c-erbB-2 staining of the cell membrane was within the range of positivity
reported in the literature for intense membranous staining - 10.6 % (16/151) vs.
9 to 16.5 %.7
450 additional breast carcinoma cases were studied to evaluate the correlation
PATHWAY® HER 2 (clone CB11) of staining with that of HercepTest, another immunohistochemical test for
aiding in the selection of patients for treatment with Herceptin. There is a
Catalog number 760-2694: 50 Test significant correlation of staining between these two tests. See section E,
Summary of Expected Results, for further information.
Caution: Federal law restricts this device to sale by or on the order
Ventana PATHWAY HER 2, in combination with Ventana Detection Kits,
of a physician, or to a clinical laboratory; and its use is restricted to,
utilizes biotinylated secondary antibodies to locate the bound PATHWAY
by, or on the order of a physician.
HER 2 primary antibody (produced by using a synthetic peptide corresponding
to a site on the internal domain of the c-erbB-2 oncoprotein). This is followed by
I. INDICATIONS AND USE
the binding of an avidin/streptavidin-enzyme conjugate to the biotin. The
complex is then visualized using a precipitating enzyme generated product.
A. Intended Use
This antibody is intended for in vitro diagnostic use. The use of Ventana pre-diluted PATHWAY HER 2 (clone CB11) and ready-to-use
detection kits, in combination with a Ventana automated slide stainer, reduces the
Ventana Medical Systems, Inc.'s (Ventana) PATHWAY HER 2 (clone CB11) is
possibility of human error and inherent variability resulting from individual reagent
a mouse monoclonal antibody intended for laboratory use for the semi-
dilution, manual pipetting, and manual reagent application.
quantitative detection of c-erbB-2 antigen in sections of formalin-fixed, paraffin-
embedded normal and neoplastic tissue on a Ventana automated C. Clinical Significance
immunohistochemistry slide staining device. It is indicated as an aid in the
Breast cancer is the most common carcinoma occurring in women, and the
assessment of breast cancer patients for whom Herceptin® treatment is
second leading cause of cancer related death. In North America, a woman’s
considered.
chance of contracting breast cancer is one in eight.23 Early detection and
Note: All of the patients in the Herceptin clinical trials were selected using a clinical appropriate treatment therapies can significantly affect overall survival.3 Small
trial assay. None of the patients in those trials were selected using PATHWAY tissue samples may be easily used in routine immunohistochemistry (IHC),
HER 2. The PATHWAY HER 2 was compared to the DAKO HercepTest® on making this technique, in combination with antibodies that detect antigens
an independent sample and found to provide acceptably concordant results. important for carcinoma interpretation, an effective tool for the pathologist in his
The actual correlation of PATHWAY HER 2 to clinical outcome has not been or her diagnosis and prognosis of disease. One important marker in breast
established. cancer today is c-erbB-2 oncoprotein (c-erbB-2).
c-erbB-2 is an intracellular membrane protein detected in the cellular
B. Summary and Explanation
membrane.7 It is closely related to EGFR and, like EGFR, has tyrosine kinase
PATHWAY HER 2 is a mouse monoclonal antibody (clone CB11) directed
activity.2 Gene amplification and the corresponding overexpression of c-erbB-2
against the internal domain of the c-erbB-2 oncoprotein. c-erbB-2 oncoprotein
has been found in a variety of tumors, including breast carcinomas.7,19
was cloned and characterized by Akiyama et al in 1986.1 It is an approximately
185 kD transmembrane glycoprotein which is structurally similar to epidermal The newly cleared therapeutic Herceptin has been shown to benefit some
growth factor receptor (EGFR). The protein is associated with tyrosine kinase breast carcinoma patients by arresting, and in some cases reversing the
activity similar to that of several growth factor receptors, and to that of the
growth of their cancer.12 The drug is a humanized monoclonal antibody that
transforming proteins of the src family. The coding sequence is consistent with
binds to HER-2/neu protein on cancer cells. Thus only patients with HER-2/neu
an extracellular binding domain and an intracellular kinase domain. This
positive breast carcinomas should benefit from treatment with Herceptin. In
suggests that c-erbB-2 may be involved in signal transduction when bound by
vitro diagnostics for the determination of HER-2/neu status in breast
a growth factor, stimulating mitogenic activity.1 carcinomas are important to aid the clinician in determination of therapy with
Herceptin.
Clone CB11 has been shown to react with a 190 kD protein from SK-BR-3 cell
lysates via Western blotting.7 SK-BR-3 is a breast carcinoma cell line which Interpretation of the results of any detection system for c-erbB-2 must take into
has a 128-fold over expression of c-erbB-2 mRNA.7 The size of the band consideration the fact that c-erbB-2 is expressed in both breast cancer tumors
identified correlates well with that reported by Akiyama et al for c-erbB-2 (185 and healthy tissue, albeit at differing levels and with different patterns of
kD).1 expression.8 Histological tissue preparations have the advantage of intact
tissue morphology to aid in the interpretation of the c-erbB-2 positivity of the
Immunohistochemistry has been used to detect specific antigens in cells or
sample. All histological tests should be interpreted by a specialist in breast
tissue since 1950.6 The use of enzymes and peroxidase as markers for cancer morphology, and/or pathology, and the results should be used in
immunohistochemistry was reported by Nakane and Pierce in 1967.18 The conjunction with other clinical and laboratory data.
increased sensitivity of the avidin-biotin-peroxidase detection system over the
enzyme labeled antibody method was documented by Hsu et al in 1981.14 D. Principles and Procedures
Ventana PATHWAY HER 2 (clone CB11) is a mouse monoclonal antibody,
The c-erbB-2 oncoprotein is expressed at a level detectable by
which binds to c-erbB-2 in paraffin-embedded tissue sections. The specific
immunohistochemistry in up to 20 percent of adenocarcinomas from various
antibody is localized by a biotin-conjugated secondary antibody formulation
sites. Between 15 and 30 percent of invasive ductal cancers are positive for
that recognizes rabbit and mouse immunoglobulins. This step is followed by the
c-erbB-2.9 Almost all cases of Paget’s disease of breast15 and up to 90 percent
addition of an avidin/streptavidin-enzyme conjugate that binds to the biotin
of cases of ductal carcinoma in situ of comedo type are positive.9 The
present on the secondary antibody. The specific antibody-secondary antibody-
immunohistochemical detection of c-erbB-2 protein overexpression is also
avidin/streptavidin-enzyme complex is then visualized with a precipitating
used as an aid in determination of patients for whom Herceptin therapy is
enzyme reaction product. Each step is incubated for a precise time and
indicated.12
temperature. At the end of each incubation step, the Ventana automated slide
stainer washes the sections to stop the reaction and remove unbound material
Staining results in normal tissues, neoplastic tissues, and 651 cases of breast
that would hinder the desired reaction in subsequent steps. It also applies
carcinoma with PATHWAY HER 2 were evaluated by Ventana. In the 78 normal
Liquid CoverslipTM, which minimizes evaporation of the aqueous reagents from
tissues tested, expression was consistent with the published literature in that
the specimen-containing slide. Results are interpreted using a light microscope
the majority was cytoplasmic. Unexpected staining patterns occurred with one
and aid in the differential diagnosis of pathophysiological processes, which
case of kidney and one case of tonsil showing cell membrane staining and one
may or may not be associated with a particular antigen.
case of thyroid exhibiting extracellular staining. Of the 19 neoplastic tissues
tested, cytoplasmic staining was seen in cancer cells of the lung, prostate,
For further information refer to the Ventana automated slide stainer Operator's
colon, bladder, cervix and ovary. 151 breast carcinomas were evaluated with
Manual.
