Human Epidermal Growth Factor Receptor 2 (HER2) Testing - …nsh.org/sites/default/files/WEB1214 Supplement Handout.pdf · 2014-12-12 · J. Weaver MAOM, HTL (ASCP), QIHC-NSH Her2

J. Weaver MAOM, HTL (ASCP), QIHC-NSH Her2 Handout

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Human Epidermal Growth Factor Receptor 2 (HER2) Testing - Validation, Application and Correlation

Her2 is encoded by the C-erbB2 gene and is one of four oncoproteins belonging to the Human Epidermal Growth Factor Receptor

(HER1-4) family of tyrosine kinases and is over-expressed in approximately 20% of invasive breast cancer cases. Over-expression of

these pathways leads to excessive activation and may contribute to more aggressive growth associated with these tumors. Her2

over-expression is associated with poor prognosis and resistance to certain chemotherapeutic agents.

Milestones in Her 2 Testing

FDA CLASSFIES IHC

ASCO/CAP, INTRO. GUIDELINES FOR HER2

DISTRIBTION OF CAP TESTING

QUESTIONAIRE

PUBLICATION OF QUESTIONAIRE

RESULTS

1998 2006 2007 2008 2009 2010 2013

PUBLICATION OF HER 2 GUIDELINES

ADDITION OF ANP.22750, 22760 REVISED ASCO/CAP HER2

GUIDELINES


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Optimization-Validation Method –Ventana Pathway® Controls ( cell line & tissue sections)

The performance of the PATHWAY HER2 (4B5) Rabbit

monoclonal Primary Antibody has been evaluated by

Ventana, through specificity, reproducibility and method

comparison studies

Deparaffinization (selected)

20 min. CC1

30 min CC1

52 min CC1

64 min CC1

Primary AB- 12 min incubation

Hematoxylin II counterstain 4

min

Bluing 4 minutes

Positive control for PATHWAY HER2 (4B5) is a known weak

HER-2/neu positive invasive breast carcinoma (for example

ductal or lobular).

PATHWAY HER2 (4B5) specificity was determined by a study

that showed no specific membrane staining for most normal

tissues.

Lot controlled cell line at all expression levels , lot controlled

Positive and negative agreement rates with two-sided score

95% confidence intervals were calculated for the six possible

pair-wise comparisons

Same slide used for the positive tissue control (ductal or

lobular invasive breast carcinoma) is the negative tissue

control. The non-staining components (surrounding stroma,

lymphoid cells and blood vessels) should demonstrate absence

of specific staining , or normal breast

Intra-run reproducibility of staining on the NexES,

BenchMark, and BenchMark XT staining instrument

platforms was determined by staining three slides each of

five breast cancer tissues with a score of 0, 1+, 2+, and 3+

HER-2 expression

Negative reagent control is used in place of the primary

antibody to evaluate nonspecific staining. The slide should be

stained with CONFIRM Negative Control Rabbit Ig. The

incubation period for the negative reagent control should

equal the primary antibody incubation period


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Optimization-Validation Method –Leica Oracle® Controls ( cell line & tissue sections)

The performance of the Oracle Primary Antibody ( CB11) has

been evaluated by Leica, through specificity, reproducibility

and method comparison studies. Staining patterns were

verified by Leica, using a large panel of normal tissues. The

Oracle system was developed to provide an alternative to the

investigational clinical trial assay (CTA) used in the Herceptin®

clinical studies. Oracle was compared to Hercep test using 431

breast tumor specimens, with 92.34% concordance.

Clone CB11 ( mouse

monoclonal) targets the Her2

oncoprotein located on the

cell membrane using the

primary antibody which is

visualized via a compact

polymer detection system.

The system has a preloaded

protocol, with HIER 25 min

with ER1(97), and IHC staining

protocol H.

A new bond cover tile should

be used with each Her2

stained slide.

60 tests ( 150 slides)

60 slides with Her2 AB

60 slides with negative reagent

15 Her2 control slides with AB

15 positive in house tissue

controls

Negative control is a ready to use mouse IgG at equivalent

concentration to the primary AB.

Oracle HER2 (CB11) specificity was determined by a study that

showed no specific membrane staining for most normal

tissues.

Lot controlled cell line at all expression levels, lot controlled.

Each lot contains 4 NBF fixed human breast cancer cell lines,

which express all staining intensities.

The slide layout provided in the IFU should be used to get the

maximum tests from each kit.

In house positive tissue controls are used and indicative of

correctly prepared tissues, ideal is a weak positive.

In house negative controls verify the specificity of the primary

antibody. Normal breast ducts unassociated with tumor act as

an internal reference.

Use of a MTB block is highly recommended.

Between laboratories reproducibility was evaluated by Leica at

three sites. Inter-observer reproducibility was evaluated using

40 randomly selected invasive breast cancer cases, agreement

87.5%.

Slide arrangement

1-Her2 control slide

2-iIn house positive control

3-In house negative control

4-Patient tissue-Her2 negative control

5-Patient tissue-Her2 primary antibody


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Validation Definitions

In Vitro Diagnostic Product (IVD)FDA Definitions

“…reagents…intended for use in diagnosis of disease…(in) specimens taken from the human body.”

– For immunohistochemistry: Class I: “…provide the pathologist with adjunctive diagnostic information…”“…minimal

potential for harm to the user.”

– Class II: “…provide prognostic or predictive data…”“…mandatory performance standards…”

Analyte Specific Reagents (ASRs)

Non-IVD diagnostic reagent

Not bundled with other materials in a kit

Not subject to FDA preclearance or special controls

Product literature makes no claims regarding use or performance

Class II IHC Tests-(results used by non-pathologists)

Stand alone diagnostic, Predictive or prognostic, Widely accepted valid scientific claims

E.g. hormone receptors in breast cancer

FDA vs. Non-FDA Approved Systems

FDA approved: May use data from manufacturer or from published reports, but MUST verify Sensitivity, Specificity

/Accuracy, Precision, Reportable Range

Non-FDA approved: Must establish accuracy, precision, analytic sensitivity, interferences & reportable range

Includes modified FDA-approved systems!


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Table 1 Summary of ASCO/CAP Selected Guidelines

2007 Recommendation 2013 Recommendation

ISH Interpretation Count at least 20 cells, pathologist must confirm counting

involves invasive component

Bright field ISH, pathologist scans entire slides

prior to counting 20 cells. Comparison needed

between normal breast and tumor cells to

manage difficult to interpret areas.

Acceptable ISH and IHC

tests

Should preferentially use FDA approved IHC,

ISH or FISH test.

IHC rejection criteria Test rejected or repeated with FISH if; controls are not as

expected, artifacts involve most of the sample, sample has strong

membrane staining in normal breast ducts.

same

IHC interpretation criteria + Her2 results requires homogenous, dark , circumferential

pattern in >30% of invasive tumor ( chicken wire pattern)

Should interpret IHC using a threshold or

more than 10% of tumor cells that show

homogeneous, dark circumferential pattern to

be 3+ positive.

Reporting requirements Report must include guideline detailed elements Same, and changes to reporting requirements

and algorithms.

Source: Guideline Summary of Her2 Recommendations, American Society of Clinical Oncologists and College of American

Pathologists, 2013.


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Descriptions of Her2 Scoring Criteria:

• IHC result of 3+ cell surface protein expression (uniform intense membrane staining of > 30% of invasive tumor cells, or FISH

result of amplified Her2 gene copy number (average of > 6 copies/nucleus or Her2/CEP ratio of > 2.2).

• Equivocal Her2- IHC scores of 2+ (complete membrane staining that is either non-uniform or weak intensity in at least 10% of

cells), or FISH scores with Her2/CEP ratios of 1.8-2.2 or average gene copy numbers between 4.o-6.o.

