positive and staining in the negative tissue control confirms the lack of antibody cross reactivity to cells
negative control tissue. Control tissue should be fresh autopsy/biopsy/surgical specimens or cellular components. The staining of normal breast is an adequate negative control
prepared and fixed as soon as possible in a manner identical to test sections. Such tissue tissue. Intact stromal and ductal elements should show no intense staining in the
may monitor all steps of the analysis, from tissue preparation through staining. Use of a membrane, indicating that staining did not occur. If the tissue is counterstained, there may
tissue section fixed or processed differently from the test specimen provides control for all be staining around the outside of the cell, i.e., the interstitial spaces. If specific staining
reagents and method steps except fixation and tissue preparation. A tissue with weak occurs in the negative tissue control, results with the patient specimen should be
positive staining is more suitable than strong positive staining for optimal quality control and considered invalid.
to detect minor levels of reagent degradation. Ideally a tissue which is known to have weak
but positive staining should be chosen to ensure that the system is sensitive to small Negative Reagent Controls
amounts of reagent degradation or problems with the IHC methodology. Generally, Nonspecific staining, if present, will have a diffuse appearance. Sporadic light staining of
however, neoplastic tissue that is positive for HER-2/neu is strongly positive due to the connective tissue may also be observed in tissue sections that are excessively formalin
nature of the pathology (overexpression). An example of a positive control for PATHWAY fixed. Intact cells should be used for interpretation of staining results, as necrotic or
HER2 (4B5) is a known weak HER-2/neu positive invasive breast carcinoma (for example degenerated cells often stain nonspecifically.
ductal or lobular). The positive staining tissue components (cytoplasmic membrane of
neoplastic cells) are used to confirm that the antibody was applied and the instrument Patient Tissue
functioned properly. Patient specimens should be examined last. Positive staining intensity should be assessed
A known weak HER-2/neu positive invasive breast carcinoma tissue may contain both within the context of any background staining of the negative reagent control. As with any
positive and negative staining cells or tissue components and may serve as both the immunohistochemical test, a negative result means that the antigen in question was not
positive and negative control tissue. detected, not that the antigen is absent in the cells or tissue assayed. The morphology of
Known positive tissue controls should be utilized only for monitoring the correct each tissue sample should also be examined utilizing a hematoxylin and eosin stained
performance of processed tissues and test reagents, and not as an aid in determining a section when interpreting any immunohistochemical result. The patient's morphologic
specific diagnosis of patient samples. findings and pertinent clinical data must be interpreted by a qualified pathologist.
A qualified pathologist who is experienced in immunohistochemical procedures must
Negative Tissue Control evaluate positive and negative controls and qualify the stained product before interpreting
The same slide used for the positive tissue control (ductal or lobular invasive breast results.
carcinoma) may be used as the negative tissue control. The non-staining components
(surrounding stroma, lymphoid cells and blood vessels) should demonstrate absence of Scoring Conventions for the Interpretation of PATHWAY HER2 (4B5)
specific staining and provide an indication of specific background staining with the primary Breast carcinomas that are considered positive for HER-2 protein overexpression must
antibody. Alternatively, normal breast tissue is an adequate negative control tissue. Use a meet threshold criteria for intensity of staining (2+ or greater on a scale of 0 to 3+) and
tissue known to be fixed, processed and embedded in a manner identical to the patient percent positive tumor cells (greater than 10%). Staining must also localize to the cellular
sample(s) with each staining run to verify the specificity of PATHWAY HER2 (4B5) for membrane. Cytoplasmic staining may still be present, but this staining is not included in the
demonstration of HER-2/neu, and to provide an indication of specific background staining determination of positivity. Three fields within the well preserved and well stained region of
(false positive staining). the tissue should be examined for intensity of staining and determination of completeness
of the cytoplasmic membrane stain. Staining that completely encircles the cytoplasmic
Negative Reagent Control membrane should be scored as an intensity of ?2+? or ?3+?. Partial staining of the
A negative reagent control must be run for every specimen to aid in the interpretation of membrane should be scored as a ?1+?. It may be necessary to examine borderline cases at
results. A negative reagent control is used in place of the primary antibody to evaluate 400X or higher magnification to discriminate between intensities of ?1+? and ?2+?. In
nonspecific staining. The slide should be stained with CONFIRM Negative Control Rabbit contrast to cases scored as an intensity of 3+, the staining scored as 2+ has a crisper and
Ig. The incubation period for the negative reagent control should equal the primary antibody more clearly delineated ring, while cases scored as 3+ exhibit a very thick outline. Below is
incubation period. a quick reference chart for staining criteria. Refer to Ventanas Scoring Guide for a more
detailed description with photographs of staining with PATHWAY HER2 (4B5).