Ventana PATHWAY HER 2 in relation to estrogen receptor expression. Strong
c-erbB-2 positivity in these cases exhibited an inverse correlation with estrogen
receptor which is consistent with published literature.2, 11, 22, 26 Intense
2006-06-02 Page 1 of 9 14160US Rev. B
16. Ventana Reaction Buffer (10X) (BenchMark Series automated slide
II. MATERIALS AND METHODS
stainers)
A. Reagents Provided
Ventana EZ PrepTM (10X) (BenchMark Series automated slide
17.
stainers)
PATHWAY HER 2 consists of one dispenser of c-erbB-2 primary antibody
Ventana Liquid CoverslipTM (Low Temperature) (ES and NexES IHC
18.
(clone CB11) and contains approximately 5 ml (50 test) of pre-diluted reagent.
automated slide stainers)
The dispenser contains approximately 3 µg (50 test) of mouse monoclonal
19. Ventana Liquid Coverslip (High Temperature) (BenchMark Series
antibody (clone CB11) directed against the internal domain of the c-erbB-2
automated slide stainers)
oncoprotein. c-erbB-2 oncoprotein was cloned and characterized by Akiyama
20. Ventana Cell Conditioning 2 (Pre-dilute) (BenchMark Series automated
et al in 1986.1 It is an approximately 185 kD transmembrane glycoprotein
slide stainers)
which is structurally similar to epidermal growth factor receptor (EGFR). Clone
21. Ventana Endogenous Biotin Blocking Kit
CB11 has been shown to react with a 190 kD protein from SK-BR-3 cell lysates
22. Ventana Antibody Diluent
(a breast carcinoma cell line which has a 128-fold over expression of c-erbB-2
23. Ventana Negative Control Mouse Ig, which contains MOPC 21, a
mRNA7) via Western blotting.7 The size of the band identified correlates well
mouse myeloma protein of subclass IgG1, kappa light chain. Negative
with that reported by Akiyama et al for the size of the purified protein.1 Two
Control Mouse Ig is nonreactive with human antigens. An equivalent
additional bands at 150 kD and 130 kD are also detected on heavily loaded
negative control may be used.
gels. It has been suggested that the 150 kD and 130 kD bands visualized
24. Mounting Medium: Pro-Texx Mounting Medium (Scientific Products Cat.
correspond to precursor or cytoplasmic versions of c-erbB-2.7, 8 No. M7635-5) or equivalent for use with DAB Detection Kit or Immu-
Mount (Shandon Cat. No. 99900402) or equivalent for use with AEC
The immunogen used to develop c-erbB-2 primary antibody, clone CB11 was a
Detection Kit.
17 amino acid synthetic peptide corresponding to the internal domain of the
25. Cover Glass (Baxter Scientific Products Cat. Nos. M6045-7, M6045-8,
protein near the C-terminus.7 The region of the c-erbB-2 protein was selected
M6045-9, M6045-10, or equivalent, depending on size of tissue)
on the basis of computer-predicted antigenicity. The antibody is produced from
26. Light microscope (20-80X)
hybridoma culture supernatants with no further purification. The antibody is
27. Staining jars or baths
diluted in 0.1 M phosphate buffered saline with 0.3 % carrier protein and
28. Timer (capable of 3-10 minute intervals)
0.05 % ProClin 300, a preservative containing the active ingredients 5-chloro-2-
29. Wash bottles
methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one (Supelco Catalog
30. Absorbent wipes
Number 4-8126). There is trace fetal calf serum, approximately 0.25 %, present
31. Hydrogen peroxide
from the stock solution.
32. Ventana Hematoxylin counterstain
Total protein concentration of the reagent is approximately 2.25 mg/ml. Specific 33. Ventana Bluing Reagent
antibody concentration is approximately 0.63 µg/ml (0.0003 % of the total
D. Storage and Handling
protein). Clone CB11 is immunoglobulin class IgG1, light chain kappa. There is
no known irrelevant antibody in the preparation. The specificity of the antibody Store PATHWAY HER 2 at 2 °C to 8 °C. Do not freeze.
was shown by immunoprecipitation to have molecular mass of 190 kD.7
Replace the cap and store dispenser in an upright position when not in use on
B. Reconstitution, Mixing, Dilution, Titration the instrument. This will insure proper reagent delivery. Use care to avoid
damaging dispensers.
Ventana PATHWAY HER 2 is optimized for use on a Ventana automated slide
stainer in combination with Ventana detection kits. No reconstitution, mixing, Ventana PATHWAY HER 2 should be allowed to stand at least 30 minutes at
dilution, or titration is required. Further dilution may result in loss of antigen room temperature prior to use. PATHWAY HER 2 must be returned to storage
staining. Any such change must be validated by the user. Differences in tissue conditions identified above immediately after use. Any storage conditions other
processing and technical procedure in the user's laboratory may produce than those specified in the package insert must be validated by the user.
significant variability of results, necessitating regular performance of "in-house"
Every Ventana PATHWAY HER 2 dispenser is expiration dated. Do not use
controls (see section IV. B. Quality Control).
reagent beyond expiration date for prescribed storage method. The product
has been designed to have 12 months dating after the date of manufacture.
C. Materials and Reagents Needed But Not Provided
The following reagents and materials may be required for staining but not E. Indications of Instability
provided with Ventana PATHWAY HER 2:
When properly stored, the reagent should be stable to the dating indicated on
1. 10 % neutral buffered formalin (Baxter Cat. No. C4320-101 or the label. Ventana has designed the PATHWAY HER 2 to have one year
equivalent, or refer to Theory and Practice of Histotechnology by stability from the date of manufacture. The user must honor the expiration date
Sheehan and Hrapchak24) on the label. There are no obvious signs to indicate instability of this product.
2. Negative tissue control slide (normal breast tissue) Therefore, positive and negative controls should be run simultaneously with
3. Positive tissue control slide (breast carcinoma tissue) unknown specimens. Positive controls assure that the specimen staining was
4. Ventana PATHWAY HER 2 Control Slides (Cat. No. S9100) carried out correctly. Negative reagent controls are used to assess non-specific
5. Microtome staining which must be taken into consideration when interpreting results.
6. Microscope slides [Baxter Cat. No. M6164-plus (silanized), or Baxter Whenever positive control material shows a decrease in staining, it is a
Polysine Cat. No. M6143, or polylysine-coated, or equivalent] possible indication of reagent instability, and Ventana Technical Consultation
Drying oven capable of maintaining a temperature of 70 °C ± 5 °C
7. Center (800-227-2155) should be contacted immediately.
8. Ventana bar code labels (Ventana Cat. No. 451-000, 451-800, or 451-
F. Specimen Collection and Preparation for Analysis
801 for negative control and any one of Cat. No. 451-001 through 451-
175 for PATHWAY HER 2) (appropriate for negative control and Formalin-fixed, paraffin-embedded tissues which have been antigen-enhanced
primary antibody being tested) are suitable for use with Ventana PATHWAY HER 2 when used with Ventana
9. Xylene (histological grade) detection kits and a Ventana automated slide stainer (See Section II. C.
10. Ethanol or reagent alcohol (histological grade) Materials and Reagents Needed, But Not Provided).
-100 % solution: Undiluted ethanol or reagent alcohol The recommended fixative is 10 % neutral buffered formalin. The amount used
-95 % solution: Mix 95 parts of ethanol or reagent alcohol with 5 parts of is 15 to 20 times the volume of tissue. No fixative will penetrate more than 2 to
deionized water 3 mm of solid tissue or 5 mm of porous tissue in a 24 hour period. A 3 mm or
-80 % solution: Mix 80 parts of ethanol or reagent alcohol with 20 parts smaller section of tissue should be fixed no less than 4 hours and no more than
of deionized water
8 hours. Fixation can be performed at room temperature (15-25 °C).24
11. Deionized/distilled water
Osseous tissues should be decalcified prior to tissue processing to facilitate
Ventana ES®, NexES® IHC, or BenchMark® Series (BenchMark,
12.
tissue cutting and prevent damage to microtome blades.24
BenchMark XT, and BenchMark LT) automated slide stainer
13. Ventana Basic DAB, AEC, or Alkaline Phosphatase Red detection kit
14. Detection kit specific software (ES automated slide stainer only)
15. Ventana APK Wash (10X) (ES and NexES IHC automated slide
stainers)
2006-06-02 Page 2 of 9 14160US Rev. B
Properly fixed and embedded tissues expressing the antigen will keep 2 years if ES and NexES IHC automated slide stainers
stored in a cool place (15-25 °C). The Clinical Laboratory Improvement Act (CLIA) of
Recommended staining protocol for the Ventana ES and NexES IHC
1988 requires in 42 CFR 493.1259(b) that "The laboratory must retain stained slides
automated slide stainers are listed in Table 1 below. These parameters can be
at least ten years from the date of examination and retain specimen blocks at least
displayed, printed and edited according to the procedure in the Ventana
two years from the date of examination".4
automated slide stainer Operator's Manual. Other operating parameters for the
Approximately 5 µm thick sections should be cut and picked up on glass slides. automated slide stainers have been preset at the factory.
