MONOCLONAL MOUSE ANTI-HUMAN PROGESTERONE RECEPTOR, CLONE PGR 636, READY-TO-USE (LINK)

K130861 · Dako North America, Inc. · MXZ · Dec 9, 2013 · Hematology

Device Facts

• Record ID: K130861
• Device Name: MONOCLONAL MOUSE ANTI-HUMAN PROGESTERONE RECEPTOR, CLONE PGR 636, READY-TO-USE (LINK)
• Applicant: Dako North America, Inc.
• Product Code: MXZ · Hematology
• Decision Date: Dec 9, 2013
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 864.1860
• Device Class: Class 2

Indications for Use

For in vitro diagnostic use Flex Monoclonal Mouse Anti-Human Progesterone Receptor, Clone PgR 636, Ready-to-Use (LINK) is intended for use in immunohistochemistry with EnVision™ FLEX +, High pH visualization kit together with the Autostainer Link 48 instrument to semi quantitatively detect human progesterone receptor in formalin fixed, paraffin embedded human breast carcinoma. This antibody labels progesterone receptor positive cells and is useful in the assessment of progesterone receptor status in human breast carcinomas. The clinical interpretation of any staining or its absence should be complemented by morphological studies using proper controls and should be evaluated within the context of the patient's clinical history and other diagnostic tests by a qualified pathologist.

Device Story

Device is an immunohistochemistry (IHC) reagent kit using monoclonal mouse anti-human progesterone receptor (clone PgR 636). Input is FFPE breast carcinoma tissue sections. Process involves heat-induced epitope retrieval (HIER), incubation with primary antibody, and application of EnVision™ FLEX+ visualization system (HRP-linked) on the Autostainer Link 48 instrument. Enzymatic conversion of DAB+ chromogen creates visible reaction product at antigen sites. Output is stained slide for light microscopy. Used in clinical pathology labs; operated by trained laboratory personnel. Pathologists interpret staining (positive if ≥1% tumor nuclei stained) to determine PR status, informing breast cancer treatment decisions. Benefits include standardized, automated PR assessment.

Clinical Evidence

Clinical performance evaluated via concordance and reproducibility studies. Concordance study (n=236) compared PgR 636 to predicate, showing 94.5% overall agreement, 95.8% positive agreement, and 93.1% negative agreement. Reproducibility study involved 21 unique breast cancer specimens evaluated across three laboratories over five days (315 total evaluations), demonstrating high inter-laboratory and inter-instrument reproducibility.

Technological Characteristics

Monoclonal mouse anti-human antibody (Clone PgR 636) in liquid buffer with stabilizing protein and 0.015 mol/L sodium azide. Target concentration 0.5 ug/mL (range 0.4-0.6 ug/mL). Optimized for automated IHC staining on Dako Autostainer Link 48. Uses EnVision™ FLEX+, High pH visualization chemistry. Detects nuclear PR protein expression.

Indications for Use

Indicated for semi-quantitative detection of human progesterone receptor (PR) in formalin-fixed, paraffin-embedded (FFPE) human breast carcinoma tissue to assist in assessing PR status.

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

• PR component of the Dako ER/PR pharmDx™ Kit (K042884)

Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

A. 510(k) Number:
k130861

B. Purpose for Submission:
Addition of a new visualization system (EnVision™ FLEX +) and a new staining platform (Autostainer Link 48) to the 510(k) cleared Dako anti-PGR (636) device

C. Measurand:
Progesterone receptor

D. Type of Test:
Immunohistochemistry

E. Applicant:
Dako North America, Inc.

F. Proprietary and Established Names:
FLEX Monoclonal Mouse Anti-Human Progesterone Receptor, Clone PGR 636, Ready-To-Use (LINK)

G. Regulatory Information:
1. Regulation section:
21 CFR §864.1860 Immunohistochemistry Reagents and Kits
2. Classification:
Class II
3. Product code:
MXZ, Immunohistochemistry Assay, Antibody, Progesterone Receptor
4. Panel:

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Pathology (88)

## H. Intended Use:

1. Intended use(s):

For *in vitro* diagnostic use

Flex Monoclonal Mouse Anti-Human Progesterone Receptor, Clone PgR 636, Ready-to-Use (LINK) is intended for use in immunohistochemistry with EnVision™ FLEX +, High pH visualization kit together with the Autostainer Link 48 instrument to semi quantitatively detect human progesterone receptor in formalin fixed, paraffin embedded human breast carcinoma. This antibody labels progesterone receptor positive cells and is useful in the assessment of progesterone receptor status in human breast carcinomas.