Unexplained Discrepancies
Unexplained discrepancies in controls should be referred to your local Ventana office Table 4. Criteria for Intensity of Cell Membrane Staining with PATHWAY HER2 (4B5).
immediately. If quality control results do not meet specifications, patient results are invalid. Staining Pattern Score (Report to HER2 Staining
See the Troubleshooting section of this insert. Identify and correct the problem, then repeat Treating Physician) Assessment
the patient samples. No membrane staining is 0 Negative
observed
Assay Verification Faint, partial staining of the 1+ Negative
Prior to initial use of an antibody or staining system in a diagnostic procedure, the specificity membrane
of the antibody should be verified by testing it on a series of tissues with known
Weak complete staining of the 2+ Positive
immunohistochemistry performance characteristics representing known positive and
membrane, greater than 10%
negative tissues (refer to the Quality Control Procedures previously outlined in this section
of cancer cells
of the product insert and to the Quality Control recommendations of the College of
Intense complete staining of 3+ Positive
American Pathologists Laboratory Accreditation Program, Anatomic Pathology Checklist,15
the membrane, greater than
or the CLSI Approved Guideline16 or both documents). These quality control procedures
10% of cancer cells
should be repeated for each new antibody lot, or whenever there is a change in assay
parameters. Breast cancer tissues with known HER2 status are suitable for assay
LIMITATIONS
verification.
General Limitations
1. Immunohistochemistry is a multiple step diagnostic process that requires specialized
Interpretation of Results
training in the selection of the appropriate reagents, tissue selections, fixation,
The Ventana automated immunostaining procedure causes a brown colored (DAB) reaction
processing, preparation of the immunohistochemistry slide, and interpretation of the
product to precipitate at the antigen sites localized by PATHWAY HER2 (4B5). A qualified
staining results.
pathologist experienced in immunohistochemical procedures must evaluate controls and
2. Tissue staining is dependent on the handling and processing of the tissue prior to
qualify the stained product before interpreting results.
staining. Improper fixation, freezing, thawing, washing, drying, heating, sectioning, or
contamination with other tissues or fluids may produce artifacts, antibody trapping, or
Positive Controls
false negative results. Inconsistent results may result from variations in fixation and
The stained positive tissue control should be examined first to ascertain that all reagents
embedding methods, or from inherent irregularities within the tissue.
are functioning properly. The presence of an appropriately colored reaction product within
3. Excessive or incomplete counterstaining may compromise proper interpretation of
the membrane of the target cells is indicative of positive reactivity. Depending on the
results.
incubation length and potency of the hematoxylin used, counterstaining will result in a pale
4. The clinical interpretation of any positive staining, or its absence, must be evaluated
to dark blue coloration of cell nuclei. Excessive or incomplete counterstaining may
within the context of clinical history, morphology and other histopathological criteria.
compromise proper interpretation of results.
The clinical interpretation of any staining, or its absence, must be complemented by
If the positive tissue control fails to demonstrate positive staining, any results with the test
morphological studies and proper controls as well as other diagnostic tests. It is the
specimens should be considered invalid.
responsibility of a qualified pathologist to be familiar with the antibodies, reagents and
methods used to interpret the stained preparation. Staining must be performed in a
Negative Tissue Controls
certified licensed laboratory under the supervision of a pathologist who is responsible
The negative tissue control should be examined after the positive tissue control to verify the
specific labeling of the target antigen by the primary antibody. The absence of specific
2007-02-14 14427EN Rev B
Page 4 of 8
for reviewing the stained slides and assuring the adequacy of positive and negative appropriately over three instrument runs and across all instrument platforms tested.
