VENTANA ER PRIMARY ANTIBODY (CLONE 6F11)

K984567 · Ventana, Inc. · MYA · Aug 12, 1999 · Hematology

Device Facts

• Record ID: K984567
• Device Name: VENTANA ER PRIMARY ANTIBODY (CLONE 6F11)
• Applicant: Ventana, Inc.
• Product Code: MYA · Hematology
• Decision Date: Aug 12, 1999
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 864.1860
• Device Class: Class 2

Intended Use

Ventana ER primary antibody (clone 6F11) is intended for laboratory use for the qualitative detection of estrogen receptor (ER) antigen in sections of formalin fixed, paraffin embedded normal and neoplastic tissue on a Ventana automated slide staining immunohistochemistry device. It is indicated as an aid in the management, prognosis and prediction of therapy outcome of breast cancer within the context of the patient's clinical history and other diagnostic tests evaluated by a qualified pathologist.

Device Story

Monoclonal antibody (clone 6F11) binds to estrogen receptor (ER) antigen in nuclear regions of normal/neoplastic tissue; used on Ventana automated slide staining devices. Process: tissue pretreatment/antigen enhancement; inhibitor application to reduce endogenous peroxidase; primary antibody incubation; biotin-conjugated secondary antibody application; streptavidin-enzyme conjugate binding; chromogenic substrate application. Output: visualized precipitating enzyme product on microscope slides. Operated by laboratory personnel; results interpreted by qualified pathologist via light microscopy. Output aids in breast cancer prognosis and therapy prediction by identifying ER-positive status. Benefits: provides standardized immunohistochemical assessment of ER status to guide clinical decision-making.

Clinical Evidence

Retrospective study of 100 breast cancer cases comparing Ventana ER IHC (clone 6F11) to SBA/DCC cytosol receptor assay. Primary endpoints: sensitivity, specificity, and concordance. Results at ≥11% tumor cell nuclei cutoff: 98.6% sensitivity (95% CI: 92.5-100%), 78.6% specificity (95% CI: 59.1-91.7%), and 93% concordance (95% CI: 86-97%). Additional specificity testing performed on 76 normal and 19 neoplastic tissues.

Technological Characteristics

Mouse anti-human ER monoclonal antibody (clone 6F11, IgG kappa). Reagent concentration ~1 mg/ml. Utilizes indirect biotin-avidin-enzyme detection system. Requires antigen enhancement (microwave/citrate buffer). Compatible with Ventana automated slide staining devices. Visualization via chromogenic enzyme substrate (DAB, AEC, or Alkaline Phosphatase).

Indications for Use

Indicated for use in the management, prognosis, and prediction of therapy outcome of breast cancer in patients. Used for qualitative detection of ER antigen in formalin-fixed, paraffin-embedded tissue sections to aid in the diagnosis of ER-positive tumors.

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

• Abbot Laboratories' ER-ICA Monoclonal, Immunocytochemical Assay for the Detection of Human Estrogen Receptor (P880026)

Submission Summary (Full Text)

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# K984567

## 510(k) SUMMARY Ventana ER Primary Antibody (Clone 6F11)

#### Submitted By:

Ventana Medical Systems, Inc. 3865 N. Business Center Drive Tucson, Arizona 85705 Tel: [phone] Fax: [phone] Contact: Judith Frederick, MS, CCRA Date: August 4, 1999

FDA/CDRH/ODE/DHHS

6 AUG 99

13 52

RECEIVED

This summary of 510(k) safety and effectiveness information is being submitted in accordance with the requirements of SMDA 1990 and 21 CFR 807.92. The assigned 510(k) number is K984567.

#### Name of the Device:

| Trade Name: | Ventana ER Primary Antibody (Clone 6F11) |
|-----------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Common Name: | Antibody for aid in the detection of estrogen receptor (ER) antigen in histological tissue sections |
| Classification: | To the best of our knowledge ER antibody for aid in the detection of ER antigen in histological tissue sections is classified as class II and codified at 21 CFR 864.1860; Medical Devices; Classification/Reclassification Immunohistochemistry Reagents and Kits, June 3, 1998. |

Predicate Device: Ventana ER primary antibody (clone 6F11) is substantially equivalent to commercially available Abbot Laboratories' ER-ICA Monoclonal, Immunocytochemical Assay for the Detection of Human Estrogen Receptor, approved by the FDA as PMA # P880026 and downclassified to class II by 21 CFR 864.1860 Immunohistochemistry Reagents and Kits on June 3, 1998.

Description: Ventana's ER Primary Antibody (clone 6F11) is a monoclonal antibody which specifically binds to estrogen receptor antigen located in the nuclear region of a variety of normal and neoplastic tissues. The dispenser contains approximately 5 ug (50 test) or 25 ug (250 test) of mouse anti-human ER monoclonal antibody. The total protein concentration of the reagent is approximately 1 mg/ml. Specific antibody concentration is approximately 1 ug/ml. Clone 6F11 is immunoglobulin class IgG, light chain kappa. There is no known irrelevant antibody in the preparation.