The slides should either be silanized or coated with a polylysine compound.
Table 1. Recommended Protocols for PATHWAY HER 2 on ES and NexES IHC
Tissue should be dried by placing the slides in a 70 °C (+/- 5°C) oven for at
automated slide stainers
least two hours, but not longer than 24 hours.24 Studies at Ventana indicate
unbaked cut tissue and cell line sections affixed to glass slides and stored at
PROCEDURE ENZYME DIGEST PRIMARY COUNTERSTAIN
room temperature are stable for 9 months. Each laboratory should validate the
TYPE INCUBATION
cut slide stability for there own procedures and environmental storage
conditions. PARAFFIN NONE 32 MIN HEMATOXYLIN
Deparaffinize by immersion in two xylene baths for five minutes ± 1 minute
The procedure for staining c-erbB-2 with PATHWAY HER 2 on the Ventana ES
each. Rehydrate and bring to water with 3 minutes ± 1 minute each in two
and NexES IHC automated slide stainers is as follows (Refer to the Operator's
baths containing 100 % ethanol, 3 minutes ± 1 minute in 95 % ethanol, 3
Manual for specific details on the operation of the Ventana automated slide
minutes ± 1 minute in 80 % ethanol, and 10 dips in water.24
stainer).
Use an appropriate antigen enhancement procedure.Error! Reference source not
1. The PATHWAY HER 2 dispenser, appropriate detection kit dispensers,
found.25
and desired accessory reagents are loaded onto the reagent tray and
placed on the Ventana automated slide stainer.
Blot-dry frosted end of processed tissue slides, ensuring that the tissue
2. Each slide must be labeled with the appropriate bar code specifying the
sections do not dry. Properly label processed slides with bar codes and place
staining procedure and PATHWAY HER 2. The slide bar codes should
in Wash Solution until ready to load on Ventana automated slide stainer.
be applied after the antigen enhancement procedure.
It is recommended that slides be loaded on the instrument within an hour, if at all 3. Load the deparaffinized/antigen-enhanced sections from 1X APK Wash
possible. They may be left in wash solution for up to 2 hours if necessary, as long as Solution pH 7.6, being careful not to allow the tissue to dry.
tissue is not allowed to dry.
Ventana PATHWAY HER 2 has been optimized to work in combination with
PATHWAY HER 2 works well with antigen enhanced, formalin-fixed, paraffin- Ventana detection kits to provide the greatest specific staining-to-background
embedded tissues. Variable results may occur as a result of special processes such ratio. The following is the sequence of events carried out by the Ventana ES
as decalcification of bone marrow preparations. and NexES IHC automated slide stainers.
III. Safety Issues 1. Inhibitor solution is applied to decrease the endogenous peroxidase
activity if a peroxidase-based detection system is used. The inhibitor on
A. Precautions
the slide is incubated with mixing for 4 minutes at 37 °C.
1. This antibody is intended for in vitro diagnostic use. 2. The slide is rinsed; the optimized PATHWAY HER 2 is applied and
2. Take reasonable precautions when handling reagents. Use disposable incubated with mixing for 4 to 32 minutes at 37 °C (see Specific
gloves when handling suspected carcinogens or toxic materials, for Limitation #3).
example xylene or formaldehyde. 3. The slide is rinsed; biotinylated secondary antibody is applied and
3. Do not smoke, eat or drink in areas where specimens or reagents are being incubated with mixing for 8 minutes at 37 °C.
handled. 4. The slide is rinsed; avidin/streptavidin-enzyme conjugate is applied and
4. Avoid contact of eyes and mucous membranes with reagents. If reagents incubated with mixing for 8 minutes at 37 °C.
come in contact with sensitive areas, wash with copious amounts of water. 5. The slide is rinsed; chromogenic enzyme substrate is added and
5. Patient specimens and all materials coming into contact with them should be incubated (the time is indicated in each Detection Kit insert) with mixing
handled as if capable of transmitting infection and disposed of with proper at 37 °C.
precautions. Never pipette by mouth and avoid contact of reagents and 6. The slide is rinsed; if a DAB Detection Kit is used, copper DAB
specimens with skin and mucous membranes. enhancer is applied with mixing for 4 minutes at 37 °C.
6. Avoid microbial contamination of reagents as this could produce incorrect 7. Counterstain (selectable): if counterstain and/or post counterstain is
results. selected, each will be applied to the slide and incubated with mixing for
7. Incubation times and temperatures other than those specified may give the selected time interval at 37 °C. Ventana Hematoxylin counterstain
erroneous results. Any such change must be validated by the user. and Bluing Reagent post counterstain are recommended for this
8. The reagents have been optimally diluted and further dilution may result in protocol, with 4 minute incubation for each.
loss of antigen staining. Any such change must be validated by the user.
9. Ventana recommended protocols are described in section IV. A. Step by BenchMark automated slide stainer
Step Procedure. Deviation from these protocols may produce erroneous
results. All protocols must be validated by the user. Users who deviate Recommended staining protocol selected in the procedure menu for the
from recommended test procedures must accept responsibility for Ventana BenchMark automated slide stainer is BMK PATHWAY HER 2. These
interpretation of patient results under these circumstances. parameters can be displayed, printed and edited according to the procedure in
the Ventana automated slide stainer Operator's Manual. Other operating
B. Toxicity of Antibody Solution parameters for the automated slide stainers have been preset at the factory.
Symptoms of overexposure to ProClin 300, the preservative in the antibody The procedure for staining c-erbB-2 with PATHWAY HER 2 on the Ventana
reagent, may include skin and eye irritation and irritation to mucous BenchMark automated slide stainer is as follows (Refer to the Operator's
membranes and upper respiratory tract. The concentration of ProClin 300 in Manual for specific details on the operation of the Ventana automated slide
this product is 0.05 %. This antibody solution does not meet the OSHA criteria stainer).
for a hazardous substance. Refer to the Material Safety Data Sheet enclosed
with each shipment of product, or call (800) 227-2155 for a copy. 1. The PATHWAY HER 2 dispenser, appropriate detection kit dispensers,
and desired accessory reagents are loaded onto the reagent tray and
IV. Instructions for Use placed on the Ventana BenchMark automated slide stainer.
2. Each slide must be labeled with the appropriate bar code specifying the
A. Step by Step Procedure
staining procedure and PATHWAY HER 2.
Ventana PATHWAY HER 2 was developed for use on a Ventana automated 3. Load the labeled slides onto the slide carousel.
slide stainer in combination with Ventana detection kits and accessories.
2006-06-02 Page 3 of 9 14160US Rev. B
Ventana PATHWAY HER 2 has been optimized to work in combination with 6. A/B Block (selectable): The slide is rinsed; Blocker A is applied and
Ventana detection kits to provide the greatest specific staining-to-background incubated with mixing for 4 minutes. The slide is rinsed; Blocker B is
ratio. The following is the sequence of events carried out by the Ventana applied and incubated with mixing for 4 minutes. This step utilizes the
BenchMark automated slide stainer. Ventana Endogenous Biotin Blocking Kit; blocking is recommended for
this assay to reduce background staining due to endogenous biotin.