• Note that patients with Her2/CEP ratios of 2.0-2.2 were formerly considered Her2+ and were eligible for Herceptin® ( 3% of

samples), data does not support excluding them at this time.

• Negative Her2- either an IHC result of 0 or 1+ for cellular protein membrane expression ( no staining or weak, incomplete

membrane staining in any proportion of tumor cells), or FISH result of Her2/CEP ratio less than 1.8 or average of fewer than 4

copies of Her2 gene.

• Equivocal results from a single test require additional action. Equivocal IHC must be confirmed by FISH. Equivocal FISH are

confirmed by enumerating additional cells or repeat assay.

• CISH amplification is defined as greater than 10 discrete copies per nucleus or as large gene copy clusters in >50% of nuclei.

Unaltered gene copy is 1-5 copies per nucleus (Tanner). SOURCE - SUMMARY of 2007 & 2013 RECOMMENDATIONS


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• IHC result of 3+ cell surface protein expression (uniform intense membrane staining of > 30% of invasive tumor cells, or FISH

result of amplified Her2 gene copy number ( average of > 6 copies/nucleus or Her2/CEP ratio of > 2.2).

• Equivocal Her2- IHC scores of 2+ (complete membrane staining that is either non-uniform or weak intensity in at least 10% of

cells), or FISH scores with Her2/CEP ratios of 1.8-2.2 or average gene copy numbers between 4.o-6.o.

• Note that patients with Her2/CEP ratios of 2.0-2.2 were formerly considered Her2+ and were eligible for Herceptin® ( 3% of

samples), data does not support excluding them at this time.

• Negative Her2- either an IHC result of 0 or 1+ for cellular protein membrane expression ( no staining or weak, incomplete

membrane staining in any proportion of tumor cells), or FISH result of Her2/CEP ratio less than 1.8 or average of fewer than 4

copies of Her2 gene.

• Equivocal results from a single test require additional action. Equivocal IHC must be confirmed by FISH. Equivocal FISH are

confirmed by enumerating additional cells or repeat assay.

• CISH amplification is defined as greater than 10 discrete copies per nucleus or as large gene copy clusters in >50% of nuclei.

Unaltered gene copy is 1-5 copies per nucleus ( Tanner). SEE HANDOUT-SUMMARY of 2007 & 2013 RECOMMENDATIONS


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EXAMPLE-DRAFT of VALIDATION SUMMARY:

“HER2 Antibody Optimization-Validation Summary”

Antibody: Her 2 Species: Rabbit Monoclonal

Clone: 4B5

Titer/Concentration: pre-diluted dispenser

Catalog #: 790-2991 Localization: membrane

Status & Intended use: This antibody is intended for in vitro diagnostic use. PATHWAY anti-HER-2/neu is a rabbit monoclonal antibody (clone 4B5) directed against the internal domain of the c-erbB-2 oncoprotein (HER2). c-erbB-2 oncoprotein was cloned and characterized by Akiyama, et al in 1986. It is an approximately 185 kD transmembrane glycoprotein which is structurally similar to epidermal growth factor receptor (EGFR). The protein is associated with tyrosine kinase activity similar to that of several growth factor receptors, and to that of the transforming proteins of the src family. The coding sequence is consistent with an extracellular binding domain and an intracellular kinase domain. This suggests that HER2 may be involved in signal transduction and stimulation of mitogenic activity. Clone 4B5 has been shown to react with a 185 kD protein from SK-BR-3 cell lysates via Western blotting. SK-BR-3 is a breast carcinoma cell line, which has a 128-fold over expression of HER2 mRNA. The size of the band identified correlates well with that reported by Akiyama et al for HER2 protein (185 kD). Immunohistochemistry has been used to detect specific antigens in cells or tissue since 1950. The use of enzymes and peroxidase as markers for immunohistochemistry was reported by Nakane and Pierce in 1967. The HER2 protein is expressed at a level detectable by immunohistochemistry in up to 20 percent of adenocarcinomas from various sites. Between 15 and 30 percent of invasive ductal cancers are positive for HER2. Almost all cases of Paget’s disease of breast, and up to 90 percent of cases of ductal carcinoma in situ of comedo type are positive. The immunohistochemical detection of HER2 protein overexpression is also used as an aid in determination of patients for whom Herceptin therapy is indicated. Staining results in normal tissues, neoplastic tissues, and 322 cases of breast carcinoma with PATHWAY HER2 (4B5) were evaluated by Ventana.

Purpose:

Immunohistochemical staining is used to determine the presence or absence of the targeted antigen in formalin-fixed, paraffin embedded

tissue. The Ventana Medical Systems, Inc.'s (Ventana) PATHWAY anti-HER-2/neu (4B5) Rabbit Monoclonal Primary Antibody (PATHWAY HER2

(4B5)) is a rabbit monoclonal antibody intended for laboratory use for the semi-quantitative detection of HER2 antigen in sections of formalin-

fixed, paraffin-embedded normal and neoplastic tissue on a VENTANA automated immunohistochemistry slide staining device. It is indicated as

an aid in the assessment of breast cancer patients for whom Herceptin treatment is considered.


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Clinical Methods: PATHWAY HER2 (4B5) is a rabbit monoclonal antibody, which binds to HER2 in paraffin-embedded tissue sections. The specific antibody can be localized by a conjugated secondary antibody formulation that recognizes rabbit immunoglobulins followed by the addition of a secondary antibody-HRP conjugate (ultraView Universal DAB Detection Kit). The specific antibody-enzyme complex is then visualized with a precipitating enzyme reaction product. Each step is incubated for a precise time and temperature. At the end of each incubation step, the VENTANA automated slide stainer washes the sections to stop the reaction and to remove unbound material that would hinder the desired reaction in subsequent steps. It also applies Liquid Coverslip, which minimizes evaporation of the aqueous reagents from the specimen slide. Clinical cases should be evaluated within the context of the performance of appropriate controls. A positive tissue control fixed and processed in the same manner as the patient specimen (for example, a weakly positive breast carcinoma) is used in addition to staining with PATHWAY HER2 (4B5), along with a slide stained with CONFIRM Negative Control Rabbit Ig. For the test to be considered valid, the positive control tissue should exhibit membrane staining of the tumor cells. These components should be negative when stained with CONFIRM Negative Control Rabbit Ig. In addition, it is recommended that a negative tissue control slide (for example, a HER-2/neu negative breast carcinoma) be included for every batch of samples processed and run on the VENTANA automated slide stainer. This negative tissue control should be stained with PATHWAY HER2 (4B5) to ensure that the antigen enhancement and other pretreatment procedures did not create false positive staining.

Required Reagents & Materials

Catalog #790-2991: PATHWAY anti-HER-2/neu (4B5) Primary Antibody contains sufficient reagent for 50 tests.

One 5 mL dispenser PATHWAY anti-HER-2/neu (4B5) Primary Antibody contains approximately 30 μg of a rabbit monoclonal antibody directed against human c-erbB-2 antigen. 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 serum, approximately 0.25 %, present from the stock solution. Total protein concentration of the reagent is approximately 16 mg/mL. Specific antibody concentration is approximately 6 μg/mL. PATHWAY anti-HER-2/neu (4B5) Primary Antibody is a rabbit IgG diluted from tissue culture supernatants. There is no known irrelevant antibody reactivity observed in this product.

Reconstitution, Mixing, Dilution, and Titration: This antibody is optimized for use on a VENTANA automated slide stainer in combination with VENTANA iVIEW DAB Detection Kit and compatible with ultraView Universal DAB Detection Kit. No reconstitution, mixing, dilution, or titration is required. The reagent is stored at 4-8 degrees Celsius when not in use.

CONFIRM Negative Control Rabbit Ig (Cat. No. 760-1029) (negative reagent control). The reagent is stored at 4-8 degrees Celsius when not in use.