controls. Users should verify between run reproducibility results by staining several sets of
5. Ventana provides antibodies and reagents at optimal dilution for use when the serial sections with low, medium and high antigen density on different days.
provided instructions are followed. Any deviation from recommended test procedures 5. BenchMark XT Inter laboratory staining and Inter-reader scoring reproducibility:
may invalidate expected results. Appropriate controls must be employed and Three laboratories, from separate institutions in the United States, participated in the
documented. Users who deviate from recommended test procedures must accept inter-laboratory reproducibility study. Cut slides of 40 neutral buffered formalin-fixed
responsibility for interpretation of patient results. invasive breast carcinoma cases [10 each from each HER-2 binning category (0-1+,
6. This product is not intended for use in flow cytometry, performance characteristics 2+, 3+)] and six (6) PATHWAY HER-2 4 in 1 Control Slides were shipped to each of
have not been determined. the sites for staining on a Ventana BenchMark XT automated slide staining device
7. Reagents may demonstrate unexpected reactions in previously untested tissues. The using the recommended staining protocol. Controls included the PATHWAY HER-2 4
possibility of unexpected reactions even in tested tissue groups cannot be completely in 1 Control Slides and a second slide of each case stained with negative Ig reagent.
eliminated because of biological variability of antigen expression in neoplasms, or No sites experienced invalid runs, based upon the performance of the controls. The
other pathological tissues.17 Contact your local Ventana office with documented results were analyzed by Ventana. Thirty-four of forty (34/40) slides exhibited similar
unexpected reactions. staining intensity across staining sites. Six samples (6/40 or 15%) varied by no more
8. Tissues from persons infected with hepatitis B virus and containing hepatitis B than 1 intensity level. Three (3/6) samples varied between 0 and 1+, which are both
surface antigen (HBsAg) may exhibit nonspecific staining with horseradish considered to be negative. Two samples (2/40 or 5%) varied between 2+ and 3+,
peroxidase.18 and one sample (1/40) varied between 1+ and 2+.
9. False positive results may be seen because of non-immunological binding of proteins 6. Inter-investigator scoring reproducibility: In all of the 40 cases (100%), a minimum of
or substrate reaction products. They may also be caused by pseudoperoxidase 2 of 3 pathologists agreed.
activity (erythrocytes), endogenous peroxidase activity (cytochrome C), or 7. Lot to Lot reproducibility was determined by automated staining of 5 breast cancer
endogenous biotin (example: liver, brain, breast, kidney) depending on the type of tissues with scores of 0, 1+, 2+, and 3+ HER2 expression with 3 lots of PATHWAY
immunostain used.19 HER2 (4B5). Stained tissues were scored on a 0 to 3+ scale by three qualified
10. As with any immunohistochemistry test, a negative result means that the antigen was readers. There was 100% agreement between lots and readers for the 3 slides and 5
not detected, not that the antigen was absent in the cells or tissue assayed. tissues stained.
8. Comparison Studies of PATHWAY HER2 (4B5) rabbit monoclonal antibody to
Specific Limitations PATHWAY HER-2 (CB11) mouse monoclonal antibody: Summary of Studies
1. The antibody has been optimized for a 32 minute incubation time in combination with Performed. A method comparison study was conducted to examine the correlation of
Ventana iVIEW DAB detection kits and the Ventana automated slide stainers. PATHWAY HER2 (4B5) to PATHWAY HER-2 (CB11) and PathVysion Her-2 FISH,
Because of variation in tissue fixation and processing, it may be necessary to both previously approved FDA diagnostic tests. Six investigators participated in the
increase or decrease the primary antibody incubation time on individual specimens. study. Two sets of three different investigators evaluated two independent cohorts
For further information on fixation variables, refer to Immunohistochemistry (Cohort 1: n=178, Cohort 2: n=144) using known breast cancer cases stained with
Principles and Advances?.20 HER-2 CB11 and HER2 4B5. FISH data was obtained from patient history. A
2. The antibody, in combination with Ventana detection kits and accessories, detects consensus score from the three readers for each antibody was created for each case
antigen that survives routine formalin fixation, tissue processing and sectioning. to reduce intra-reader variability known to exist with HER-2 scoring.22, 23, 24 A total of
Users who deviate from recommended test procedures are responsible for 322 cases were evaluated. The Slides stained with PATHWAY HER-2 (CB11) were
interpretation and validation of patient results. processed and stained according to the manufacturers instructions specified in the
3. Bone marrow was not tested for specificity. The user should determine appropriate Ventana CB11 package insert. There was an average of approximately one year
staining in the above tissues prior to interpretation of staining information. between staining and reading of the CB11 stained slides. Since scores from one of
the six readers was outside of the confidence interval data from the two cohorts are
presented as follows:
SUMMARY OF EXPECTED RESULTS
The performance of the PATHWAY HER2 (4B5) Primary Antibody was evaluated through
specificity, reproducibility and method comparison studies. All staining was performed using Table 5. Cohort 1-Consensus IHC Scores of Three Pathologists:
the iVIEW detection protocol listed above on a Benchmark XT Automated Stainer unless
CB11
otherwise specified.