1. Deparaffinization (selectable): EZ Prep is applied and incubated for Rinse step prior to Biotinylated Ig secondary antibody dispense is
12 minutes at 76 °C. On-instrument deparaffinization is recommended. omitted if this step is selected.
2. Cell conditioning: The slide is rinsed; Cell Conditioning 2 (pre-dilute) is 7. The slide is rinsed; Biotinylated Ig secondary antibody is applied and
applied and incubated with mixing for 32 minutes at 95 °C. incubated with mixing for 8 minutes at 37 °C.
3. The slide is rinsed; Inhibitor solution is applied to decrease the 8. The slide is rinsed; avidin-HRPO enzyme conjugate is applied and
endogenous peroxidase activity. The inhibitor on the slide is incubated incubated with mixing for 8 minutes at 37 °C.
with mixing for 4 minutes at 42 °C. 9. The slide is rinsed; DAB and DAB H2O2 are added and incubated
4. The slide is rinsed; the optimized PATHWAY HER 2 is applied and together for 8 minutes with mixing at 37 °C.
incubated with mixing for 32 minutes at 42 °C (see Specific 10. The slide is rinsed; copper DAB enhancer is applied with mixing for
Limitation #3). 4 minutes at 37 °C.
5. A/B Block (selectable): The slide is rinsed; Blocker A is applied and 11. Counterstain (selectable): if counterstain and/or post counterstain is
incubated with mixing for 4 minutes. The slide is rinsed; Blocker B is selected, each will be applied to the slide and incubated with mixing for
applied and incubated with mixing for 4 minutes. This step utilizes the the selected time interval at 37 °C. Ventana Hematoxylin counterstain
Ventana Endogenous Biotin Blocking Kit; blocking is recommended for and Bluing Reagent post counterstain are recommended for this
this assay to reduce background staining due to endogenous biotin. protocol, with 4 minute incubation for each.
Rinse step prior to Biotinylated Ig secondary antibody dispense is 12. NeuVision Counterstain (selectable): do not select.
omitted if this step is selected.
6. The slide is rinsed; Biotinylated Ig secondary antibody is applied and
Post-staining Procedure
incubated with mixing for 8 minutes at 42 °C.
7. The slide is rinsed; avidin-HRPO enzyme conjugate is applied and Slides may be removed from the instrument, rinsed in detergent solution,
incubated with mixing for 8 minutes at 42 °C. dehydrated, cleared, (do not dehydrate or clear for AEC chromogen) and
coverslipped using a mounting medium described in Section II C item 24 and cover
8. The slide is rinsed; DAB and DAB H2O2 are added and incubated
glass described in Section II C item 25. The stained slides should be read within two
together for 8 minutes with mixing at 42 °C.
to three days of staining and should be stable for up to two weeks if properly stored
9. The slide is rinsed; copper DAB enhancer is applied with mixing for
at room temperature (15-25 °C).
4 minutes at 42 °C.
10. Counterstain (selectable): if counterstain and/or post counterstain is
B. Quality Control Procedures
selected, each will be applied to the slide and incubated with mixing for
the selected time interval at 42 °C. Differences in tissue processing and technical procedures in the user’s
laboratory may produce significant variability in results, necessitating regular
BenchMark XT and BenchMark LT automated slide stainers
performance of in house controls in addition to the following procedures.
Consult the quality control guidelines of “Special report: Quality control in
Recommended staining protocol selected in the procedure menu for the
Immunohistochemistry�10 and/or the Proposed NCCLS guideline for IHC.20
Ventana BenchMark XT and BenchMark LT automated slide stainers is
XT PATHWAY HER-2 V.1. These parameters can be displayed, printed and 1. Cell Line Controls
edited according to the procedure in the Ventana automated slide stainer
Ventana has available as a separate product formalin-fixed cell line
Operator's Manual. Other operating parameters for the automated slide
controls embedded in paraffin, sectioned and placed on charged slides
stainers have been preset at the factory.
(catalog # S9100). Cell line controls may be useful for a preliminary
The procedure for staining c-erbB-2 with PATHWAY HER 2 on the Ventana validation of the processing method used for staining slides with
BenchMark XT and BenchMark LT automated slide stainer is as follows (Refer PATHWAY HER 2. Ventana has available three cell line controls
to the Operator's Manual for specific details on the operation of the Ventana characterized by in situ hybridization for gene copy number and by
automated slide stainer). Scatchard analysis for receptor content. When processed and stained
appropriately, the cell lines should stain as described in Table 2. If the
1. The PATHWAY HER 2 dispenser, appropriate detection kit dispensers,
indicated staining, especially the 1+ staining in greater than 10% of the
and desired accessory reagents are loaded onto the reagent tray and
cells, is not evident in the Level 2 cell line control, the staining of the
placed on the Ventana BenchMark automated slide stainer.
tissues should be repeated.
2. Each slide must be labeled with the appropriate bar code specifying the
staining procedure and PATHWAY HER 2. Table 2. Characterization of c-erbB-2 in Cell Line Controls
3. Load the labeled slides onto the slide carousel.
Control Gene Copy # IHC Staining % Cells
Cell Line: Receptor #
Slide: (Avg.) Intensity Staining
Ventana PATHWAY HER 2 has been optimized to work in combination with
Ventana detection kits to provide the greatest specific staining-to-background Level 1 MDA-MB- 2.5 8.45E+04 0 100%
ratio. The following is the sequence of events carried out by the Ventana 468
BenchMark XT and BenchMark LT automated slide stainer.
Level 2 T-47D 4.8 1.13E+05 1+ >10%
1. Deparaffinization (selectable): EZ Prep is applied and incubated for
12 minutes at 77 °C. On-instrument deparaffinization is recommended. Level 3 SK-BR-3 17 2.02E+05 3+ 100%
2. Cell conditioning: The slide is rinsed; Cell Conditioning 2 (pre-dilute) is
2. Positive Tissue Control
applied and incubated with mixing for 36 minutes at 95 °C.
3. The slide is rinsed; Inhibitor solution is applied to decrease the A positive control tissue fixed and processed in the same manner as
endogenous peroxidase activity. The inhibitor on the slide is incubated the patient specimens must be run for each set of test conditions and
with mixing for 4 minutes at 37 °C. with every PATHWAY HER 2 staining procedure performed by the
4. NeuVision Blocking Reagent (selectable): do not select. instrument. This tissue should contain both positive staining cell/tissue
5. The slide is rinsed; the optimized PATHWAY HER 2 is applied and components and negative cell/tissue components and serve as both the
incubated with mixing for 32 minutes at 37 °C (see Specific positive and negative control tissue. Control tissues should be fresh
Limitation #3). autopsy/biopsy/surgical specimens prepared and fixed as soon as
possible in a manner identical to test sections. Such tissues may monitor
all steps of the analysis, from tissue preparation through staining. Use of a
tissue section fixed or processed differently from the test specimen provides
control for all reagents and method steps except fixation and tissue
processing.