Microscope slides, such as Superfrost Plus

Positive and negative tissue controls (invasive breast carcinoma and normal breast tissue)

Bar code labels (appropriate for negative reagent control and primary antibody being tested)

Xylene (histological grade)

Ethanol or reagent alcohol (histological grade)


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100% solution: undiluted ethanol or reagent alcohol, 95% solution: mix 95 parts ethanol or reagent alcohol with 5 parts of deionized water, 80% solution: mix 80 parts ethanol or reagent alcohol with 20 parts deionized water

Deionized or distilled water

Clinical Utility: Breast cancer is the most common carcinoma occurring in women, and the second leading cause of cancer related death. In North America, a woman’s chance of contracting breast cancer is one in eight. Early detection and appropriate treatment therapies can significantly affect overall survival. Small tissue samples may be easily used in routine immunohistochemistry (IHC), making this technique, in combination with antibodies that detect antigens important for carcinoma interpretation, an effective tool for the pathologist in their diagnosis and prognosis of disease. One important marker in breast cancer today is c-erbB-2 oncoprotein (HER2). HER2 is an intracellular membrane protein detected in the cellular membrane. It is closely related to EGFR and, like EGFR, has tyrosine kinase activity. Gene amplification and the corresponding overexpression of c-erbB-2 has been found in a variety of tumors, including breast carcinomas. The therapeutic drug Herceptin has been shown to benefit some breast carcinoma patients by arresting, and in some cases reversing the growth of their cancer. The drug is a humanized monoclonal antibody that binds to HER2 protein on cancer cells. Thus only patients with HER-2/neu positive breast carcinomas should benefit from treatment with Herceptin. In vitro diagnostics for the determination of HER2 status in breast carcinomas are important to aid the clinician in determination of therapy with Herceptin. Interpretation of the results of any detection system for HER2 must take into consideration the fact that HER2 is expressed in both breast cancer tumors and healthy tissue, albeit at differing levels and with different patterns of expression. Histological tissue preparations have the advantage of intact tissue morphology to aid in the interpretation of the HER2 positivity of the sample. All histological tests should be interpreted by a specialist in breast cancer morphology, and/or pathology, and the results should be complemented by morphological studies and proper controls and used in conjunction with other clinical and laboratory data.

Specimen Type:

The Her2 staining protocol has been optimized by Ventana on site by a technical staining specialist, and the in house performance characteristics

have been verified and validated for use on non-decalcified, formalin-fixed, paraffin embedded tissue sections prepared in house, under current

testing conditions, with tissue section prepared by following standard histologic procedures as described in the routine histology procedure

manual.

Limits of Detection:

The limits of detection have been determined by multiple pre analytic and analytic factors, including the concentration of the antigen in the

tissue being examined. The limit of detection, sensitivity and specificity is evaluated by the Pathologist by examination of a panel of stained

tissue sections known to express or lack the target antigen.


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Parameters of Performance & Quality Control Procedures, using Cell Line System Controls & %NBF fixed Tissues Ventana provided, separate lot controlled slides consisting of four formalin-fixed cell line controls embedded in paraffin, sectioned and placed on a single charged slide (catalog # 781-2991). PATHWAY HER-2 4 in 1 Control Slides are used for a preliminary validation of the processing method used for staining slides with PATHWAY HER2 (4B5), and for performance controls across staining runs. These four cell line controls are characterized by in situ hybridization for gene copy number. When processed and stained appropriately, the cell lines should stain as described in the PATHWAY Her-2 4 in 1 Control Slide package insert. If the indicated staining is not evident in the appropriate cores, especially the 1+ and 2+ controls, the staining of the tissues should be repeated. Table 1. Characteristics of PATHWAY HER-2 4 in 1 Control Slides HER2

IHC Score

Cell Line HER2/Chr17 Ratio*

0 MDA-MB-231 1.11

1+ T47D 1.12

2+ MDA-MB-453 2.66

3+ BT-474 5.53

Tissue Controls for Each Staining Run: When sufficient material is available, multiple tissue controls with known positive at all expression levels and tissues with known negative

expression are utilized for optimal run to run and lot to lot quality control. When sufficient material is not available, single tissue sections

reflecting positive and negative staining patterns are run together or in parallel as described within quality control procedures for tissue &

reagent performance control and control for lot to lot verification.

Positive Tissue Control A positive control tissue fixed and processed in the same manner as the patient specimens must be run for each set of test conditions and with every PATHWAY HER2 2012-03-16 4 / 11 14427US Rev F staining procedure performed. An example of a positive control for PATHWAY HER2 (4B5) is a known weak HER-2/neu positive invasive breast carcinoma (for example 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 functioned properly. A known weak HER-2/neu positive invasive breast carcinoma tissue may contain both positive and negative staining cells or tissue components and may serve as both the positive and negative control tissue. Known positive tissue controls should be utilized only for monitoring the correct performance of processed tissues and test reagents, and not as an aid in determining a specific diagnosis of patient samples.


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Negative Tissue Control The same slide used for the positive tissue control (ductal or lobular invasive breast carcinoma) may be used as the negative tissue control. The non-staining components (surrounding stroma, lymphoid cells and blood vessels) should demonstrate absence of specific staining and provide an indication of specific background staining with the primary antibody. Alternatively, normal breast tissue is an adequate negative control tissue. Use a tissue known to be fixed, processed and embedded in a manner identical to the patient sample(s) with each staining run to verify the specificity of PATHWAY HER2 (4B5) for demonstration of HER-2/neu, and to provide an indication of specific background staining (false positive staining). Negative Reagent Control A negative reagent control must be run for every specimen to aid in the interpretation of results. A negative reagent control is used in place of the primary antibody to evaluate nonspecific staining. The slide should be stained with CONFIRM Negative Control Rabbit Ig. The incubation

period for the negative reagent control should equal the primary antibody incubation period. A negative reagent control is used in place of the primary antibody to evaluate nonspecific staining. The slide should be stained with CONFIRM Negative Control Rabbit Ig. The incubation period for the negative reagent control should equal the primary antibody incubation period. Records of assessment of individual staining results and pathologist interpretation are tabulated and retained with the validation staining run sheets and validation records. Single control tissue sections are identified for use in lot to lot assessments of new antibody lots of the same vendor and clone within immunohistochemistry quality control records. A minimum of one known positive and one known negative control is stained parallel to the previous lot before use of the new lot in patient testing. General Limitations & Interferences Immunohistochemistry is a multi-step process that requires specialized training in the selection and use of reagents, protocol variables, tissue

selection, fixation and processing requirements. Staining results achieved are highly dependent of the handling and processing of tissue prior to

immunohistochemistry staining. Improper fixation, freezing, thawing, washing, drying, heating, sectioning or contamination from other tissue or

fluids is likely to produce artifacts, aberrant staining, or false negative results. The clinical interpretation of immunohistochemical staining is

performed only by highly trained and experienced pathologists who evaluate these results in the context of the patient’s tissue morphology,

clinical history, appropriate controls and other diagnostic information.