The clinical interpretation of any staining or its absence should be complemented by morphological studies using proper controls and should be evaluated within the context of the patient's clinical history and other diagnostic tests by a qualified pathologist.

2. Indication(s) for use:

Same as above

3. Special conditions for use statement(s):

Prescription use only

4. Special instrument requirements:

Autostainer Link 48 stainer instrument

## I. Device Description:

Dako FLEX Monoclonal Mouse Anti-Human Progesterone Receptor, Clone PgR 636, Ready-to-Use (Link) antibody is utilized to semi-quantitatively detect human progesterone receptor (PR) in formalin-fixed, paraffin-embedded human breast carcinoma. This product is pre-diluted and optimized for use with the Dako Autostainer Link 48 automated slide staining platform and the FLEX + visualization system. Anti-PR, Clone PgR 636 is provided in liquid form in a buffer containing stabilizing protein and 0.015 mol/L sodium azide. The target concentration of Anti-PR, Clone PgR 636 is 0.5 µg/mL; the acceptable concentration range is 0.4 µg/mL to 0.6 µg/mL.

## J. Substantial Equivalence Information:

1. Predicate device name(s) and 510(k) number(s):

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Dako ER/PR pharmDx™ Kit, k042884

## 2. Comparison with predicate:

| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Intended Use | Detection of progesterone receptor | Same |
| Antibody Type | Monoclonal, mouse origin | Same |
| Isotype | IgG1, kappa | Same |
| Tissue Type | Formalin-fixed, paraffin embedded breast tissue | Same |
| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| PR Clone | PgR 636 | PgR 1294 |
| Visualization System | ENVISION™ FLEX+ | ENVISION™+ |
| Staining Platform | Dako Autostainer | Autostainer Link 48 |
| Interpretation of results | ASCO/CAP Method; Positive = ≥1% positive staining cells | Allred Method; 3-8 = Positive (>1-10% positive staining cells) |

## K. Standard/Guidance Document Referenced (if applicable):

None

## L. Test Principle:

Dako FLEX Monoclonal Mouse Anti-Human Progesterone Receptor, Clone PgR 636, Ready-to-use (Link) Antibody specifically binds to PR proteins located in the cell nucleus of PR-expressing cells. It is optimized for use on formalin-fixed paraffin embedded breast tissues. Automated immunohistochemical staining is performed on routinely processed, formalin-fixed, paraffin-embedded (FFPE) specimens using a specific heat-induced epitope retrieval (HIER) method and incubation with the primary mouse monoclonal antibody PR, Clone PgR 636. The procedure employs a ready-to-use horseradish peroxidase (HRP)-linked visualization reagent. The enzymatic conversion of the added DAB+ chromogen results in the formation of a visible reaction product at the antigen site. The tissue slide may then be counterstained with hematoxylin stain and coverslipped. The slide is visualized with a light microscope and scored as positive if ≥1% of tumor cell shows nuclei staining of any intensity or negative if <1% nuclei tumor cell shows nuclei staining of any intensity.

## M. Performance Characteristics (if/when applicable):

1. Analytical performance:

a. Precision/Reproducibility:

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Intra-run, inter-run and inter-instrument/operator precision: Studies to assess the intra-run, inter-run and inter-instrument/operator precision were conducted using the Flex Monoclonal Mouse Anti-Human Progesterone Receptor, Clone PgR 636, Ready-to-Use device on the Dako Autostainer Link 48 instrument, using the recommended staining procedure with EnVision™ FLEX+ Visualization System and PT Link pretreatment module. In each of these studies, 12 different paraffin embedded tissue blocks of human breast carcinoma were evaluated. The sample set represented the dynamic range of PR protein expression including 4 cases around the cut-off. Three sections from each block were stained by one operator for intra-run precision study, one section from each tissue block was stained by one operator on each of five non-consecutive days for inter-run precision study, and a total of 3 sections from each tissue block were stained on three different Autostainer instruments by 3 different operators for inter-instrument precision study. Each run included one section from each block that was stained with a negative control reagent. Positive staining was defined as $\geq 1\%$ of tumor nuclei stained at any intensity. For all the studies, staining score for each tissue block stained with Anti-PR, Clone PgR 636 did not differ within the runs, across the different runs or instruments, and the slides stained with the negative control reagent were reported to have negative staining.