4B5 3 2 0, 1 Total
1. Specificity: PATHWAY HER2 (4B5) specificity was determined by a study that
showed no specific membrane staining for most normal tissues. Staining results were 3 29 24 5 58
as follows: adrenal (0/3), breast (0/3), cerebellum (0/3), cerebrum (0/3), cervix (0/3), 2 2 13 17 32
colon (0/3), esophagus (1/3), heart (0/2), kidney (0/3), liver (0/3), lung (0/3), 0, 1 0 0 53 53
mesothelial cells (0/3), ovary (0/3), pancreas (0/3), parathyroid (1/3, focal membrane
Total 31 37 75 143
staining), peripheral nerve (1/3), pituitary (0/2), prostate (1/3), salivary gland (0/3),
skeletal muscle (0/3), skin (0/3), small intestine (0/3), spleen (0/3), stomach (0/3),
Cohort 1: Performance characteristics for 3 x 3 Presentation
testis (0/3), thymus (0/2), thyroid (0/3), tonsil (2/3 focal staining of surface epithelial
Overall agreement is 29+13+53/143=66.4% (95% C.I. = 38.6%, 59.7%)
cells), and uterus (0/3).
Cohort 1: Performance characteristics for 2 x 2 Presentation (HER-2 antibody
PATHWAY HER2 (4B5) specificity was also determined by a study that showed no
positive (2+ and 3+) and negative (0+ and 1+) scores are combined).
specific membrane staining in most neoplastic tissues. Staining results were as
Positive percent agreement is 29+2+24+13/31+37 =100% (95% C.I. %= 97.5%
follows: breast cancer (1/4), carcinoid (0/2), colon cancer (1/3), hepatocellular cancer
- 100%)
(0/5), leiomyoma (0/2), lung cancer (0/2), lymphoma (0/3), melanoma (0/2), ovarian
Negative percent agreement is 53/75 = 70.7% (95% C.I. = 58.5% - 80.1%)
cancer (1/2), pancreatic cancer (0/3), prostate cancer (0/3), renal cell cancer (0/5),
Overall agreement is 29+24+2+13+53/143=84.7% (95% C.I. = 78.2% - 90.0)
sarcoma (0/2), stomach cancer (0/3), thyroid cancer (0/3), and undifferentiated
cancer (0/1).
Table 6. Cohort 2- Consensus IHC Scores of Three Pathologists:
Positive staining in tonsilar epithelieum, esophageal epithelium, prostate, peripheral
CB11
nerve, parathyroid, breast cancer, colon, and ovarian cancer are consistent with
4B5 3 2 0, 1 Total
published literature regarding expression of HER-2/neu.