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Specific Limitations 1. Immunohistochemistry is a multiple step diagnostic process that requires specialized training in the selection of the appropriate reagents, tissue selections, fixation, processing, preparation of the immunohistochemistry slide, and interpretation of the staining results. 2. Tissue staining is dependent on the handling and processing of the tissue prior to staining. Improper fixation, freezing, thawing, washing, drying, heating, sectioning, or contamination with other tissues or fluids may produce artifacts, antibody trapping, or false negative results. Inconsistent results may result from variations in fixation and embedding methods, or from inherent irregularities within the tissue. 3. Excessive or incomplete counterstaining may compromise proper interpretation of results. 4. The clinical interpretation of any positive staining, or its absence, must be evaluated within the context of clinical history, morphology and other histopathological criteria. The clinical interpretation of any staining, or its absence, must be complemented by morphological studies and proper controls as well as other diagnostic tests. It is the 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 certified licensed laboratory under the supervision of a pathologist who is responsible for reviewing the stained slides and assuring the adequacy of positive and negative controls. 5. Ventana provides antibodies and reagents at optimal dilution for use when the provided instructions are followed. Any deviation from recommended test procedures may invalidate expected results. Appropriate controls must be employed and documented. Users who deviate from recommended test procedures must accept responsibility for interpretation of patient results. 6. This product is not intended for use in flow cytometry, performance characteristics have not been determined. 7. Reagents may demonstrate unexpected reactions in previously untested tissues. The possibility of unexpected reactions even in tested tissue groups cannot be completely eliminated because of biological variability of antigen expression in neoplasms, or other pathological tissues.


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8. Tissues from persons infected with hepatitis B virus and containing hepatitis B surface antigen (HBsAg) may exhibit nonspecific staining with horseradish peroxidase. 9. False positive results may be seen because of non-immunological binding of proteins or substrate reaction products. They may also be caused by pseudoperoxidase activity (erythrocytes), endogenous peroxidase activity (cytochrome C), or endogenous biotin (example: liver, brain, breast, kidney) depending on the type of immunostain used. 10. As with any immunohistochemistry test, a negative result means that the antigen was not detected, not that the antigen was absent in the cells or tissue assayed.

Specific Limitations of the Pathway System

The antibody has been optimized as indicated for VENTANA platforms and detection chemistries. Because of variation in tissue fixation and processing, it may be necessary to increase or decrease the primary antibody incubation time on individual specimens.

The antibody, in combination with VENTANA detection kits and accessories, detects antigen that survives routine formalin fixation, tissue processing and sectioning. Immunohistochemical testing has been validated for use with 10% neutral buffered formalin-fixed tissues. Cold ischemic and fixation times are recorded for each breast tissue sample on which Her2 testing is performed and recorded within the final pathology report.

Bone marrow was not tested for specificity.

Interpretation of results The VENTANA automated immunostaining procedure causes a brown colored (DAB) reaction product to precipitate at the antigen sites localized

by PATHWAY HER2 (4B5). A qualified pathologist experienced in immunohistochemical procedures must evaluate controls and qualify the

stained product before interpreting results.

Scoring Conventions for the Interpretation of PATHWAY HER2 (4B5) Breast carcinomas that are considered positive for HER-2 protein overexpression must meet threshold criteria for intensity of staining (2+ or greater on a scale of 0 to 3+) and percent positive tumor cells (greater than 10%). Staining must also localize to the cellular membrane. Cytoplasmic staining may still be present, but this staining is not included in the determination of positivity. Three fields within the well


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preserved and well stained region of the tissue should be examined for intensity of staining and determination of completeness of the cytoplasmic membrane stain. Staining that completely encircles the cytoplasmic membrane should be scored as an intensity of “2+” or “3+”. Partial staining of the membrane should be scored as a “1+”. It may be necessary to examine borderline cases at 400X or higher magnification to discriminate between intensities of “1+” and “2+”. In contrast to cases scored as an intensity of 3+, the staining scored as 2+ has a crisper and more clearly delineated ring, while cases scored as 3+ exhibits a very thick outline. Refer to VENTANA Interpretation Guide for PATHWAY HER-2/neu (4B5) for a more detailed description with photographs of staining with PATHWAY HER2 (4B5).

Table 2. Criteria for Intensity and Pattern of Cell Membrane Staining with PATHWAY HER2 (4B5)

Staining Pattern

Score (Report to

Treating Physician)

HER2 Staining Assessment

No membrane staining is

observed

0

Negative

Faint, partial staining of the

membrane in any proportion of

the cancer cells

1+

Negative

Weak complete staining of the

membrane, greater than 10% of

cancer cells

2+

Weakly Positive

Intense complete staining of the

membrane, greater than 10% of

cancer cells

3+

Positive

Ventana –Published Concordance

The performance of the PATHWAY HER2 (4B5) Primary Antibody has been evaluated by Ventana, through specificity, reproducibility and method

comparison studies.

Specificity: PATHWAY HER2 (4B5) specificity was determined by a study that showed no specific membrane staining for most normal tissues. Staining results were as follows: adrenal (0/3), breast (0/3), cerebellum (0/3), cerebrum (0/3), cervix (0/3), colon (0/3), esophagus (1/3), heart (0/2), kidney (0/3), liver (0/3), lung (0/3), mesothelial cells (0/3), ovary (0/3), pancreas (0/3), parathyroid (1/3, focal membrane staining),


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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), testis (0/3), thymus (0/2), thyroid (0/3), tonsil (2/3 focal staining of surface epithelial cells), and uterus (0/3). PATHWAY HER2 (4B5) specificity was also determined by a study that showed no specific membrane staining in most neoplastic tissues. Staining results were as follows: breast cancer (1/4), carcinoid (0/2), colon cancer (1/3), hepatocellular cancer (0/5), leiomyoma (0/2), lung cancer (0/2), lymphoma (0/3), melanoma (0/2), ovarian cancer (1/2), pancreatic cancer (0/3), prostate cancer (0/3), renal cell cancer (0/5), sarcoma (0/2), stomach cancer (0/3), thyroid cancer (0/3), and undifferentiated cancer (0/1). Positive staining in tonsilar epithelieum, esophageal epithelium, prostate, peripheral nerve, parathyroid, breast cancer, colon, and ovarian cancer are consistent with published literature regarding expression of HER-2/neu. Sensitivity: Sensitivity is dependent upon the preservation of the antigen. Any improper tissue handling during fixation, sectioning, embedding or storage which alters antigenicity weakens HER-2/neu protein detection by PATHWAY HER2 (4B5) and may generate false negative results. Intra-run reproducibility of staining on the NexES, BenchMark, and BenchMark XT staining instrument platforms was determined by staining three slides each of five breast cancer tissues with a score of 0, 1+, 2+, and 3+ HER-2 expression. For each case, three of 3 slides stained appropriately within a run and for all instrument platforms tested. Users verify within run reproducibility results by staining several sets of serial sections with low, medium and high antigen density in a single run. Inter-run and inter-platform reproducibility of staining was determined by staining three slides each of five breast cancer tissues with scores of 0, 1+, 2+, and 3+ HER-2 expression on three different instrument runs across the NexES, BenchMark, and BenchMark XT instrument platforms. For each case, nine of 9 slides stained appropriately over three instrument runs and across all instrument platforms tested. Users should verify between run reproducibility results by staining several sets of serial sections with low, medium and high antigen density on different days. Comparisons studies of three laboratories, from separate institutions in the United States, participated in the inter-laboratory reproducibility study. Cut slides of 40 neutral buffered formalin-fixed invasive breast carcinoma cases [10 each from each HER-2 binning category (0-1+, 2+, 3+)] and six (6) PATHWAY HER-2 4 in 1 Control Slides were shipped to each of the sites for staining on a VENTANA BenchMark XT automated slide staining device using the recommended staining protocol. Controls included the PATHWAY HER-2 4 in 1 Control Slides and a second slide of each case stained with negative Ig reagent. No sites experienced invalid runs, based upon the performance of the controls. The results were analyzed by Ventana. Thirty-four of forty (34/40) slides exhibited similar staining intensity across staining sites. Six samples (6/40 or 15%) varied by no more than 1 intensity level. Three (3/6) samples varied between 0 and 1+, which are both considered to be negative. Two samples (2/40 or 5%) varied between 2+ and 3+, and one sample (1/40) varied between 1+ and 2+.

In all of the 40 cases (100%), a minimum of 2 of 3 pathologists agreed.