Inter-Site Reproducibility: Three sites with one investigator each evaluated tissue sections from a set of 21 different specimens with approximately equal distribution of PR positive and negatives cases with three specimens around the cut-off. Each site performed staining on 5 non-consecutive days over at least 20 days. Study slides were blinded and randomized prior to evaluation by the site pathologist. Average positive percent agreement (APPA) and average negative percent agreement (APNA) between sites were calculated and the results are summarized in the tables below.

Site 1 vs. Site 2

| | Site 1 | | | |
| --- | --- | --- | --- | --- |
| Site 2 | | Positive | Negative | Total |
| | Positive | 46 | 4 | 50 |
| | Negative | 0 | 55 | 55 |
| | Total | 46 | 59 | 105 |

APPA - 95.7%; APNA - 94.7%

Site 1 vs. Site 3

| | Site 1 | | | |
| --- | --- | --- | --- | --- |
| Site 3 | | Positive | Negative | Total |
| | Positive | 45 | 0 | 45 |
| | Negative | 1 | 59 | 60 |
| | Total | 46 | 59 | 105 |

APPA - 99.2%; APNA - 98.9%

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Site 2 vs. Site 3

| | Site 2 | | | |
| --- | --- | --- | --- | --- |
| Site 3 | | Positive | Negative | Total |
| | Positive | 45 | 0 | 45 |
| | Negative | 5 | 55 | 60 |
| | Total | 50 | 50 | 105 |

APPA - 96.5%; APNA - 95.8%

Lot to Lot Reproducibility: The Lot-to-lot reproducibility of the Anti-PR, Clone PgR 636 primary antibody was evaluated across three manufactured lots of antibody, using 30 human breast carcinoma tissue samples that exhibited differing levels of PR expression. The sample set represented the dynamic range of PR protein expression including 11 specimens around the cut-off. Samples were tested in triplicate for each lot for a total of 9 serial sections per specimen. Slides were excluded from analysis due to tissue loss or inadequate invasive carcinoma. Scores for samples from each block showed 100% agreement within and across lots.

Lot 1 vs Lot 2 Reproducibility

| | LOT 1 | | |
| --- | --- | --- | --- |
| LOT 2 | Negative | Positive | Total |
| Negative | 87 | 0 | 87 |
| Positive | 0 | 168 | 168 |
| Total | 87 | 168 | 255 |
| | | lower CI | upper CI |
| --- | --- | --- | --- |
| APNA | 100.00% | 97.840 | 100.000 |
| APPA | 100.00% | 98.870 | 100.000 |
| Total Percent Agreement | 100.00% | 98.516 | 100.000 |

Lot 1 vs. Lot 3 Reproducibility

| | LOT 1 | | |
| --- | --- | --- | --- |
| LOT 3 | Negative | Positive | Total |
| Negative | 87 | 0 | 87 |
| Positive | 0 | 166 | 166 |
| Total | 87 | 166 | 253 |
| | | lower CI | upper CI |
| --- | --- | --- | --- |
| APNA | 100.00% | 97.840 | 100.000 |
| APPA | 100.00% | 98.856 | 100.000 |
| Total Percent Agreement | 100.00% | 98.504 | 100.000 |

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Lot 2 vs. Lot 3 Reproducibility

| | LOT 2 | | |
| --- | --- | --- | --- |
| LOT 3 | Negative | Positive | Total |
| Negative | 90 | 0 | 90 |
| Positive | 0 | 168 | 168 |
| total | 90 | 168 | 258 |
| | | lower CI | upper CI |
| --- | --- | --- | --- |
| APNA | 100.00% | 97.910 | 100.000 |
| APPA | 100.00% | 98.870 | 100.000 |
| Total Percent Agreement | 100.00% | 98.533 | 100.000 |

b. Linearity/assay reportable range:

Not applicable

c. Traceability, Stability, Expected values (controls, calibrators, or methods):

Stability: Real-time stability tests using three lots of the device and simulated ship-stress tests using one lot of the device were conducted to determine the shelf life of the reagent. Based on the testing, the current product shelf-life is set at 18 months.