3 72 1 0 73
2. Sensitivity: Sensitivity is dependent upon the preservation of the antigen. Any
2 1 12 5 18
improper tissue handling during fixation, sectioning, embedding or storage which
alters antigenicity weakens HER-2/neu protein detection by PATHWAY HER2 (4B5) 0, 1 0 7 80 87
and may generate false negative results. Total 73 20 85 178
3. Intra run reproducibility of staining on the NexES, BenchMark, and BenchMark XT
staining instrument platforms was determined by staining three slides each of five Cohort 2: Performance characteristics for 3 x 3 Presentation
breast cancer tissues with a score of 0, 1+, 2+, and 3+ HER-2 expression. For each Overall agreement is 72+12+80/178=92.1% (95% C.I. = 80.1%, 93.1%)
case, three of 3 slides stained appropriately within a run and for all instrument Cohort 2: Performance characteristics for 2 x 2 Presentation (HER-2 antibody
platforms tested. Users should verify within run reproducibility results by staining positive (2+ and 3+) and negative (0+ and 1+) scores are combined).
several sets of serial sections with low, medium and high antigen density in a single Positive percent agreement is 72+12+1+1/73+20 = 92.5% (95% C.I. = 85.2% -
run. 96.9%)
4. Inter run and inter-platform reproducibility of staining was determined by staining Negative percent agreement is 80/85 = 94.1% (95% C.I. = 86.8% - 98.1%)
three slides each of five breast cancer tissues with scores of 0, 1+, 2+, and 3+ HER- Overall agreement is 72+12+1+1+80/178=93.3% (95% C.I. = 88.5% - 96.4%)
2 expression on three different instrument runs across the NexES, BenchMark, and
BenchMark XT instrument platforms. For each case, nine of 9 slides stained
2007-02-14 14427EN Rev B
Page 5 of 8
Table 7. Cohort 1- Consensus CB11 IHC Scores of Three Table 11. Cohort 1: 4B5 Scoring for the Three Pathologists
Pathologists Compared to FISH
Investigator 1 Investigator 2 Investigator 3
FISH
CB11 Positive Negative Total HER2
3 32 0 32 Score 4B5 Score 4B5 Score 4B5 Score
2 32 5 37 3 72 70 73
0, 1 22 53 75
2 22 19 18
Total 86 58 144
0,1 80 89 87
Cohort 1: Performance characteristics for CB11 and FISH, 2 x 2 Presentation
(where scores of 2 and 3 are considered positive) Total 174 178 178
Positive percent agreement is 32+32/ 86= 74.4% (95% C.I. = 63.8% - 83.2%)
Note: A total of 3 samples varied by more than one grade level (i.e.
Negative percent agreement is 53/58 = 91.4% (95% C.I. = 80.9% - 97.1%)
0, 2+) when evaluated by the three pathologists.
Overall agreement is 32+32+53/144=81.2% (95% C.I. = 73.9% - 87.2%)
Sample 1: One pathologist scored 2+, two pathologists scored 0+.
Sample 2: One pathologist scored 0+ two pathologists scored 2+
Table 8. Cohort 1- Consensus 4B5 IHC Scores of Three
Sample 3: One pathologist scored 0+, the second scored 1+, and
Pathologists Compared to FISH
the third scored 2+
FISH
4B5 Positive Negative Total
Table 12. Cohort 1: CB11 Scoring for the Three Pathologists
3 55 3 58
2 25 8 33 Investigator 1 Investigator 2 Investigator 3
0, 1 6 47 53 HER2
Total 86 58 144 Score CB11 Score CB11 Score CB11 Score
Cohort 1: Performance characteristics for 4B5 and FISH, 2 x 2 Presentation
3 72 75 73
(where scores of 2 and 3 are considered positive)
2 22 22 18
Positive percent agreement is 55+25/ 86= 93.0% (95% C.I. = 87.9% - 96.3%)
Negative percent agreement is 47/58 = 81.0% (95% C.I. = 73.4% - 86.0%) 0,1 80 81 87
Overall agreement is 55+25+47/144=88.2% (95% C.I. = 82.1% - 92.2%)
Total 174 178 178
Note: A total of 1 sample varied by more than one grade level (i.e.1 -
Table 9. Cohort 2- Consensus CB11 IHC Scores of Three
3+) when evaluated by the three pathologists.