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Lot-to-Lot reproducibility was determined by automated staining of 5 breast cancer tissues with scores of 0, 1+, 2+, and 3+ HER2 expression with 3 lots of PATHWAY HER2 (4B5). Stained tissues were scored on a 0 to 3+ scale by three qualified readers. There was 100% agreement between lots and readers for the 3 slides and 5 tissues stained. Comparison Studies to other clones were performed by review of PATHWAY HER2 (4B5) rabbit monoclonal antibody to PATHWAY HER-2 (CB11) mouse monoclonal antibody: Summary of Studies Performed. A method comparison study was conducted to examine the correlation of PATHWAY HER2 (4B5) to PATHWAY HER-2 (CB11) and PathVysion Her-2 FISH, both previously approved FDA diagnostic tests. Six investigators participated in the study. Two sets of three different investigators evaluated two independent cohorts (Cohort 1: n=144, Cohort 2: n=178) using known breast cancer cases stained with HER-2 CB11 and HER2 4B5. FISH data was obtained from patient history. A consensus score from the three readers for each antibody was created for each case to reduce intra-reader variability known to exist with HER-2 scoring.22,23,24 A total of 322 cases were evaluated. The slides stained with PATHWAY HER-2 (CB11) were processed and stained according to the manufacturer’s instructions specified in the VENTANA CB11 package insert. Inter-pathologist Reproducibility of Detection Comparison Study Specimens: Positive and negative agreement rates with two-sided score 95% confidence intervals were calculated for the six possible pair-wise comparisons between readers for each method.

Inter-pathologist Reproducibility of Detection Comparison Study Specimens: Positive and negative agreement rates with two-sided score 95% confidence intervals were calculated for the six possible pairwise comparisons between readers for each method. Conclusion: Data from the above studies indicated that the PATHWAY HER2 (4B5) primary antibody was specific and reproducible in its ability to locate appropriate membrane staining for normal and neoplastic tissues. The method comparison data demonstrated that PATHWAY HER2 (4B5) primary antibody is indicated as an aid in the assessment of breast cancer patients for whom Herceptin treatment is considered. Complete statistical data on comparison studies are contained within the Pathway Her2 specifications sheet.


18

Table 3. Verification-Validation of PATHWAY HER2 (4B5), Breast Tissue Case # DOB Sex Tissue Pathology Fixation % Tumor Score

S14-1361 3-27-1939 F Invasive mammary CA with lobular features 28 hours

S14-1531 10-18-1945 F Invasive ductal carcinoma 8 hours

S14-1390 12-28-1942 F Invasive poorly differentiated ductal 6.5 hours

S14-1450 8-26-1948 F Invasive moderately differentiated ductal 29 hours

S14-1728 2-23-1956 F Invasive poorly differentiated ductal 9.5 hours


S14-2098 12-28-1942 F Poorly differentiated ductal carcinoma 6 hours

S14-2153 3-21-1932 F Invasive moderately ductal carcinoma 7 hours

S14-2294 10-22-1974 F Invasive ductal adenocarcinoma 27 hours

S14-4879 8-28-1958 F Invasive mammary carcinoma 8 hours


S14-4276 5-3-1935 F Invasive ductal adenocarcinoma 27 hours

S14-4282 6-30-1956 F Invasive mammary carcinoma 25.5 hours


S14-4624 9-27-1937 F Invasive ductal with extensive intraductal component 18 hours

S14-4681 2-18-1961 F Invasive ductal carcinoma with lobular features 48 hours


S14-00014 10-29-1958 F Invasive ductal Adenocarcinoma and ductal in situ 7.5 hours



S14-00748 9-27-1954 F Poorly differentiated metastatic carcinoma 28 hours

S14-01216 5-17-1957 F Invasive poorly differentiated ductal 30 hours





S14-02661 6-22-1936 F Invasive ductal carcinoma 27.5 hours

S14-04025 4-23-1931 F Invasive ductal carcinoma UK

S14-04242 11-13-1957 F Invasive mucinous carcinoma 3 hours

S14-08466 7-1-1941 F Invasive ductal 27 hours


S14-09150 5-18-1960 F Invasive well differentiated ductal carcinoma 9 hours


S14-09570 2-14-1929 F Invasive solid duct carcinoma, with associated ductal carcinoma in situ 58 hours

S14-00376 9-1-1954 F Infiltrating ductal carcinoma UK


Dates of slide staining of individual slides for inter-run reproducibility comparison are shown in the Ultra run reports. Reagent lots for each run are also

recorded with the stain run reports.


19

Concordance

Summary Statement:

The optimization and validation results for the PATHWAY HER2 (4B5) have been reviewed. It has been determined that the performance of the

assay under our laboratory conditions, reliably displays the expected pattern of reactivity in normal and diseased tissues consistent with its

intended use. It is determined to be suitable for patient testing.

Approved: _____________________________________ Date: ________________________

Diagnostic % specificity = 100 x the number of diseased patients with a negative test (divided by) the total number of patients without the disease Diagnostic % sensitivity = 100 x the number of diseased patients with a positive test (divided by) the total number of patients tested Precision is assessed by repeated staining of same positive/negative controls


20

DRAFT EXAMPLE OF IHC STAINING PROCEDURE, LEICA ORACLE STAINING SYSTEM®

Title: Bond Oracle HER2 IHC Staining System Author: Joelle Weaver MAOM, HTL (ASCP) QIHC

Date 5/27/2014

Principle

Immunohistochemical staining techniques are used to demonstrate the presence of antigens in cells and tissues. Immunohistochemistry is a

multi-step procedure based on the attractive forces between antibodies and antigens producing visualization using ordinary light microscopy.

Immunohistochemistry is widely utilized within surgical pathology and serves as an adjunctive and diagnostic clinical tool.

Purpose

This document outlines the procedure for Bond Oracle Her2 IHC staining system, product code TA9145, for use in the staining of 60 tests (150

slides). The Bond Staining run can be completed in approximately 3 hours 20 minutes excluding tissue section-slide preparation time.

Description and Intended Use

The Bond Oracle Her2 staining system is a semi-quantitative immunohistochemical assay to determine Her2 (human epidermal growth factor

receptor 2) Oncoprotein status in breast cancer tissues processed for examination by a pathologist. The results of the Bond Oracle system are

indicated as an aid in the assessment of patients for whom Herceptin® (Trastuzmab) treatment is being considered. The Bond Oracle system

contains the mouse monoclonal anti-Her2 antibody, clone CB11. Clone CB11 (Novocastra) is directed against the internal domain of the Her2

Oncoprotein. The Her2 Oncoprotein is expressed at levels detectable by immunohistochemistry in up to 20% of adenocarcinomas from various

sites.

Software Upgrade

Use of the Oracle staining system requires software version 5.0 (BDD 51, software 5.0, CPV # 21.201.A, service tag # 555D441), of the Bond

operating system. A Software upgrade has been performed by a Leica certified engineer with assistance from a Leica antibody staining specialist

on 4/21/14, Leica instrument # M21187. Migration of staining protocols, inventory and other stored database information has been verified in a

validation-performance test run using the validated CD3 stain protocol and ( 15) suitable tonsil tissue sections. Results of the test run were

satisfactory with no staining errors, software errors and all slides staining appropriately. Records of the upgrade verification and test slides are

retained in the validation records located on shelf 2 I room # 306. Training of the technical staff on the software was completed during installation

by the Leica staining specialist.


21

Specimens

This procedure applies only to specimens fixed in neutral buffered formalin (10% NBF) when used for clinical-diagnostic use. Only validated stain

protocols are used for staining patient tissue sections. Tissue sections should be sectioned at 3-4 microns thickness and placed on appropriate

control slides. All control materials for use in immunohistochemical assays are prepared with FFPE tissues. Smears or other cell preparations

which have been fixed in 100% ethanol or methanol may be immunostained with tissue staining procedures, but the results obtained have not

been validated for clinical use.

