DAKOCYTOMATION ER/PR PHARMDX KIT

K042884 · Dakocytomation California, Inc. · MXZ · Feb 15, 2005 · Hematology

Device Facts

• Record ID: K042884
• Device Name: DAKOCYTOMATION ER/PR PHARMDX KIT
• Applicant: Dakocytomation California, Inc.
• Product Code: MXZ · Hematology
• Decision Date: Feb 15, 2005
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 864.1860
• Device Class: Class 2

Indications for Use

The DakoCytomation ER/PR pharmDx™ assay is an immunohistochemical (IHC) kit system to identify human estrogen receptor (ER) and human progesterone receptor (PR) expression in breast cancer tissues routinely processed and paraffin-embedded for histological evaluation. ER/PR pharmDx specifically detects the ER alpha protein as well as the PR located in the cell nucleus of ER and/or PR-expressing cells. ER/PR pharmDx is indicated as an aid in identifying patients eligible for treatment with antihormonal or aromatase inhibitor therapies, as well as an aid in the prognosis and management of breast cancer. The DakoCytomation Monoclonal Mouse Anti-Human Progesterone Receptor, clone PgR 1294, is intended for laboratory use as a semi-quantitative detection of progesterone receptor by light microscopy in routinely processed normal and pathological human paraffinembedded tissue. This antibody is indicated for use as an aid in the management, prognosis and prediction of outcome of breast cancer. The clinical interpretation of any positive staining or its absence should be complemented by morphological and histological studies with proper controls. Evaluations should be made within the context of the patient's clinical history and other diagnostic tests by a qualified individual.

Device Story

IHC kit system identifies ER and PR expression in formalin-fixed, paraffin-embedded breast tissue; utilizes monoclonal mouse anti-human ER cocktail (clones 1D5/ER-2-123) and PR antibody (clone PgR 1294). Two-step staining procedure uses dextran-based reagent linked to secondary goat anti-mouse antibodies and horseradish peroxidase; enzymatic conversion of chromogen creates visible reaction product at antigen site. Performed manually or on Dako Autostainer in clinical laboratories by pathologists. Results interpreted via light microscopy using Allred scoring method (PS + IS > 2 = positive). Assists clinicians in determining patient eligibility for endocrine therapies and predicting disease-free/overall survival.

Clinical Evidence

Bench testing only. Performance characteristics evaluated include specificity, sensitivity, reproducibility, and concordance. Concordance testing between the DakoCytomation ER/PR pharmDx kit and the reference Allred method demonstrated 99% concordance.

Technological Characteristics

Immunohistochemical (IHC) kit system; utilizes monoclonal mouse anti-human antibodies (Clone PgR 1294). Detection via immunoenzymatic staining for light microscopy visualization. Optimized for manual and automated staining platforms. Semi-quantitative detection of nuclear protein expression.

Indications for Use

Indicated for patients with breast cancer to identify eligibility for antihormonal or aromatase inhibitor therapies and as an aid in prognosis and management. Used on routinely processed, paraffin-embedded tissue samples.

Regulatory Classification

Identification

Immunohistochemistry test systems (IHC's) are in vitro diagnostic devices consisting of polyclonal or monoclonal antibodies labeled with directions for use and performance claims, which may be packaged with ancillary reagents in kits. Their intended use is to identify, by immunological techniques, antigens in tissues or cytologic specimens. Similar devices intended for use with flow cytometry devices are not considered IHC's.

Special Controls

(2) Class II (special control, guidance document: “FDA Guidance for Submission of Immunohistochemistry Applications to the FDA,” Center for Devices and Radiologic Health, 1998). These IHC's are intended for the detection and/or measurement of certain target analytes in order to provide prognostic or predictive data that are not directly confirmed by routine histopathologic internal and external control specimens. These IHC's provide the pathologist with information that is ordinarily reported as independent diagnostic information to the ordering clinician, and the claims associated with these data are widely accepted and supported by valid scientific evidence. Examples of class II IHC's are those intended for semiquantitative measurement of an analyte, such as hormone receptors in breast cancer.