2006-06-02 Page 4 of 9 14160US Rev. B
A tissue with weak positive staining is more suitable than strong positive 6. Assay Verification
staining for optimal quality control and to detect minor levels of reagent
Prior to initial use of this antibody in the user's laboratory or if there is a
degradation. Ideally, a tissue which is known to have weak, but positive
change of lot number, the specificity of the antibody should be verified by
staining should be chosen to ensure that the system is sensitive to small
staining a number of positive and negative tissues with known performance
amounts of reagent degradation or problems with the IHC methodology.
characteristics. Refer to the quality control procedures previously outlined in
Generally, however, neoplastic tissue that is positive for c-erbB-2 is strongly
this section of the product insert and to the quality control recommendations
positive due to the nature of the pathology (overexpression). An example of
of the CAP certification program for immunohistochemistry5 and/or the
tissue to use as a positive control with PATHWAY HER 2 is intraductal
NCCLS IHC guideline.20 These quality control tests should be repeated for
adenocarcinoma of the breast demonstrating positivity for c-erbB-2. The
each new lot or whenever there is a change of lot number of one of the
positive staining cells/tissue components (cell membrane staining of
reagents in a matched set or a change in assay parameters. Quality control
ductal cells) are used to confirm that PATHWAY HER 2 was applied
cannot be meaningfully performed on an individual reagent in isolation since
and the instrument functioned properly.
the matched reagents, along with a defined assay protocol, must be tested
Known positive tissue controls should only be utilized for monitoring the in unison before using a kit for diagnostic purposes. Tissues listed in the
correct performance of processed tissues and test reagents, NOT as an aid Summary of Expected Results are suitable for assay verification.
in formulating a specific diagnosis of patient samples. If the positive tissue
Assay verification on a daily basis may be accomplished through the
controls fail to demonstrate positive staining, results with the test specimens
proper use of the above mentioned positive and negative controls, as
should be considered invalid.
described in this section (see Table 3 below). In addition, it is
3. Negative Tissue Control recommended that on a monthly basis, the c-erbB-2 positive tissue
control be stained and compared to the same tissue control stained the
Use a tissue control known to be fixed, processed and embedded in a
previous month. Comparison of controls stained at monthly intervals
manner identical to the patient sample(s) with each staining run to verify
serves to monitor the assay stability, sensitivity, specificity, and
the specificity of PATHWAY HER 2 for demonstration of c-erbB-2, and
reproducibility.
to provide an indication of specific background staining (false positive
staining). Also, the variety of different cell types in most tissue sections All quality control requirements should be performed in conformance with
can be used by the laboratorian as internal negative control sites to local, state and/or federal regulations or accreditation requirements.
verify PATHWAY HER 2 performance specifications. For example, the
Table 3. The Purpose of Daily Quality Control
same tissue used for the positive tissue control (intraductal
adenocarcinoma of the breast) may be used as the negative tissue Tissue: Fixed and processed Specific antibody and Nonspecific Antibody* or
control. The non-staining components (surrounding stroma, lymphoid cells like patient sample secondary antibody Ventana Antibody Diluent
and blood vessels) should demonstrate absence of specific staining, and (#251-018) plus same
provide an indication of specific background staining. Alternatively, normal secondary antibody as used
breast tissue is an adequate negative control tissue. If specific staining with specific antibody
occurs in the negative tissue control sites, results with the patient specimens
Positive control: Controls all steps of the Detection of non-specific
should be considered invalid.
tissue or cells containing analysis. Validates reagent background staining.
target antigen to be detected and procedures used for
4. Nonspecific Negative Reagent Control
(could be located in patient staining
A negative reagent control must be run from every tissue block stained tissue). The ideal control is
on the Ventana automated slide stainer to aid in the interpretation of weakly positive staining tissue
each patient result. A negative reagent control is used in place of the to be most sensitive to
primary antibody to evaluate nonspecific staining and allow better antibody degradation.
interpretation of specific staining at the antigen site. This provides an
Negative Control: Tissue or Detection of unintended Detection of non-specific
indication of nonspecific background staining for each slide. In place of
cells to be negative (could be antibody cross-reactivity to background staining
the primary antibody, stain the slide with Ventana CONFIRM Negative
located in patient tissue or cells/cellular components
Control Mouse Ig, a mouse myeloma protein (IgG1, kappa) directed
positive control tissue)
against an antigen not found in human specimens, or with an
Patient Tissue Detection of specific staining Detection of non-specific
appropriate substitute that has been produced in the same way as the
background staining
primary antibody and in the same matrix solution. The mouse myeloma
protein is the ideal negative control because it is nonspecific, produced *= Same source and type as the specific antibody but not directed against any human
in the same way as the primary antibody and of murine origin of the antigen. To detect non-specific antibody binding, e.g., binding of Fc portion of antibody by the
same isotype. If an alternative negative reagent control is used, dilute to tissue.
the same dilution as the primary antibody/antiserum in Ventana
C. Interpretation of Staining
Antibody Diluent. Approximately 0.25 % fetal calf serum is retained in
PATHWAY HER 2. Addition of 0.25 % fetal calf serum in Ventana Scoring conventions for the interpretation of PATHWAY HER 2:
Antibody Diluent is also suitable for use as a nonspecific negative
Breast carcinomas that are considered positive for c-erbB-2 must meet a
reagent control. Diluent alone may be used as a less desirable
threshold criteria for intensity of staining (2+ or greater on a scale of 0 to 3+)
alternative to the previously described negative reagent controls. The
and percent positive tumor cells (greater than 10 %). Staining must also
incubation period for the negative reagent control should correspond to
localize to the cellular membrane. Cytoplasmic staining may still be present,
that of the primary antibody.
but this staining is not included in the determination of positivity. Three fields
When panels of several antibodies are used on serial sections, the within the well-preserved and well-stained region of the tissue should be
negatively staining areas of one slide may serve as a negative/non-specific examined for intensity of staining and determination of completeness of the
binding background control for other antibodies. cytoplasmic membranous stain. Staining that completely encircles the
cytoplasmic membrane should be scored as an intensity of �2+� or �3+�. Partial
To differentiate endogenous enzyme activity or nonspecific binding of
staining of the membrane should be scored as a �1+�. It may be necessary to
enzymes from specific immunoreactivity, additional patient tissues may be
examine borderline cases at 400X or higher magnification to discriminate
stained exclusively with substrate-chromogen or enzyme complexes (avidin-
between intensities of �1+� and �2+�. In contrast to cases scored as an intensity
biotin, streptavidin) and substrate-chromogen, respectively.
of 3+, the staining scored as 2+ has a crisper and more clearly delineated ring,
5. Unexplained Discrepancies while cases scored as 3+ exhibit a very thick outline. Below is a quick
reference chart for staining criteria. Refer to Ventana PATHWAY HER 2
Unexplained discrepancies in control results should be referred to Scoring Guide for a more detailed description with photographs of staining with
Ventana Technical Consultation Center (800-227-2155) immediately. If PATHWAY HER 2.
quality control results do not meet specifications, patient results are
invalid. See Troubleshooting, section of this insert. Identify and correct
the problem, then repeat the patient samples
2006-06-02 Page 5 of 9 14160US Rev. B
Table 4. Criteria for Intensity of Cell Membrane Staining with PATHWAY HER 2 antigen in question was not detected, not that the antigen is absent in the
cells/tissue assayed. If necessary use a panel of antibodies to aid in the
Staining Pattern Score c-erbB-2 Staining
identification of false negative reactions (see Section IV. B. Quality Control
(Report to treating Assessment
Procedures). The morphology of each tissue sample should also be examined
physician)
utilizing a hematoxylin and eosin stained section when interpreting any
No membrane staining is observed 0 Negative immunohistochemical result. The patient's morphologic findings and pertinent
Faint, partial staining of the membrane 1+ Negative clinical data must be interpreted by a qualified pathologist. Refer to Summary
and Explanation, Limitations, and Summary of Expected Results for specific
Weak complete staining of the 2+ Positive
information regarding immunoreactivity.
membrane, greater than 10% of cancer
cells D. Limitations
Intense complete staining of the 3+ Positive
General Limitations:
membrane, greater than 10% of cancer
cells 1. Immunohistochemistry (IHC) is a multistep diagnostic process that consists
of specialized training in the selection of the appropriate reagents; tissue
Interpretation of staining within the context of controls: selection, fixation, processing; preparation of the IHC slide; and
interpretation of the staining results.