Decalcified Tissue

When staining is performed on decalcified tissue samples (especially breast tissue immunohistochemistry for Er, Pr, and Her 2); or if Fluorescent

in-situ hybridization (FISH) or chromogenic in-situ hybridization (CISH) are performed on decalcified samples, the following disclaimer is included

in the pathology report. “This assay has not been validated on decalcified tissues. Results should be interpreted with caution given the likelihood

of false negativity on decalcified specimens”.

Safety

The chromogen, 3, 3’-Diaminobenzidine (DAB), is a known carcinogen and appropriate personal protective equipment is required when handling

this reagent. Sodium Azide is a commonly used preservation of antibodies in solution. It is a known carcinogen. Xylene is a strong solvent that

acts as a neurotoxin, avoid direct skin contact and use non-latex gloves when using this reagent. Use the expected care needed to safely handle

microtome blades. Dispose of used blades in the blade dispenser or sharp’s container. Broken slides must be disposed of in the sharp’s

container.

Regulation

Manufacturers of commercial immunohistochemistry assay reagents, kits, instruments and computer software are federally regulated with

requirements based on risk level classification. In Vitro Diagnostic use devices (IVD), which includes many manufactured products for use in

immunohistochemistry, are under the authority of 21CFR 864.1860. Many of the antibodies used by laboratories for immunohistochemistry may

also be classified as analyte specific reagents (ASR) by the Food & Drug Administration, (FDA). For Class I analyte-specific reagents (ASRs) the

patient report contain the federally regulated disclaimer; “This test was developed and its performance characteristics determined by this


22

laboratory. It has not been cleared or approved by the U.S. Food and Drug Administration”. The Bond Oracle Her2 system is approved by the

FDA for IVD use.

Dilution and Preparation

All reagents are formulated specifically for use with this assay and lot numbers are specific for each Bond Oracle Her2 staining system. No

changes are necessary, and changes from this manufacturer’s configuration will invalidate the assay. The pretreatment and staining protocol are

predetermined and cannot be altered by the user (IHC protocol H, ER1 for 25 minutes at 97 degrees Celsius).

The Bond Oracle staining system (TA9145) contains components required to complete immunohistochemistry on formalin-fixed, paraffin-

embedded tissue sections. The tissue sections are incubated in the RTU primary Her2 antibody (clone CB11) using the detection by compact

polymer technology. The enzymatic conversion of the subsequently added chromogen (DAB), results in the formation of a visible reaction

product at the antigenic sites. The results are interpreted by a qualified pathologist via light microscopy. Bond Polymer Refine Detection uses a

novel polymerization technology to prepare polymeric horse radish peroxidase linked antibody conjugates. This type of detection does not use

streptavidin and biotin, so non-specific staining is not produced from endogenous biotin.

Kit Components

The kit provides sufficient reagents to stain 150 slides (60 test slides, 60 negative controls, 15 vendor supplied control cell line slides, and 15 in

house positive tissue controls). The number of tests is based on the standard usage and provides for a maximum of 15 runs. Optimal usage is

obtained when at least 4 slides are stained simultaneously on each slide staining tray.


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Component Description

Her2 control slides ( 15) Sections of FFPE human breast cancer cell lines that demonstrate

Her2 expression at 0, 1+, 2+ and 3+.

Her2 primary antibody, 13.5ml RTU, affinity purified mouse monoclonal IgG antibody, clone CB11

Her2 negative control, 9ml RTU mouse IgG at a concentration equivalent to the concentration of

the Her2 primary

Peroxide block, 22.5ml 3-4% hydrogen peroxide

Post primary,22.5ml Rabbit-anti-mouse IgG

Polymer,22.5ml Poly-HRP goat, anti-rabbit IgG

DAB part 1, 2.25ml Chromogen in 0.1% hydrogen peroxide in stabilizer solution

DAB part 2, 22.5ml (x2) Diluted in 0.1% hydrogen peroxide ( v/v)

Hematoxylin, 22.5ml 0.1% Hematoxylin

All reagents supplied within the Oracle kit are formulated specifically for use with this assay and lot numbers are specific for each Bond Oracle

staining system. The kit components are not sold separately. No substitutions are made to any component or stain results are considered

invalid. The kit is stored at 2-8 degrees Celsius when not in use. Return to refrigeration immediately after use. Do not freeze.

Other Reagents and Required Materials

Immuno-staining instrumentation Leica Bond Max serial # M211887, Refine detection kit catalog # DS9800, primary antibody ( RTU or

concentrated) ,antigen retrieval solution 1 catalog # AR9961& antigen retrieval solution # 2 catalog # AR9640, Bond Dewax solution catalog #

AR92222, Bond Wash buffer catalog # AR9590 , xylene Stat lab # 8400-1 reagent absolute ethanol Stat lab # 6900-1, mounting media Cytoseal

XL American Mastertech # 822639, glass slides ( various vendors, must be positively charged) , glass cover slips, antibody diluent #AR 9352,

Scilogix calibrated pipettes, control tissue and/or blocks, distilled water, cover tiles # S21.2001 and slide racks for the Leica Bond Max

immunostainer.


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Storage Conditions for Reagents not within the Oracle Kit

Antibody Diluent AR9352, refrigerate at 2-8ᴏ C. Concentrated and ready-to-use primary antibodies refrigerate at 2-8ᴏ C. Refine detection DS9800,

Antigen retrieval solution 1 AR9661 & antigen retrieval solution 2 AR 9640, Bond Wash solution AR9590 for long term storage at 2-8ᴏ C. Bond

Dewax solution AR9222 is stored at room temperature. Reagent ethanol and xylene used with slide preparation is stored in flammable storage at

room temperature. Temperature fluctuations are monitored continuously during performance of technical procedures, and corrective measures

are taken as appropriate. Temperature and humidity are recorded at least once daily on the quality control logs. Corrective actions are made

when conditions fall outside of acceptable ranges. Corrective actions are recorded in the quality control records.

Refine Detection Leica DS9800 2-8 degrees Celsius when not is

use

Bond Wash Leica AR9590 2-8 degrees Celsius when not is

use

Bond Retrieval Solution 1 Leica AR9961 2-8 degrees Celsius when not is

use

Bond Retrieval Solution 2 Leica AR9640 2-8 degrees Celsius when not is

use

Bond Dewax Leica AR9222 Room temperature

All RTU and Concentrated

Antibodies

Individual order numbers for each (

see procedure and inventory)

2-8 degrees Celsius when not is

use


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Slide Layout

New Bond universal covertiles (product code S21.2001) are used with each slide stained by the Bond Oracle staining system. The slide layout

depicted below results in optimal performance and use of the Oracle Her2 system so that the full 60 tests are obtained.

Slide position Slide description Reagent Tissue type

1 Case 1 Her2 negative control Test




5 Case 1 Her2 primary antibody Test




9 Her2 control slide Her2 primary antibody Positive

10 In house tissue control Her2 primary antibody Positive


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Instructions for assigning cases using version 5.0 of the Bond software for staining with the Oracle system using the optimal slide layout depicted above

1. On the bond instrument, ensure that the bulk and hazardous waste containers have enough capacity to perform the needed staining runs.

2. Ensure there is adequate alcohol, distilled water, Bond Dewax, Bond epitope solution 1, and Bond wash in the bulk reagent containers to perform the staining runs.

3. Ensure that a clean Bond mixing station is installed.

4. Turn on the PC associated with the Bond advanced staining system and open and log on to the Bond software, username is “PGXLadmin”, and password is “PGXL123”.

5. For a new Bond Oracle Her2 system, scan the reagent tray barcodes with the handheld scanner to enter the system into the Bond reagent inventory.