Intended Use: Ventana ER primary antibody (clone 6F11) is intended for laboratory use for the qualitative detection of estrogen receptor (ER) antigen in sections of formalin fixed, paraffin embedded normal and neoplastic tissue on a Ventana automated slide staining immunohistochemistry device. It is indicated as an aid in the management, prognosis and

Ventana Medical Systems, Inc.

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prediction of therapy outcome of breast cancer within the context of the patient's clinical history and other diagnostic tests evaluated by a qualified pathologist.

Technology of the device and predicate device: The demonstration of antigens in tissue and cells by immunostaining is a process involving first the binding of an antibody to an antigen of interest and second, visualization of the bound primary antibody by an indirect biotin-avidin system coupled to an enzyme. Ventana ER primary antibody (clone 6F11) is located by a biotin conjugated secondary antibody formulation which recognizes rabbit or mouse immunoglobulins. This step is followed by the addition of a streptavidin-enzyme conjugate which binds to the biotin on the secondary antibody. The primary antibody-secondary antibody-avidin enzyme complex is visualized by using a precipitating enzyme generated product. Ventana ER Primary Antibody (clone 6F11) was developed for use on Ventana's automated slide staining devices. Briefly, the procedure is as follows:

- 1. Pretreatment of tissues or cells on a microscope slide with an antigen-enhancement procedure is required.
- 2. An inhibitor solution is added to the slide on which antigen-enhanced tissues or cells are affixed. The inhibitor reduces the endogenous peroxidase activity in the tissue. This step takes 4 minutes at 37° C. The slide is washed with buffer and covered with liquid coverslip.
- 3. Optimized ER Primary Antibody (clone 6F11) is applied, mixed and incubated for 4 to 32 minutes at 37° C. The slide is washed with buffer and covered with liquid coverslip.
- 4. Streptavidin-enzyme reagent is added to the slide, mixed and incubated at 37° C. (The time of incubation is indicated in each detection kit insert.) The slide is washed with buffer and covered with liquid coverslip.
- 5. The chromogenic enzyme substrate is applied to the slide and mixed. The solutions are incubated with mixing at 37° C. (The time of incubation is indicated in each detection kit insert.) The slide is washed and ready for counterstaining, copper enhancement, or can be prepared for microscopic reading immediately.

Abbott ER-ICA Monoclonal detects estrogen receptor in frozen tissue sections affixed to glass slides in a technically similar fashion to the immunostaining process used for staining paraffin embedded tissue. The frozen sections are fixed in formaldehyde, methanol and acetone, then treated with Blocking Reagent to prevent nonspecific binding. The primary antibody (the Ig fraction of a rat monoclonal antibody specific for ER receptor) is then added to the tissue. Visualization of the bound primary antibody is via an indirect peroxidase anti-peroxidase (PAP) system. ER-ICA Monoclonal is located by a goat anti-rat bridging antibody. Anti-Rat PAP complex which recognizes the bridging antibody is then added. This step is followed by the addition of a precipitating enzyme generated product (hydrogen peroxidase) and a chromogen substrate solution, containing DAB. The resulting insoluble reddish brown precipitation allows visualization of the bound primary antibody ?. The two devices are similar in that both systems specifically bind to ER proteins in breast carcinoma tissues, both employ similar detection chemistry principles for visualization of the product, and both aid in the diagnosis and prognosis of breast carcinoma.

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#### Performance Data:

1. Tissue Specificity: The staining patterns of 76 normal tissues were examined. These included adrenal, bone marrow, breast, cerebrum, cervix, colon, endometrium, esophagus, heart, kidney, liver, lung, mesothelium, ovary, pancreas, peripheral nerve, prostate, salivary gland, skeletal muscle, skin, small intestine, spleen, stomach, testis, thyroid, tonsil, and thymus. Tissues not tested as recommended in "FDA Guidance for Submission of Immunohistochemistry Applications to the FDA", were cerebellum, pituitary, and parathyroid. Staining was considered to be appropriate, with the exception of the unexpected negative staining of one case of breast tissue, and one case of endometrium. The pathologist noted however, that in the case of the breast, the tissue was poorly preserved, so the negative staining may be due to fixation. Positive nuclear staining was found in the lobular and ductal cells of the breast (2 of 3 cases), epithelia and fibromuscular cells of the cervix (3 of 3 cases), the glandular epithelia, stromal and smooth muscle cells of the endometrium (2 of 3 cases), and the surface epithelium and stromal cells of the ovary (3 of 3 cases). All other tissues were negative.

The staining patterns of 19 neoplastic tissues were also evaluated by a qualified pathologist. Neoplastic tissues examined were lung, prostate, colon, lymphoma, bladder, stomach, cervix, and ovary. Positive nuclear staining occurred in the tumor tissue of both cases of ovarian cancer (100% of tumor cell nuclei stained) and in the surrounding normal tissue of two cases of cervical cancer. All other tissues were negative.