Pathologists Compared to FISH
Sample 1: One pathologist scored 1+, the second scored 2+, and
FISH
the third scored 3+
CB11 Positive Negative Total
3 72 1 73
2 13 7 20
0, 1 8 77 85 Table 13. Cohort 2: 4B5 Scoring for the Three Pathologists
Total 93 85 178
Investigator 4 Investigator 5 Investigator 6
Cohort 2: Performance characteristics for CB11 and FISH, 2 x 2 Presentation
HER2
(where scores of 2 and 3 are considered positive)
Score 4B5 Score 4B5 Score 4B5 Score
Positive percent agreement is 72+13/ 93= 91.3% (95% C.I. = 85.0% - 96.7%)
Negative percent agreement is 77/85 = 90.6% (95% C.I. = 83.9% - 96.3%) 3 59 65 50
Overall agreement is 72+13+77/178=91.0% (95% C.I. = 86.5% - 94.9%)
2 30 28 39
Table 10. Cohort 2- Consensus 4B5 IHC Scores of Three 0,1 52 51 55
Pathologists: Compared to FISH
Total 141 144 144
FISH
Note: A total of 6 samples varied by more than one grade level (e.g.
4B5 Positive Negative Total
0, 3+) when evaluated by the three pathologists.
3 72 1 73
Sample 1: One pathologist scored 0+, the second scored 0+, and
2 11 7 18
the third scored 2+
0, 1 10 77 87
Sample 2: One pathologist scored 1+, the second scored 1+, and
Total 93 85 178 the third scored 3+
Cohort 2: Performance characteristics for 4B5 and FISH, 2 x 2 Presentation Sample 3: One pathologist scored 0+, the second scored 2+, and
(where scores of 2 and 3 are considered positive) the third pathologist scored 2+
Positive percent agreement is 72+11/ 93= 89.2% (95% C.I. = 82.5% - 95.1%) Sample 4 and 5: One pathologist scored 0+, the second scored 2+,
Negative percent agreement is 77/85 = 90.6% (95% C.I. = 84.0% - 96.4%) and the third scored 2+
Overall agreement is 72+11+77/178=90.0% (95% C.I. = 85.4% - 93.6%) Sample 6: One pathologist scored 0+, the second scored 3+, and
the third scored 3+
Inter-pathologist Reproducibility of Comparison Studies Specimens
Since it is well known that different pathologists may have different interpretations of
immunohistochemistry slides, three pathologists were employed for each of the two cohorts
(for a total of 6 pathologists) to read all samples. A two-out-of-three rule was used to
adjudicate the final results. Below is a summary of the variable results obtained by the three
pathologists of the comparison study samples for each cohort.
2007-02-14 14427EN Rev B
Page 6 of 8
5. Hsu, S. M. et al. Use of avidin-biotin-peroxidase complex (ABC) in
Table 14. Cohort 2: CB11 Scoring for the three Pathologists
immunoperoxidase techniques: A comparison between ABC and unlabeled antibody
Investigator Investigator
(PAP) procedures. J. Histochem. Cytochem. 29: 577-580, 1981.
4 5 Investigator 6
6. Dickson, R. B. and Lippman, M. E. Genes, Oncogenes, and Hormones. Boston,
HER2 Kluwer Academic Publishers, 1992.
Score CB11 Score CB11 Score CB11 Score 7. Keatings, L. et al. c-erbB-2 oncoprotein expression in mammary and extramammary
Pagets disease: an immunohistochemical study. Histopathology. 17: 234-247, 1990.
3 31 37 28
8. Herceptin (Trastuzamab) Package Insert. February 2005.
2 38 32 47
9. Roche, P.C. Immunohistochemical stains for breast cancer. Mayo Clin. Proc. 69: 57-
0,1 75 75 69 58, 1994.
10. Charpin, C. et al. c-erbB-2 oncoprotein detected by automated quantitative
immunocytochemistry in breast carcinomas correlates with patients' overall and
disease-free survival. Br. J. Cancer. 75: 1667-1673, 1997.
Total 144 144 144 11. Corbett, I. P. et al. NCL-4B5: A new monoclonal antibody recognizing the internal
Note: A total of 8 samples varied by more than one grade level (i.e.. domain of the c erbB 2 oncogene protein, effective for use on formalin fixed, paraffin-
0 - 2+) when evaluated by the three Pathologists. embedded tissue. J. Pathol. 161: 15-25, 1990.