Predicate Devices

• Ventana ER, clone 6F11 (K984567)
• DakoCytomation ER, clone 1D5 (K993957)
• Dako Cytomation PR, clone PgR 636 (K020023)

Submission Summary (Full Text)

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510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION
DECISION SUMMARY
DEVICE ONLY TEMPLATE

A. 510(k) Number:
k042884

B. Purpose for Submission:
Marketing this test in the U.S.

C. Analyte:
Estrogen and Progesterone Receptors on formalin-fixed paraffin-embedded breast cancer specimens

D. Type of Test:
Immunohistochemical test for the semi-quantitative detection of estrogen and progesterone receptors

E. Applicant:
DakoCytomation California, Inc.

F. Proprietary and Established Names:
DakoCytomation ER/PR pharmDx™ Kit
Monoclonal Mouse Anti-Human Progesterone Receptor, Clone: PgR 1294

G. Regulatory Information:
1. Regulation section:
21 CFR §864.1860 Immunohistochemistry reagents and kits
2. Classification:
Class II
3. Product Code:
MYA, MXZ
4. Panel:
Pathology 88

H. Intended Use:
1. Intended use(s):

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The DakoCytomation ER/PR pharmDx™ assay is a semi-quantitative immunohistochemical (IHC) kit system to identify estrogen receptor (ER) and progesterone receptor (PR) expression in normal and neoplastic tissues routinely processed and paraffin-embedded for histological evaluation. ER/PR pharmDx specifically detects the ER alpha protein as well as the PR protein located in the cell nucleus of ER and PR-expressing cells respectively.

The DakoCytomation Monoclonal Mouse Anti-Human Progesterone Receptor, clone PgR 1294, is intended for laboratory use as a semi-quantitative detection of progesterone receptor by light microscopy in routinely processed normal and pathological human paraffin-embedded tissue. This antibody is indicated for use as an aid in the management, prognosis and prediction of outcome of breast cancer.

2. Indication(s) for use:
ER/PR pharmDx is indicated as an aid in identifying patients eligible for treatment with anti-hormonal or aromatase inhibitor therapies, as well as an aid in the prognosis and management of breast cancer.

3. Special condition for use statement(s):
4. Special instrument Requirements:
The automated kit is for use with the Dako Autostainer.

I. Device Description:

The DakoCytomation ER/PR pharmDx™ assay is a semiquantitative immunohistochemical (IHC) kit system to identify estrogen receptor (ER) and progesterone receptor (PR) expression in normal and neoplastic tissues routinely processed and paraffin-embedded for histological expressing cells respectively. ER/PR pharmDx assay is available in two configurations, both manual and automated and is optimized for use with DakoCytomation detection systems. The kit contains enough materials to perform a maximum of 50 tests and 10 individual staining runs. It consists of the materials listed below:

DakoCytomation Monoclonal Mouse Anti-Human Estrogen Receptor Cocktail, clones 1D5 and ER-2-123. The ER cocktail specifically binds to estrogen receptor antigen located in the nuclear region of a variety of normal and neoplastic tissues. It contains monoclonal mouse IgG1 and IgG2a anti-human antibodies to human ER in tissue culture supernatant in a Tris-HCL buffer, pH 7.2, containing stabilizing protein and 0.015 mol/L sodium azide as preservative.

DakoCytomation Monoclonal Mouse Anti-Human Progesterone Receptor, clone PgR 1294. The PR antibody specifically binds to progesterone receptor antigen located in the nuclear region of a variety of normal and neoplastic tissues. It contains monoclonal IgG1 anti-human antibodies to human PR in tissue culture supernatant in a Tris-HCL buffer, pH 7.2, containing stabilizing protein and 0.015 mol/L sodium azide as preservative. It will also be available as a concentrate.

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DakoCytomation Negative Control Reagent. It contains monoclonal mouse IgG1 and IgG2a antibody in tissue culture supernatant in a Tris-HCL buffer, containing stabilizing protein and 0.015 mol/L sodium azide.

Control Slides. Each slide contains sections of two pelleted, formalin-fixed, paraffin-embedded cell lines, which represent a moderate level of ER or PR protein expression (dependent on primary antibody applied to slide) and a negative cell line.

## J. Substantial Equivalence Information:

1. Predicate device name(s):
ER: Ventana ER, clone 6F11
DakoCytomation ER, clone 1D5
PR: Dako Cytomation PR, clone PgR 636

2. Predicate K number(s):
K984567
K993957
K020023

3. Comparison with predicate:

Estrogen Receptor

| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Intended Use | Semi-quantitative detection of estrogen receptor | Semi-quantitative detection of estrogen receptor |
| Antibody type | Monoclonal, mouse origin | Monoclonal, mouse origin |
| Technology | Immunohistochemistry | Immunohistochemistry |
| Tissue Type | Formalin-fixed, paraffin embedded breast tissue | Formalin-fixed, paraffin embedded breast tissue |
| Differences | | |
| Item | Device | Predicate |
| Clone | 1D5/ER-2-123 | 1D5 and 6F11 |
| Interpretation of results | Allred Method; 3-8 = positive (>1-10% positive staining cells) | Positive = > 11% positive staining cells |