Controls: Positive and negative tissue controls should be run simultaneously using the same protocol as the patient specimens in each staining run. Ideally the positive controls should include a low PR-expressing breast carcinoma tissue. In addition, each case should also have a negative reagent control slide included in the staining run.

d. Detection limit:

Not applicable

e. Analytical specificity:

Three sections from 30 tissue types for a total of 90 sections were stained with the Flex Monoclonal Mouse Anti-Human Progesterone Receptor, Clone PgR 636, Ready-to-Use device on the Dako Autostainer Link 48, using the recommended staining procedure with EnVision™ FLEX+ Visualization System and PT Link pre-treatment module. Staining results were available for 88 of 90 tissues. Results from two samples were not available due to tissue loss. The cell types that most commonly demonstrated nuclear positivity were epithelial cells, stromal cells, interstitial cells and inflammatory cells. Nuclear PR expression was also observed in meningial cells, adrenal glomerular cells, lymphoid cells, and pancreatic islets of langerhans. Myocytes of the heart demonstrated a peri-nuclear staining pattern. Cytoplasmic

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immunoreactivity was also observed in adrenal glomerular cells, mesothelial cells, alveolar cells, islets of langerhans and follicular cells.

f. Assay cut-off:

A positive staining result is defined as ≥1% of tumor cell nuclei staining of any intensity and negative result is <1% nuclei tumor cell nuclei staining of any intensity.

2. Comparison studies:

a. Method comparison with predicate device:

This study was a three-site, blinded, randomized, comparative study evaluating the performance of the PR clone, PgR 636 device to the PR component of the ER/PR pharmDx™ device on FFPE single tissue sections from invasive breast cancer cases. Staining was performed on the Dako Autostainer Link 48 instrument and EnVision™ Flex + visualization system. A total of 258 unique specimens representing the range of PR expression were used in this study with equal number of PR negative and PR positive specimens being present. Appropriate positive and negative control slides were used. Stained slides were interpreted by pathologists and the intensity and the proportion of cells staining were recorded. Eighteen slides were excluded from analysis due to insufficient or absence of invasive tumor or tissue shredding. A total of 240 specimens were available for analysis. PR expression in DCIS also was evaluated. Slides were scored based on the scoring method specified in the instructions for use for the predicate device as well as the ASCO/CAP scoring method which is the scoring method for the test device. The results of the method comparison study are presented in the below tables.

Comparison of ASCO/CAP Scores of RTU PgR Clone PgR 636 Antibody vs PR Component of ER/PR pharmDx Kit

| | PR Component of ER/PR pharmDx Kit | | | |
| --- | --- | --- | --- | --- |
| | | Positive | Negative | Total |
| PgR Clone PgR 636 Antibody | Positive | 119 | 8 | 127 |
| | Negative | 1 | 112 | 113 |
| | Total | 120 | 120 | 240 |

PPA = 99.2% (95%CI: 93.2 - 98.1%); NPA = 93.3% (95%CI: 92.8 - 98.0%)
OPA = 96.3% (95%CI: 93.0 - 98.0%)

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Comparison of Allred Scores of RTU PgR Clone PgR 636 Antibody vs PR Component of ER/PR pharmDx Kit

| | PR Component of ER/PR pharmDx Kit | | | |
| --- | --- | --- | --- | --- |
| | | Positive | Negative | Total |
| RTU PgR Clone PgR 636 Antibody | Positive | 123 | 11 | 134 |
| | Negative | 1 | 105 | 106 |
| | Total | 124 | 116 | 240 |

PPA = 99.2% (95%CI: 92.0 – 97.3); NPA = 90.5% (95%CI: 90.8 – 96.9)
OPA = 95.0% (95%CI: 91.5 – 97.1)

b. Matrix comparison:

Not applicable

3. Clinical studies:

a. Clinical Sensitivity:

Not applicable

b. Clinical specificity:

Not applicable

c. Other clinical supportive data (when a. and b. are not applicable):

None

4. Clinical cut-off:

A positive result is defined as nuclear staining of ≥1% of tumor cells at any stain intensity.

5. Expected values/Reference range:

Not applicable

N. Proposed Labeling:

The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.

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O. Conclusion:

The submitted information in this premarket notification is complete and supports a substantial equivalence decision.

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