6. Go to the slide set up screen and click “Add Case”.

7. Enter the details for the first case in the required fields.

8. Ensure the dispense volume is set to 150µl and the preparation protocol is “Dewax”, click “OK”. 9. With the case highlighted in the slide set up screen, click “Add slide”.

10. First, add the patient test slides. Ensure that the tissue type is set to Test tissue.

11. Confirm the dispense volume is set to 150µl and the preparation protocol is “Dewax”, click “OK”. 12. Select the staining mode values, Single and Oracle (do not click “Oracle control” for test slides). 13. Select the process “IHC”.


27

14. Select Her2 negative control from the marker list. The protocols tab defaults to the correct staining protocol, (IHC protocol H) and HIER protocol (HIER25 minutes with ER1 (97)).

15. Click “Add slide”. The negative control slide is created.

16. Still in the add slide dialog, select Her2 Primary Antibody from the marker list. Default protocols and all other settings stay the same.

17. Click “Add slide”, and the test slide is created.

18. Repeat the above steps for each test case/slide until all the slides have been created for a staining run.

19. Next, to create the Her2 control slide, add it to the last case or create a new case for control slides (depending on the number of slides to be stained on this run).

20. A Her2 control slide must be included in each staining run (each slide tray) to validate the assay. 21. In the “Add slide” dialog set the tissue type to Positive tissue. 22. Click Oracle control. 23. Select the lot number of the Her2 control slide in the Lot number list (drop down). The lot number is inscribed on each slide and also on

the slide mailer.

24. The Her2 control slides must come from the same lot as the lot from the Bond Oracle system. 25. Select Her2 primary antibody from the marker list. Retain dispense volume, staining mode, process and protocol settings.

26. Click “Add slide” to the Her2 control slide.

27. Add a positive in house tissue control slide. 28. Deselect Oracle control.


28

29. Select Her2 primary antibody from the marker list, Retain dispense volume, staining mode, process and protocol settings. The tissue type remains Positive tissue.

30. Click “Add slide”. This completes slide creation.

31. Print the slide labels. All Oracle slides are identified with “OC” on the slide label in addition to the other slide information and patient identifiers. The control slide label contains the Oracle Her2 lot number.

32. Label each slide carefully cross checking patient identification and slide identification.

33. Open the lids of all the Bond Oracle reagent components and load the reagent tray onto the Bond. 34. Place the slides in the layout order described previously to optimize reagent usage.

35. Apply new covertiles to each slide.

36. Load the slide tray onto the Bond and press the Load/Unload button.

37. Confirm that the slides have been correctly scanned, and that there are not alert messages present. 38. Verify bulk reagents and click the RUN (play) button on the system status screen.

39. Ensure that the tray indicator field displays “Proc (OK)” and that the batch number and finish time are displayed indicating the run has been accepted and initiated.

40. When the run is complete, (blinking green light), again press the Load/Unload button, allow the slide tray to lift completely, and remove the slides trays from the Bond.

41. Remove covertiles, rinse slides, dehydrate, clear and coverslip.


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Pretreatment and Antigen Retrieval

Slides are drained thoroughly of excess water before assembling for staining runs. The standard pretreatment for IHC and ISH slides is the pre-

programmed default “Bake and Dewax” pretreatment protocol. This slide pretreatment will be applied to all slides unless specifically deselected

by the user. The Bond Oracle staining system uses IHC staining protocol H, with epitope retrieval solution 1 for 25 minutes. The staining protocol

and antigen retrieval have been optimized and predetermined by Leica Microsystems for use on the Bond staining instrumentation. End users do

not edit or alter the Oracle staining protocol. The use of HIER pretreatment on formalin-fixed, paraffin embedded tissue restores epitopes which

have been modified by formalin fixation, allowing improved accessibility of the primary antibody to the epitope. Bond epitope retrieval solution

1 contains Citrate based buffer at an approximate pH of 5.9-6.1.

Detection

Bond Polymer Refine Detection uses a novel polymerization technology to prepare polymeric horse radish peroxidase linked antibody

conjugates. This type of detection does not use streptavidin and biotin, so non-specific staining is not produced from endogenous biotin.

Specimen Preparation and Slide Identity

The slide is hand labeled at the time of preparation. The procedure for slide preparation is described in the “Leica Bond Immunostaining

Procedure” HP-56.

A customized, reagent resistant is then applied over the hand labeling. The slide label for this facility has been programmed into the Bond

software. The PGXL default slide label contains the unique test of accession number assigned to the tissue sample, the patient’s name, the slide

identifier (unique number assigned in the stain run), the primary marker, the run date, the stain protocol, and enzyme or HIER applied. Bond

Oracle slide labels are designated with the inscription “OC”. An additional free text comment section is available for any additional information

desired.

Assay Verification

The staining performance of the staining system has been verified as to antibody staining specificity by testing on a series of in house prepared

and tissue samples of known Her2 positive and negative profiles. Parallel test slides have been concurrently run with appropriate control tissues

of human breast cancer, including commercially characterized controls, in house prepared samples, and tissue microarray (TMA) slides with

characterized immunoreactivity.


30

Optimization-Verification date: 4/25/14-Optimized Staining Protocol for Her2 Oncoprotein

Staining Protocol is “IHC Protocol H” with HIER using antigen retrieval 1 for 25 minutes incubation time.

1. Heat and Dewax

2. Peroxide block – 5 minutes at ambient temperature

3. Bond wash, 3 rinses at ambient temperature

4. Primary- 8 minutes

5. Polymer detection- 30 minutes at ambient temperature


7. Post primary-10minutes are ambient temperature


9. Polymer-10 minutes at ambient temperature


11. Deionized water

12. Mixed DAB Oracle – 10 minutes at ambient temperature

13. Deionized water rinse



16. Hematoxylin- 5 minutes


18. Bond wash rinse ( bluing)


20. Dehydrate, clear and coverslip ( off line)

ER1 lot 10084, Dewax lot 24961, control slide lot 25002


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Control Tissues

Differences in tissue fixation, processing and embedding may produce variability in results, which makes it necessary to include in house controls

in addition to the Her2 control slides provided by Leica Microsystems with the Oracle staining system. Control tissue used for in house controls is

biopsy or surgical specimens which have been formalin-fixed, processed in house and paraffin embedded using standard in house procedures in

the same manner as the patient test samples.

Her2 control slide-Her2 primary antibody

Each of the supplied Her2 control slides contains four formalin-fixed, paraffin-embedded human breast cancer cell line cores with staining

intensity scores of 0, 1+, 2+, and 3+. One control is included in each test run. The Her2 control slide verifies performance but not tissue

preparation steps.

In House Positive Control-Her2 Primary Antibody

In house positive controls are biopsy or surgical specimens fixed, processed, and embedded according to in house procedures as expediently as

possible to preserve antigenicity. Pre-cut controls are stored under refrigeration (2-8 degrees Celsius), and used within 6 weeks. Controls are

prepared in the same manner as patient test samples, and contain tissue elements which are weakly positive to detect changes in sensitivity.

Positive tissue controls assess proper tissue preparation and staining method. At least one positive control is included with each test run. If the

positive control fails to demonstrate appropriate staining reaction, the run and results obtained on patient test sections should be considered

invalid.

In House Negative Control-Her2 Primary Antibody

Apply the supplied Her2 negative control reagent in place of the Her2 primary antibody to a corresponding section for each patient test to

evaluate non-specific staining and provide for accurate interpretation of the specific Her2 staining at the antigenic site.

Patient Tissue- Her2 Primary Antibody

Positive staining intensity is assessed by a qualified pathologist within the context of any non-specific background staining.