Progesterone Receptor

| Item | Device | Predicate |
| --- | --- | --- |
| Intended Use | Semi-quantitative detection of progesterone receptor | Semi-quantitative detection of progesterone receptor |
| Antibody type | Monoclonal, mouse origin | Monoclonal, mouse origin |
| Technology | Immunohistochemistry | Immunohistochemistry |
| Tissue Type | Formalin-fixed, paraffin embedded breast tissue | Formalin-fixed, paraffin embedded breast tissue |
| Differences | | |

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| Item | Device | Predicate |
| --- | --- | --- |
| Clone | PgR 1294 | PgR 636 |
| Interpretation of results | Allred Method; 3-8 = positive (>1-10% positive staining cells) | Positive = > 11% positive staining cells |

K. Standard/Guidance Document Referenced (if applicable):
“In Vitro Diagnostic Devices: Guidance for the Preparation of 510(k) Submissions”
“Guidance for Industry: Guidance for Submission of Immunohistochemistry Applications to the FDA”

L. Test Principle:
The ER/PR pharmDx assay consists of a two-step immunohistochemistry staining procedure for routinely processed, paraffin-embedded specimens. Immunohistochemistry is a well established, widely accepted laboratory methodology. The assay detects the presence of Estrogen receptor (ER) and Progesterone receptor (PR) through first, the binding of an antibody to the antigen (ER or PR) of interest and second, visualization of the bound primary antibody through a reagent based on dextran technology. This reagent consists of both secondary goat anti-mouse antibody molecules and horseradish peroxidase molecules linked to a common dextran polymer backbone, thus eliminating the need for sequential application of link antibody and peroxidase conjugate. The enzymatic conversion of the subsequently added chromogen results in formation of a visible reaction product at the antigen site. The specimen is then counterstained and coverslipped. Results are interpreted using a light microscope. Control slides containing formalin-fixed, paraffin-embedded human cell lines are provided for quality control of the kit reagent performance.

M. Performance Characteristics (if/when applicable):
1. Analytical performance:
a. Precision/Reproducibility:
Three paraffin embedded tissues representative of antigen expression in breast carcinoma were selected for staining. Eight serial sections from each tissue were prepared for a total of 24 slides. A range of expression levels was utilized to demonstrate reproducibility across sensitivity levels as represented by staining intensity. Staining intensity should no vary more than one grade within one tissue across the different staining.
Intra-run Reproducibility
Nine randomized tissue slides from 3 different tissue blocks containing breast carcinoma were stained during one procedure with the ER/PR pharmDx assay. Staining intensity among the 3 slides from each block was evaluated. The staining pattern was reported as nuclear. Background was reported as 0 and the slides tested with the negative reagent control were reported to have 0 staining. Staining

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intensity was equivalent (within $\frac{1}{2}$ grade) between the repeated staining of any of the three slides for each antibody.

**Inter-run Reproducibility**

The staining intensity across three days using the same reagents and the same tissues was compared for the ER/PR pharmDx assay.

Testing was not performed on consecutive days. For each tissue staining intensity across the different days remained the same. The staining pattern was reported as nuclear, background was reported as 0, and the slides tested with the negative reagent control were reported to have 0 staining.

b. **Linearity/assay reportable range:**

N/A

c. **Traceability (controls, calibrators, or method):**

ER/PR pharmDx Control slides are supplied with the kit. Each of the control slides contains two pelleted, formalin-fixed, paraffin-embedded human cell lines, one that stains with ER and PR antibodies and one that does not stain. Two slides should be stained with each staining procedure, on with the anti-ER antibody cocktail and one with the anti-PR antibody. These slides are to be used to indicate the validity of the staining run. They should not be used as an aid in the interpretation of patient results. The pathologist is responsible for running additional positive and negative controls and assuring that the ER/PR pharmDx assay is performing properly.

d. **Detection limit:**

N/A

e. **Analytical specificity:**

A total of 90 formalin-fixed and paraffin-embedded tissues covering a wide range of normal human tissue types were tested with the ER/PR pharmDx assay. The antibodies demonstrated negative immunoreactivity with most tissues. Positive immunoreactivity was noted with some normal tissues which are typically positive, like uterus, ovary, and ductal epithelial cells of the breast.

f. **Assay cut-off:**

An Allred score of greater than 2 (corresponding to as few as $1\%$ to $10\%$ weakly positive cells) was used to define ER and PR positivity on the basis of a univariate cut-point analysis of all possible scores and disease-free survival (DFS) in patients receiving any adjuvant endocrine therapy. $^{1,2}$