The Ventana automated immunostaining procedure causes a colored reaction
2. Tissue staining is dependent on the handling and processing of the tissue
product to precipitate at the antigen sites localized PATHWAY HER 2 (Refer to
prior to staining. Improper fixation, freezing, thawing, washing, drying,
the appropriate Ventana detection kit package insert for the expected color.) A
heating, sectioning or contamination with other tissues or fluids may produce
qualified pathologist who is experienced in immunohistochemical procedures
artifacts, antibody trapping, or false negative results. Inconsistent results
must evaluate positive and negative controls and qualify the stained product
may be due to variations in fixation and embedding methods, or to inherent
before interpreting results.
irregularities within the tissue.17
Positive Tissue Control: The positive tissue control stained with Ventana 3. Excessive or incomplete counterstaining may compromise proper
PATHWAY HER 2 should be examined first to ascertain that all reagents are interpretation of results.
functioning properly. The presence of rose red (3-amino-9-ethylcarbazole, 4. Unexpected negative staining in tumors may be due to loss of expression of
AEC), bright pink (fast red) or reddish-brown (3,3'-diaminobenzidine antigen or loss of the gene(s) coding the antigen as a tumor dedifferentiates.
tetrahydrochloride, DAB) reaction product with the target cells� membranes is Unexpected positive staining in tumors may be from expression of an
indicative of positive reactivity. PATHWAY HER 2 shows more intense staining antigen not usually expressed in normal cells or persistence or acquisition of
in ductal cells of breast carcinoma on the cell surface membrane, with an antigen in a dedifferentiated tumor that develops morphologic and
occasional lesser cytoplasmic staining. It is imperative that only the intense immunohistochemical markers associated with another cell lineage.
membrane, and not cytoplasmic staining, be considered positive if a false- Histopathologic classification of tumors is not an exact science and some
positive interpretation is to be avoided.7 Staining in breast carcinoma is found literature reports of unexpected staining are controversial.
only in a subset of cases (approximately 20 %). Morphologically, staining is 5. The clinical interpretation of any positive or negative staining should be
found in ductal cells in a cell membrane pattern. Ductal cells sometimes react evaluated within the context of clinical presentation, morphology and other
less intensely in a cytoplasmic pattern with the antibody. The surrounding histopathological criteria. The clinical interpretation of any positive or
stroma, lymphoid cells and blood vessels should be unreactive. If the positive negative staining should be complemented by morphological studies using
tissue control fails to demonstrate positive staining, any results with the test proper positive and negative internal and external controls as well as other
specimens should be considered invalid. diagnostic tests. It is the responsibility of a qualified pathologist who is
familiar with the proper use of IHC antibodies, reagents and methods, to
The color of the reaction product may vary if substrate chromogens other than
interpret all of the steps used to prepare and interpret the final IHC
those stated are used. Refer to substrate package inserts for expected color
preparation.
reactions. Further, metachromasia may be observed in variations of the
6. This product is not intended for use in flow cytometry. Performance
method of staining.16 characteristics have not been determined for flow cytometry.
7. Tissues from persons infected with hepatitis B virus and containing hepatitis
Depending on the incubation length and potency of the hematoxylin used,
B surface antigen (HBsAg) may exhibit nonspecific staining with horseradish
counterstaining will result in a pale to dark blue coloration of the cell nuclei.
peroxidase.21
Excessive or incomplete counterstaining may compromise proper interpretation
8. Reagents may demonstrate unexpected reactions in previously untested
of results.
tissues. The possibility of unexpected reactions even in tested tissue groups
Negative Tissue Control: The negative tissue control should be examined after cannot be completely eliminated due to biological variability of antigen
the positive tissue control to verify the specific labeling of the target antigen by expression in neoplasms, or other pathological tissues.13 Contact Ventana
the primary antibody. The absence of specific staining in the negative tissue Technical Consultation Center (800-227-2155) with documented
control confirms the lack of antibody cross-reactivity to cells/cellular unexpected reaction(s).
components. The staining of normal breast is an adequate negative control 9. Normal/nonimmune sera from the same animal source as secondary
tissue. Intact stromal and duct elements should show no intense staining in the antisera used in blocking steps may cause false negative or positive results
membrane indicating that staining did not occur. If the tissue is counterstained, due to auto-antibodies or natural antibodies.
there may be staining around the outside of the cell, i.e. the interstitial spaces. 10. False positive results may be seen due to non-immunological binding of
If specific staining occurs in the negative tissue control, results with the patient proteins or substrate reaction products. They may also be caused by
specimen should be considered invalid. pseudoperoxidase activity (erythrocytes), endogenous peroxidase activity
(cytochrome C), or endogenous biotin (e.g. liver, brain, breast, kidney)
Nonspecific staining, if present, will have a diffuse appearance. Sporadic light
depending on the type of immunostain used.17
staining of connective tissue may also be observed in sections from
excessively formalin-fixed tissues. Use intact cells for interpretation of staining Specific Limitations:
results. Necrotic or degenerated cells will often stain nonspecifically.17
1. Ventana PATHWAY HER 2 has been optimized for a 32 minute
Patient Tissue: Patient specimens should be examined last. Positive staining incubation time with antigen-enhanced tissue, in combination with
intensity should be assessed within the context of any nonspecific background Ventana detection kits and Ventana automated slide stainers. Due to
staining of the negative reagent control. Strong membrane staining is a much variation in tissue processing, the time of incubation depends upon the
more reliable indicator of the presence of clinically significant c-erbB-2 than is degree of tissue fixation and/or the effectiveness of antigen
cytoplasmic staining only.7 The neoplastic cells of breast carcinomas show no enhancement and must be determined empirically. It may be necessary
reactivity in the majority of cases (approximately 80 %). Certain subsets of to increase or decrease PATHWAY HER 2 incubation time on individual
breast carcinomas exhibit higher frequencies of c-erbB-2 over-expression. specimens. Users who deviate from recommended test procedures
Almost all cases of Paget’s disease of breast15 and up to 90 percent of cases must accept responsibility for interpretation of patient results under
of ductal carcinoma in situ of comedo type are positive.9 c-erbB-2 may be these circumstances.
2. Ventana PATHWAY HER 2, in combination with Ventana detection kits
detected among other neoplasms, particularly adenocarcinomas of the ovary,
and accessories, demonstrates c-erbB-2 antigen that survives routine
stomach, and bladder in frequencies similar to that of breast cancer (20 %).9
tissue fixation, processing and sectioning.
As with any immunohistochemical test, a negative result means that the
2006-06-02 Page 6 of 9 14160US Rev. B
3. Primary antibody incubation time depends on the degree of tissue events (18 %). Thus there can be some difficulty distinguishing a 2+
fixation and may range from 4 to 32 minutes. Ventana recommends 32 from a 3+ reaction.
minutes for use with its detection kits. For further information about
Intra-laboratory, intra-technician and inter-technique staining
fixation variables refer to Immunomicroscopy: A Diagnostic Tool for the
reproducibility: The same three laboratories that participated in the
Surgical Pathologist.25
inter-laboratory reproducibility study participated in an intra-laboratory
4. The following tissues have not been tested: parathyroid.
(technician A vs. technician B) and intra-technician (technician A vs.
5. False negative cases may result from various factors, including true
technician A, 1 week apart) study, utilizing the same four breast
antigen decrease, loss or structural change during tumor
carcinoma cases and cell line controls. The slides were returned to
"dedifferentiation" or terminal differentiation artifactual change during
Ventana and masked, then sent for evaluation by a qualified
fixation or processing. As with any immunohistochemical test, a
pathologist. At all three sites intra- and inter-technician staining of all the
negative result means that the antigen was not detected, not that the
slides stained with similar staining intensity (varied by no more than 1
antigen was absent in the cells/tissues assayed.
intensity level). No cases varied from clinically positive to clinically
6. Neoplastic tissue is the recommended positive control tissue. While
negative between the sites. Evaluating 3 intra-laboratory comparisons
many normal human tissues react positively with PATHWAY HER 2, the
(technician A run 1 vs. technician B) of the 2 cases staining positively,
staining pattern in normal tissues is generally cytoplasmic while
variation between 3+ intensity and 2+ intensity occurred one time out of
neoplastic tissues, such as breast carcinoma show intense
a total of 16 staining events (6 %). Evaluating 3 intra-technician
membranous staining.7 comparisons (technician A vs. technician A, 1 week apart) of the 2
7. c-erbB-2 may be detected among other neoplasms, particularly cases staining positively, variation between 3+ intensity and 2+ intensity
adenocarcinomas of the ovary, stomach, and bladder in frequencies similar occurred three times out of a total of 14 staining events (21 %). Thus
to that of breast cancer (20 %).9 there can be some difficulty distinguishing a 2+ from a 3+ reaction.