Samples 1-6: one pathologist scored 0+, the second scored 1+, and 12. Nicholson, R.I., et al. Relationship between EGF-R, c-erbB-2 protein expression and
the third scored 2+ Ki67 immunostaining in breast cancer hormone sensitivity. Eur. J. Cancer. 29A:
Samples 7 and 8: one pathologist scored 0+, the second scored 2+, 1018-1023, 1993.
and the third scored 2+ 13. DePotter, C. R. et al. The expression of the neu oncogene product in breast lesions
and in normal fetal and adult human tissues. Histopathology. 15: 351-362, 1989.
Following is a tabulation of the ranges of percent agreements across pairs of pathologists 14. Sheehan DC, Hrapchak BB. Theory and practice of histotechnology, 2nd Edition. The
(three pairs for each cohort). C.V. Mosby Company, St. Louis, 1980.
15. College of American Pathologists Laboratory Accreditation Program, Anatomic
Table 15. RANGES OF 2X2* AGREEMENTS FOR THE THREE PATHOLOGISTS Pathology Checklist, 2001.
16. CLSI. Quality Assurance for Immunocytochemistry: Approved Guideline. CLSI
Overall Positive Negative
document MM4-A- (ISBN 1-56238-396-5). CLSI, 940 West Valley Road, Suite 1400,
Percent Percent Percent
Wayne, PA 19087-1898 USA, 1999.
Agreement Agreement Agreement
17. Herman GE, Elfont EA. The taming of immunohistochemistry: the new era of quality
4B5 vs. CB11
control. Biotech Histochem 66(4): 194-199, 1991.
Cohort 1 82.6 ? 86.9% 97.3 ? 100.0% 68.0% - 75.4%
18. Omata M, Liew CT, Ashcavai M, Peters RL. Nonimmunologic binding of horseradish
Cohort 2 88.2 ? 95.5% 87.6 ? 95.6% 86.1 ? 95.4%
peroxidase to hepatitis B surface antigen. A possible source of error in
4B5 vs. FISH
immunohistochemistry. Am J Clin Pathol 73(5): 626-32, 1980.
Cohort 1 86.8 ? 88.2% 90.7 ? 94.2% 79.3 ? 81.0%
19. Nadji M, Morales AR. Immunoperoxidase: part 1. The technique and its pitfalls. Lab
Cohort 2 87.4 ? 89.9% 88.2 ? 90.0% 84.5 ? 91.8%
Med 14: 767, 1983.
CB11 vs. FISH
20. Roche PC, Hsi ED. Immunohistochemistry-Principles and Advances. Manual of
Cohort 1 79.9 ? 84.0% 73.3 ? 80.2% 89.7 ? 89.7%
Clinical Laboratory Immunology, 6th edition. (NR Rose Ed.) ASM Press, 2002.
Cohort 2 84.8% - 93.3% 86.7 ? 92.5% 82.7 ? 94.1%
21. Department of Health, Education and Welfare, National Institute of Occupational
* 0, 1+ = Negative. 2+ and 3+ = Positive Safety and Health, Rockville, MD. Procedures for the decontamination of plumbing
systems containing copper and/or lead azides.? DHHS (NIOSH) Publ No. 78-127,
Conclusion: Data from these studies indicated that the PATHWAY HER2 (4B5) Current 13. August 16, 1976
primary antibody was specific and reproducible in its ability to locate appropriate 22. Thomson TA, Hayes MM, Spinelli JJ, Hiland E, Sawrenko C, Phillip D, et al. HER-
membrane staining for normal and neoplastic tissues. The method comparison data 2/neu in breast cancer: interobserver variability and performance of
demonstrated that PATHWAY HER2 (4B5) primary antibody is indicated as an aid in immunohistochemistry with 4 antibodies compared with fluorescent in situ
速
the assessment of breast cancer patients for whom Herceptin treatment is hybridization, Mod Pathol 2001;14:1079?86.
considered. 23. Kay EW, Walsh CJ, Cassidy M, Curran B, Leader M. C-erbB-2 immunostaining:
problems with interpretation. J Clin Pathol, 1994;47:816?22.