32

Sample Description Primary AB Staining Her2 Negative control

Her2 control slide Supplied by Leica with the

Oracle system

Controls stain procedure, and

indicates validity of reagent

performance

Detection of non-

specific background

staining

In house positive

control tissue

Tissue with target antigen-

best if weak + to define

subtle changes in primary

AB sensitivity

Controls all steps of the assay.

Validates tissue preparation &

Bond Oracle stain

performance.

In house negative

control

Tissue expected to be

negative

Detection of non-specific

antibody cross-reactivity with

cells/tissue components.

Cell Line Data (Used in Oracle System Control Slides)

Cell line Bond Oracle Profile Her2 receptor

load/cell

Her2 Gene Amplification Status

Her2 copy number Her2; CEP17 Gene

ratio

SK-BR-3 3+ 4.3 x 105 13.35 3.55

MDA-MB-453 2+ 1.4 x 105 5.73 2.05

MDA-MB-175 1+ 6.3 x 104 3.33 1.20

MDA-MB-231 0 9.3 x 103 3.15 1.13

Her2 receptor load analysis assessed by flow cytometry, Her2 gene amplification status assessed by dual probe FISH.


33

Process Quality Control

Routine quality control measures are essential to the success of any immunohistochemical procedure. Performance of the assay is verified by

internal quality control and competency measurement, and externally by correlation and proficiency testing challenges. Key processing

variables that may adversely affect staining quality are listed below with their method of quality control.

Monitor Potential Effect Process Control

Temperatures of processors,

embedding centers, slide ovens,

water bath

Excessive heat may be

detrimental to antigenicity. High

heat at the water bath may

distort tissue morphology

Temperatures of all heated equipment are monitored and recorded

daily

pH of wash solutions Incorrect pH of solutions may

affect staining reactions.

HIER are pre-prepared, the pH of each new batch of wash buffer is

verified and recorded

Checklist for protocol, set up Incorrect protocol will invalidate

staining run

The instrument software does not allow edits to the Oracle staining

protocol, other stain protocols are set as “preferred” and can be

edited only by administrators

Reagent expirations Expiration is set when reagent is

scanned into the system.

The software will not allow expired reagents to be used.

Lot to lot variability Inconsistent, aberrant stain

results

Lot to lot verification and recording is performed with each new

reagent kit, detection kit or antibody lot

Fixation variables Fixation must be optimal for

preservation of tissue proteins

and antigen sites.

Fixation times are tracked and recorded ; included within the report. A

disclaimer is added when fixation cannot be verified or when it falls

outside of ASCO/CAP guidelines.

Figure 1Quality MGMT in Immunohistochemistry, page 96. Nakhleh


34

Interpretation of Staining

Only membrane staining pattern and intensity are evaluated by bright-field microscopy by an experience and qualified pathologist. Cytoplasmic

staining is considered as non-specific staining and it not included in the assessment of membrane stain intensity. Refer to HP-112 “Required

Scoring and Reporting Elements for Predictive Markers” for more in depth discussion of scoring and reporting procedures according to

ASCO/CAP guidelines.

Her2 Results by Immunohistochemistry

Result Criteria

Negative ( score 0) No staining, or incomplete, faint/barely perceptible

membrane staining in ≤ 10% of invasive tumor cells

Negative ( score 1+) Incomplete, faint/barely perceptible membrane

staining in ˃ 10% of invasive tumor cells

Equivocal ( score 2+) Incomplete and/or weak to moderate

circumferential membrane staining in ˃ 10% of

tumor cells or complete, intense, circumferential

membrane staining in ≤ 10% of invasive tumor cells

Positive ( score 3+) Complete, intense, circumferential membrane

staining in ˃ 10% of invasive tumor cells

For equivocal result, a reflex in situ hybridization test (same specimen) or new test (new specimen if available is initiated, using

immunohistochemistry or in situ hybridization).


35

FAQs (answers provided by CAP)

Q: Why were changes made to the Her2 guidelines?

A: Numerous papers were published which raised issues about the original recommendations. The update was created to address concerns,

continue to improve standardizartion and accuracy and bring together the recommendations of both ASCO & CAP.

Q: Do the guidelines for Her2 IHC apply to use in gastric cancer?

A: No, but you do need to separately validate.

Q: Is PT participation required for all sites who do Her2 testing?

A: Yes

Q: What PT materials does CAP offer?

A: Her2 by immunohistochemistry (HER2). Her2 by FISH (CYG), Her2 by Bright field (ISH2).

Q: If we previously validated our her2, do we need to revalidate them to be compliant with the new guidelines?

A: No, but the required number of challenges must have been performed and documentation must be retained.

References

ANP. 22969 Predictive Markers, Report Elements. College of American Pathologists, anatomic pathology checklist, 2013.

Ashok, M. Analysis of Her2 Testing in Breast Cancer; Disparities, cost effectiveness and patterns of care. Proquest Dissertion Thesis, 2009.

ANP. 23003 Receptor reporting. College of American Pathologists, anatomic pathology checklist, 2013.

Bond Oracle HER2 IHC Staining System-Instructions for Use. Leica Microsystems, 2014.

Clinical Laboratory Standards Institute, CLSI. Quality Assurance for Immunocytochemistry: Approved Guideline. CLSI document MM4-A- (ISBN 1-56238-396-5). CLSI, 940 West Valley Road, Suite 1400, Wayne, PA 19087-1898 USA, 1999.


36

Genetech; FDA submission, STN: 103792\5008\0. Trastuzumab, Herceptin®. Submission for final approval, April 2, 2001.

Hardy, L. et al. Immunohistochemistry Validation Procedures and Practices. Archives of Pathology and Laboratory Medicine, vol. 137, January

2013.

Hammone, et al. American Society for Clinical Oncology/College of American Pathologists, Guidelines Recommendations for

Immunohistochemical testing of Estrogen and Progesterone Receptors in Breast Cancer. Arch of Pathology and Laboratory Medicine, vol. 134,

June 2010.

Hartman. A. et al. Determination of Her2/Neu Status. Archives of Pathology and Laboratory Medicine, vol. 138, Apri 2014.

I-Tien Yeh, MD, Measuring HER-2 in Breast Cancer Immunohistochemistry, FISH, or ELISA?

Nakhleh, R. Quality Management in Anatomic Pathology-Promoting Patient Safety through Systems Improvement and Error Reduction. College

of American Pathologists, 2005.

Template for Reporting Results of Biomarker Testing of Specimens from Patients with Carcinoma of the Breast. The College of American Pathologists. December 2013. Summary of ASCO/CAP HER2 Guideline Recommendations-Optimal Algorithm for HER2 Testing. Wolf, Hammond, Schwartz, et al., American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. Arch Pathol Lab Med. 2007; 131: 18-43. Clinical Laboratory Standards Institute, (2011). Quality Assurance for Design Control and Implementationof Immunohistochemistry Assays-

Approved Guideline, 2nd Edition. American Society of Clinical Oncology. Update to Guidelines for Breast Cancer. Retrieved 4/2/13. http://www.cancer.net/publications-and-resources/what-know-ascos-guidelines/what-know-ascos-guideline-follow-care-breast-cancer College of American Pathologists, Prognostic Factors in Breast Cancer. http://www.thefreelibrary.com/Prognostic+Factors+in+Breast+Cancer.-a064972306 ,

http://www.cancer.net/publications-and-resources/what-know-ascos-guidelines/what-know-ascos-guideline-follow-care-breast-cancer

http://www.thefreelibrary.com/Prognostic+Factors+in+Breast+Cancer.-a064972306

http://www.thefreelibrary.com/Prognostic+Factors+in+Breast+Cancer.-a064972306%20,

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Human Epidermal Growth Factor Receptor 2 (HER2) Testing - …nsh.org/sites/default/files/WEB1214 Supplement Handout.pdf · 2014-12-12 · J. Weaver MAOM, HTL (ASCP), QIHC-NSH Her2