2. **Comparison studies:**

a. **Method comparison with predicate device:**

Immunoreactivity of the assay was tested on 210 tissues (75% ER+ and 25% ER-) and 204 (64% PR + and 36% PR-) assembled in tissue arrays. Testing consisted of staining the specimens using the Allred procedure (anti-ER clone 6F11 and anti-PR clone PgR 1294) compared to the ER/PR pharmDx procedure and evaluation of both

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using the Allred scoring method. Slides for the Allred procedure were stained at Baylor College of Medicine, Houston, Tx. Slides for the ER/PR pharmDX assay were stained manually at DakoCytomation. Staining was assessed as a proportion score (PS) and an intensity score (IS) in the malignant tissue. Positive or negative status was determined based on the total Allred score (PS + IS > 2 = positive). Sensitivity of the ER test method was 98.75% while the specificity was 100%. Sensitivity of the PR test method was 98.46% while the specificity was 100%. Results, in 2 X 2 tables below, reveal concordance of the ER/PR pharmDx assay to the Allred method for positive/negative hormone receptor of 99% for each receptor.

2 x 2 table of DakoCytomation ER test results compared to Allred Procedure ER test result

| Allred Procedure ER Test Result | | | |
| --- | --- | --- | --- |
| DAKO Cytomation ER test result | Positive n | Negative n | Total N (%) |
| Positive | 158 | 0 | 158 (74.5%) |
| Negative | 2 | 52 | 54 (25.5) |
| Total | 160 | 52 | 212 |

2 x 2 table of DakoCytomation PR test results compared to Allred Procedure PR test result

| Allred Procedure PR Test Result | | | |
| --- | --- | --- | --- |
| DAKO Cytomation PR test result | Positive n | Negative n | Total N (%) |
| Positive | 128 | 0 | 128 (62.7%) |
| Negative | 2 | 74 | 54 (37.3) |
| Total | 130 | 74 | 204 |

Additional published studies compare the Allred procedure (anti-ER clone 6F11 and anti-PR clone PgR 1294) and Ligand Binding Assay

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(LBA), a well-established reference methodology and the capability of each method to predict disease free survival. $^{1,2}$ The ER study evaluated 1,982 primary breast cancers while the PR study evaluated 1,294 primary breast cancers. ER and PR positivity was scored by the Allred scoring method (0-8) and compared to LBA (with a cutoff positivity value of 3 and 5 fmol/mg for ER and PR, respectively). Each method was then compared to clinical outcome.

An Allred score of greater than 2 (corresponding to as few as 1% to 10% weakly positive cells) was used to define ER and PR positivity on the basis of a univariate cut-point analysis of all possible scores and disease-free survival (DFS) in patients receiving any adjuvant endocrine therapy. Using the Allred method, 70.5% of all tumors were determined to be ER positive compared with 78.9% by LBA. Also by the Allred method, 54% of tumors were determined to be PR positive compared to 56% by LBA. The concordance of the ER and PR to the LBA was 85.5% and 86%, respectively. Among patients who received adjuvant endocrine therapy, ER and PR was a strong predictor of both disease-free and overall survival. The best cutoff for both end points was a total score >2.

The concordance of anti-PR clone PgR 1294, anti-PR clone PgR 636, and Dextran Coated Charcoal (DCC) in 30 breast cancer tissues was also tested. Immunostaining was considered positive when at least 5% of the nuclei from three randomly selected fields a section were stained. Specimens were considered PR positive by the biochemical DCC assay when progestin binding values were ≥10 fmol/mg. Of the 30 specimens evaluated by IHC, 76.7% (23/30) were positive with clone PgR 1294. These results were 100% concordant with clone PgR 636. When clone PgR 1294 was compared to the DCC assay, concordance was 90% (27/30).

$^{1}$ Harvey JM, Clark, GM, Osborne CK, Allred DC. Estrogen receptor status by immunohistochemistry is superior to the ligand-binding assay for predicting response to adjuvant endocrine therapy in breast cancer. J Clin Oncol 1999; 17:1474
$^{2}$ Mohsin SK, Weiss H, Havighurst T, Clark GM, Berardo M, Roanh LD, To TV, Love RL, Allred DC. Progesterone receptor by immunohistochemistry and clinical outcome in breast cancer: a validation study. Mod Pathol 2004;17:1545

b. Matrix comparison:

3. Clinical studies:

a. Clinical sensitivity: N/A

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b. Clinical specificity:
N/A

c. Other clinical supportive data (when a and b are not applicable):
4. Clinical cut-off:
N/A
5. Expected values/Reference range:
N/A

N. Conclusion:
The submitted material in this premarket notification is complete and support a substantial equivalence decision.

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