TROUBLESHOOTING 24. Michael Bilous, M.A., M.B., Ch.B., F.R.C.P.A., Mitch Dowsett, Ph.D., Wedad Hanna,
1. If the positive control exhibits weaker staining than expected, other positive controls M.D., Jorma Isola, M.D., Ph.D., Annette Lebeau, M.D., Aberlardo Moreno, M.D.,
run during the same instrument run should be checked to determine if it is because of Fr辿d辿rique Penault-Llorca, M.D., Ph.D., Josef R端schoff, M.D., Gorana Tomasic,
the primary antibody or one of the common secondary reagents. M.D., Marc van de Vijver, M.D., Ph.D. Current Perspectives on HER2 Testing: A
2. If the positive control is negative, it should be checked to ensure that the slide has the Review of National Testing Guidelines. Mod Pathol 2003; 16:173-182.
proper bar code label. If the slide is labeled properly, other positive controls run on
the same instrument run should be checked to determine if it is because of the INTELLECTUAL PROPERTY
primary antibody or one of the common secondary reagents. Tissues may have been
CONFIRM?, EZ Prep?, iVIEW?, Liquid Coverslip? , ultraView? and VIAS? are
improperly collected, fixed or deparaffinized. The proper procedure should be
trademarks of Ventana Medical Systems, Inc. PATHWAY速, BenchMark速, NexES速 IHC,
followed for collection, storage and fixation.
and Ventana速 are registered trademarks of Ventana Medical Systems, Inc.
3. If all of the paraffin has not been removed, there may be no staining. The
deparaffinization procedure should be repeated.
4. If specific antibody staining is too intense, the run should be repeated with incubation Superfrost? is a trademark of Erie Scientific Company.
time shortened by 4 minute intervals until the desired stain intensity is achieved.
5. If tissue sections wash off the slide, slides should be checked to ensure that they are Herceptin速 is a registered trademark of Genentech, Inc.
positively charged.
6. For corrective action, refer to the Step By Step Procedure section, the automated PathVysion速 is a registered trademark of Abbott Molecular.
slide stainer Operators Manual or contact your local Ventana office.
Tissue -Tek速 is a registered trademark of Sakura Finetek, Inc.
REFERENCES
1. Akiyama, T. et al. The product of the human c-erbB-2 Gene: A 185-kilodalton Permount速 is a registered trademark of Fisher Scientific Company.
glycoprotein with tyrosine kinase activity. Science 232: 1644-1646, 1986.
2. Kraus MH, Popescu NC, Amsbaugh C, King RC. Overexpression of EGF receptor-
related proto-oncogene erbB-2 in human mammary tumour cell lines by different Ventana grants to Purchaser a single use only license under the following patents: U.S.
molecular mechanisms. EMBO 6 : 605-610, 1987. Pat. Nos. 6045 759, 6192 945, 6416 713, 6945 128 and foreign counterparts. Other patents
3. Coons, A. H., and M. H. Kaplan. Localization of antigen in tissue cells. II. pending.
Improvements in method of detection of antigen by means of fluorescent antibody. J.
Exp. Med. 91: 1-13, 1950.
4. Nakane, P.K., and G.B. Pierce, Jr. Enzyme labeled antibodies: Preparations and
applications for the localizations of antigens. J. Histochem. Cytochem. 14: 929-931,
1967.
2007-02-14 14427EN Rev B
Page 7 of 8
CONTACT INFORMATION
North America
Ventana Medical Systems, Inc.
1910 E. Innovation Park Drive
Tucson, Arizona 85755
U.S.A.
+1 (520) 887 2155
(800) 227 2155 (U.S.)
Europe
Ventana Medical Systems, S.A.
Parc dInnovation ? BP 30144
Rue G. de Kaysersberg
F - 67404 Illkirch CEDEX
France
+33 (0) 3 90 40 52 00
EC REP
MDCI Ltd.
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Ventana Japan K.K.
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?220-8135腑絅綏罔羌絽?
茱水冴帥帥2-2-1
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+81 (0) 45-228-5071
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Ventana Medical Systems Pty Ltd
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+61 (0) 3 9431 6064
2007-02-14 14427EN Rev B
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