8. Not all breast carcinomas are expected to be positive with PATHWAY
Additionally these sites were invited to use their own in-house
HER 2. Only 9-16.5 % have been seen to be positive with Clone CB11.7
pretreatment methods with the study cases to evaluate inter-technique
9. The degree of c-erbB-2 protein overexpression may be an important
reproducibility. Again there were no differences of more than one
predictor of response to therapy with Herceptin. Data from clinical trials of
intensity level between methodologies and no variation occurred that
Herceptin suggests that the beneficial treatment effects were primarily
changed the clinical interpretation of the results. Evaluating 3 intra-
limited to patients whose tumor’s overexpression of c-erbB-2 protein
technique comparisons (technician A Run 2 Ventana method vs.
was 3+.12
technician A Run 2 in-house method) of the 2 cases staining positively,
variation between 3+ intensity and 2+ intensity occurred two times out
E. Summary of Expected Results
of a total of 14 staining events (14 %). Thus staining with an in-house
1. Reproducibility
method was equivalent to the Ventana staining method.
Intra-run reproducibility of staining with Ventana PATHWAY HER 2 was
Intra-investigator and inter-investigator scoring reproducibility: Five
determined by staining 20 slides containing sections from the same
investigators participated in a scoring reproducibility study. Each
neutral buffered formalin-fixed tissue. The procedure included
investigator was sent the same set of twelve neutral buffered formalin-
pretreatment of the slides using antigen enhancement in an acidic
fixed breast carcinoma cases pre-stained with hematoxylin and eosin
buffer. Slides were then stained using a protocol consisting of 32 minute
(H&E), PATHWAY HER 2, and negative Ig reagent. The procedure for
incubation of the primary antibody, Ventana's Basic DAB detection kit,
preparing the slides used in this study included pretreatment of the
and counterstaining with hematoxylin and bluing reagent. 20 of 20
slides using antigen enhancement in an acidic buffer. Slides were then
slides stained positively. All slides stained with similar staining intensity.
stained using a protocol consisting of 32 minute incubation of the
Users should verify within run reproducibility results by staining several
primary antibody (PATHWAY HER 2 or negative Ig reagent), Ventana's
sets of serial sections with low, medium, and high antigen density in a
Basic DAB detection kit, and counterstaining with hematoxylin and
single run.
bluing reagent. Investigators were provided with a scoring guide with
photographs of breast carcinoma cases stained with PATHWAY HER 2,
Inter-run reproducibility of staining with Ventana PATHWAY HER 2 was
and the study protocol with directions for scoring. Investigators were
determined by staining slides containing sections from the same neutral
also instructed to call Ventana if they had questions. Investigators
buffered formalin-fixed tissue on 20 different runs. The procedure
scored the cases twice with a minimum of 4 weeks� interval between
included pretreatment of the slides using antigen enhancement in an
scoring. Scores were evaluated for intra-investigator and inter-
acidic buffer. Slides were then stained using a protocol consisting of 32
investigator scoring reproducibility. One investigator missed (forgot to
minute incubation of the primary antibody, Ventana's Basic DAB
score) one case for round 1 evaluation, which left 74 staining events for
detection kit, and counterstaining with hematoxylin and bluing reagent.
reproducibility evaluations. Intra-investigator reproducibility was
All slides stained with similar staining intensity. Users should verify
clinically consistent (positive versus negative) for 72 of the 74 staining
between run reproducibility results by staining several sets of serial
interpretations - 97 %. Out of 34 intra-investigator evaluations of 2+ and
sections with low, medium, and high antigen density on different days.
3+ positively staining cases/cell lines, there were 5 (15 %) instances of
Inter-laboratory staining reproducibility: Three laboratories, from 2+/3+ intra-investigator scoring variability. Thus an investigator trained
separate institutions in the United States, participated in the inter- with the scoring materials provided was able to consistently interpret
laboratory reproducibility study. Cut slides of four neutral buffered PATHWAY HER 2 staining of breast carcinoma cases and cell line
formalin-fixed breast carcinoma cases and the three neutral buffered controls, although there was still some variation in the interpretation of
formalin-fixed cell line controls were shipped to the sites for staining on 2+ and 3+ staining. Inter-investigator reproducibility was evaluated for
a Ventana automated slide staining device. The slides were returned to both round 1 and round 2. In round 1, 71 of 74 interpretations were
Ventana and masked, then sent for evaluation by a qualified clinically consistent among the five investigators - 96 %. In round 1, out
pathologist. of 34 inter-investigator evaluations of 2+ and 3+ positively staining
cases/cell lines, there were 4 (12 %) instances of 2+/3+ inter-
All three sites performed identical pretreatment procedures and staining
investigator scoring variability. In round 2, 70 of 75 interpretations were
protocols. The procedure included pretreatment of the slides using
clinically consistent among the five investigators�93 %, and out of 35
antigen enhancement in an acidic buffer. Slides were then stained
inter-investigator evaluations of 2+ and 3+ positively staining cases/cell
using a protocol consisting of 32 minute incubation of the primary
lines there was one instance (3 %), of 2+/3+ inter-investigator scoring
antibody, Ventana's Basic DAB detection kit, and counterstaining with
variability. Thus investigators at different institutions trained with the
hematoxylin and bluing reagent. All slides stained with similar staining
scoring materials provided were able to consistently interpret
intensity (varied by no more than 1 intensity level). No cases varied
PATHWAY HER 2 staining of breast carcinoma cases and cell line
from clinically positive to clinically negative between the sites and no
controls, although there was still some variation in the interpretation of
sites experienced invalid runs, based upon the performance of the
2+ and 3+ staining. This variability, however, declined with investigator
controls. Controls included the three cell lines, a positive tissue control
experience; by round two there was only one instance of 2+/3+ inter-
located on the same slide as the test case, and a second slide of each
investigator variability.
case stained with negative Ig reagent. Evaluating 3 inter-laboratory
comparisons of the 2 cases staining positively, variation between 3+ Lot-to-lot reproducibility: To test the lot-to-lot variation of the PATHWAY
intensity and 2+ intensity occurred four times out of a total of 22 staining HER 2, the three PATHWAY HER 2 cell line controls and two neutral
2006-06-02 Page 7 of 9 14160US Rev. B
buffered formalin-fixed known positive breast carcinoma cases were run Table 6. 3 X 3 Concordance of Ventana PATHWAY HER 2 and HercepTest
with three different GMP lots. The procedure for preparing the slides
HercepTest
used in this study included pretreatment of the slides using antigen
enhancement in an acidic buffer. Slides were then stained using a
Negatives 2+ 3+ Total:
PATHWAY HER 2 �
protocol consisting of 32 minute incubation of the primary antibody
(PATHWAY HER 2 or negative Ig reagent), Ventana's Basic DAB Negatives 282 14 3 299
detection kit, and counterstaining with hematoxylin and bluing reagent.
Lot 2 exhibited a lower percentage of cells staining in the 1+ cell line 2+ 13 24 13 50
control (T-47D), and a drop from 3+ to 2+ for one breast carcinoma
3+ 4 14 83 101
case. All other tissues and cell lines stained uniformly with the three lots
tested. No tissues or cell lines tested changed from clinically positive or
Total: 299 52 99 450
clinically negative when stained with the three different lots of antibody.
Concordance = 86.4 %
Thus lot-to-lot staining results are very consistent with c-erbB-2 primary
95% Confidence Interval = 82.9 % to 89.5 %
antibody.
p < 0001
2. Immunoreactivity:
The observed agreement was 86.4 % (389/450) with 95 % confidence
Immunoreactivity of PATHWAY HER 2 was demonstrated by a clinical interval of 82.9 % to 89.5 %. The null hypothesis that agreement was no
study that showed appropriate staining in breast carcinoma tissue, and greater than 75 % (one-sided hypothesis) was rejected with p < 0.0001.
in-house specificity panels of normal and neoplastic tissues. The clinical
Clinical concordance (the likelihood of a patient to be selected for
study was designed to evaluate the appropriateness of PATHWAY HER
treatment) between PATHWAY HER 2 and the previously approved
2 as an aid in the assessment of patients for whom Herceptin treatment
diagnostic as an aid for determining Herceptin therapy, HercepTest is
is considered.
92.4 % (95 % CI = 89.6 % - 94.7 %). The ability of each test to
3. Appropriate performance for use as an aid in the assessment of distinguish negatives, intermediate positives (2+) and strong positives
Herceptin therapy. (3+) is also in strong agreement, 86.4 % concordance (95 %
CI= 82.9 % - 89.5%). The data generated in this study demonstrate that
This study examined the suitability of PATHWAY HER 2 for use as an
PATHWAY HER 2 is indicated as an aid in determining Herceptin
aid in determination of treatment for Herceptin therapy. A comparative
therapy based upon its high concordance of staining results with
protocol was designed to examine the correlation of performance
HercepTest, a previously approved diagnostic test for this indication.
between PATHWAY HER 2 and HercepTest, a previously approved
FDA diagnostic for this indication. As such, this assay was set as the 4. Specificity Panels
gold standard. Three investigators participated in the study. Each
Ventana also tested normal and neoplastic tissues in which formalin-
investigator evaluated the archived neutral buffered formalin-fixed,
fixed, paraffin-embedded tissues were stained with PATHWAY HER 2.
paraffin-embedded breast carcinoma tissue blocks available at their
The procedure used for preparing the slides stained with PATHWAY
institutions for HER-2/neu status and stratified their cases into positive
HER 2 included pretreatment of the slides using antigen enhancement
and negative sub-populations. 75 cases were randomly selected from
in an acidic buffer. Slides were then stained using a protocol consisting
each pool for a total of 150 cases per site, 450 cases for the study.
of 32 minute incubation of the primary antibody (PATHWAY HER 2 or
The slides stained with HercepTest were processed and stained negative Ig reagent), Ventana's Basic DAB detection kit, and
according to the manufacturer’s instructions specified in the package counterstaining with hematoxylin and bluing reagent. The seventy-eight
insert. The procedure used for preparing the slides stained with normal tissues examined included adrenal, bone marrow, breast,
PATHWAY HER 2 included pretreatment of the slides using antigen cerebrum, cerebellum, cervix, colon, endometrium, esophagus, heart,
enhancement in an acidic buffer. Slides were then stained using a kidney, liver, lung, mesothelium, ovary, pancreas, peripheral nerve,
protocol consisting of 32 minute incubation of the primary antibody prostate, salivary gland, skeletal muscle, skin, small intestine, spleen,
(PATHWAY HER 2 or negative Ig reagent), Ventana's Basic DAB stomach, testis, thyroid, tonsil, and thymus. Tissues not tested were
detection kit, and counterstaining with hematoxylin and bluing reagent. parathyroid. Cytoplasmic staining was observed in the following tissues:
colon (1 of 3 cases), endometrium (1 of 3 cases), kidney (1 of 2 cases),
Results: Data were analyzed for concordance, positivity, and
pancreas (2 of 3 cases), pituitary (1 of 2 cases), prostate (2 of 3 cases),
negativity. Concordance was evaluated in two formats: binary (positive
small intestine (2 of 3 cases), stomach (2 of 3 cases), thyroid (3 of 3
vs. negative), and 3 X 3 (3+, 2+ > 10 %, negatives - 2+ < 10 %, 1+, 0).
cases), tonsil (3 of 3 cases), and thymus (1 of 1 case). One case of
kidney and one case of tonsil showed cell membrane staining, and one
Table 5. Clinical Agreement of Ventana PATHWAY HER 2 and HercepTest
case of thyroid exhibited extracellular staining. The cell membrane
HercepTest HercepTest
Total staining of the tonsil was limited to the squamous epithelium, a
Negative Positive
secondary component.
PATHWAY
The nineteen neoplastic tissues examined were lung, prostate, colon,
HER 2 282 17 299
lymphoma, bladder, stomach, cervix, and ovary. Cytoplasmic staining
Negative
was observed in cancer cells of the lung (3 of 3 cases), prostate (2 of 3
PATHWAY
cases), colon (3 of 3 cases), bladder (1 of 1 case), cervix (2 of 3 cases),
HER 2 17 134 151
and ovary (2 of 2 cases) and was not considered to be a positive result.
Positive
The sensitivity of c-erbB-2 histochemistry is dependent upon the
Total: 299 151 450
preservation of the antigen. Any improper tissue handling during
Concordance = 92.4 %
fixation, sectioning, embedding, or storage which alters the antigenicity
95% Confidence Interval = 89.6 % to 94.7 %
weakens the c-erbB-2 detection by Ventana's PATHWAY HER 2 and
p < 0.0001
may generate false negative results.
The observed agreement between the two tests was 92.4 % (416/450).
F. Troubleshooting
The exact 95 % confidence interval was 89.6 % to 94.7 %. The null
hypothesis that agreement was no greater than 75% (one-sided 1. If the c-erbB-2 positive control exhibits weaker staining than expected,
hypothesis) was rejected with p < 0.0001. The kappa statistic (chance- check other positive controls run during the same instrument run to
corrected measure of agreement) was 0.83; the null hypothesis that determine if it is due to the primary antibody or one of the common
agreement is no better than chance was rejected with p < 0.0001. secondary reagents. Call Ventana Technical Consultation Center
McNemar’s Test of the hypothesis that the proportion positive by (800-227-2155).
PATHWAY HER 2 was equal to the proportion positive by HercepTest 2. If the c-erbB-2 positive control is negative, check to ensure that the
could not be rejected with p = 1.00. Treating HercepTest as the slide has the proper bar code label. If the slide is labeled properly,
standard, the sensitivity of PATHWAY HER 2 was 88.7 % (134/151) check other positive controls run during the same instrument run to
with 95 % confidence interval of 82.6 % to 93.3 %. The specificity was determine if it is due to the primary antibody or one of the common
94.3 % (282/299) with 95 % confidence interval of 91.1 % to 96.7 %. secondary reagents. Tissues may have been improperly collected,
2006-06-02 Page 8 of 9 14160US Rev. B
fixed, or deparaffinized. Follow proper procedure for collection, storage, 22. Quenel, N. et al. The prognostic value of c-erbB-2 in primary breast
and fixation. Call Ventana Technical Consultation Center carcinomas: A study on 942 cases. Breast Cancer Res. And Treatment.
(800-227-2155). 35: 283-291, 1995.
3. If excessive background staining occurs, it may be due to residual 23. Roche, P.C. Immunohistochemical stains for breast cancer. Mayo Clin.
paraffin. If this is the case, repeat deparaffinization procedure. Proc. 69: 57-58, 1994.
Alternatively, high levels of endogenous biotin may be present. Pre- 24. Sheehan, D.C., and B.B. Hrapchak. Theory and Practice of
incubate tissue with biotin blocking reagents. (Endogenous Biotin Blocking Histotechnology. 1980. The C.V. Mosby Company, St. Louis.
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5. If tissue sections wash off slide, check to be sure slides are silanized or breast cancer. J. Clin. Oncol. 8: 1120-1128, 1989.
coated with polylysine or equivalent material. Refer to the Ventana
automated slide stainer Operator’s Manual for corrective action or contact INTELLECTUAL PROPERTY
Ventana Technical Consultation Center (800-227-2155). Liquid CoverslipTM, EZ PrepTM, and CONFIRMTM are trademarks of Ventana Medical
Systems, Inc.; ES®, NexES® IHC, BenchMark®, PATHWAY® and Ventana® are
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