Prior to staining with Ventana ER primary antibody (Clone 6F11), the dried, deparaffinized sections used for the specificity studies were treated for antigen enhancement. Slides containing the tissue sections were placed in citrate buffer (pH 5.75) and heated under pressure in a microwave for approximately 30 minutes. The slides were allowed to stand for an additional 30 minutes (approximate), then transferred to fresh citrate buffer until ready to run on the Ventana automated slide stainer. Optimized ER Primary Antibody (clone 6F11) was applied, mixed and incubated 32 minutes at 37° C. Endogenous biotin was blocked using Ventana Medical Systems' Endogenous Biotin Blocking Kit (catalogue number 250-050). Detection used was Ventana's DAB Kit (catalogue number 250-001). DAB Detection includes: 1) An inhibitor solution, added to reduce the endogenous peroxidase activity in the tissue. 2) Universal biotinylated secondary antibody. 3) Avidin-HRPO. 4 and 5) Basic DAB solution and hydrogen peroxide. The two reagents are applied together and mixed on the specimen slide. And 6) Copper enhancement. The incubation time for each reagent was pre-programmed at Ventana.

2. Validation of Assay Performance by Comparison with an FDA approved chemical cytosol receptor assay for ER, serum binding assay/ dextran coated charcoal (SBA/DCC): The safety and effectiveness of Ventana's ER Primary Antibody (clone 6F11) have been examined through clinical testing by Ventana Medical Systems, Inc. Ventana verified that ER Primary Antibody (clone 6F11) detects ER in breast carcinoma tissue in an equivalent manner to SBA/DCC by examining 100 cases of breast cancer for expression of ER. The cases were randomly chosen from an archived collection of breast carcinoma biopsies (300 cases total) at one investigative site. The study was retrospective in design, using archived tissue samples embedded in paraffin

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blocks that had concurrent ER SBA/DCC analyses in the patient file. For antigen enhancement, the investigator used a 10 minute autoclave steam heating procedure and the Biogenex Citra antigen retrieval solution. The staining protocol was identical to that used in the specificity studies.

Quality control procedures included a positive tissue control (uterus) placed on every slide run in addition to the case tissue. Each case had two slides stained with Ventana ER Primary Antibody (Clone 6F11) and one slide with Ventana Negative Control Ig (catalogue # 250-2014). For the test to be considered valid, the positive control tissue was expected to exhibit nuclear staining of the endometrial glands and stroma. These components were to be negative when stained with Negative Control Ig. In addition, a negative tissue control slide (ER-negative breast carcinoma) was included for every batch of samples processed and run on the Ventana automated slide staining device. This negative tissue control was stained with ER Primary Antibody (Clone 6F11) to ensure that the antigen enhancement and other treatment procedures did not create false positive staining. The study cases were evaluated within the context of the performance of the controls.

Staining of ER primary antibody (clone 6F11) was assessed both as a percentage of cells stained and by staining intensity. Scoring of tumor cell percentages was estimated and categorized into the following cell count ranges: 0, 1-10, 11-25, 26-75, and over 75 percent. If the scorer was uncertain of the category, 100 tumor cells in each of three fields at 40X magnification was counted and the values averaged for a true count percentage. The emphasis in data analysis was on percent positive cells rather than stain intensity. Scoring of the benign elements was also performed to aid in discrepancy resolution. The pathologist scoring the IHC was masked to the results of the ER SBA/DCC. Results were as follows:

| Cut off for % Positive Cells | TP | TN | FP | FN | TOTAL |
|------------------------------|----|----|----|----|-------|
| Raw ER ≥1% | 71 | 15 | 12 | 2 | 100 |
| Raw ER ≥ 11% | 71 | 21 | 6 | 2 | 100 |
| Resolved ER ≥1% | 71 | 16 | 12 | 1 | 100 |
| Resolved ER > 11% | 71 | 22 | 6 | 1 | 100 |

## Table 1: ER IHC vs. ER SBA/DCC

Scoring Criteria: ER IHC positivity was compared to ER SBA/DCC at different cut-off values to determine the cut-off value for ER Primary Antibody (Clone 6F11) that best agreed with the SBA/DCC results. Sensitivity, specificity, and concordance of the ER test, relative to that of the ER SBA/DCC test. improved using a cut-off value of > 11% compared to > 1%. Ventana therefore recommends evaluating slides with a cut-off for positivity of > 11% positive staining of tumor cell nuclei. Cell intensity was variable and was not used as a criteria for positivity. ER SBA/DCC was scored as positive at greater than or equal to 10 femtomoles (fm).

Discrepancy Resolution: Because SBA/DCC cannot distinguish between positivity of pathological and benign elements, the protocol allowed for the resolution of false negative ER

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IHC to true negative if benign elements were positive and pathological elements did not stain. The results of these analyses were noted as "Resolved". A possible explanation for the negative SBA/DCC results of the 6 cases is estrogen receptor degradation due to poor tissue handling during sample collection'. The one false negative ER-IHC case may have been due to sample over-fixation.

Agreement: ER IHC with Ventana's ER Primary Antibody (Clone 6F11) showed the best agreement with ER SBA/DCC (x2 = 67.82, P < 0.0001) when ≥ 11% of tumor cell nuclei were scored as positive. 93% of the cases were concordant, with a 95% confidence interval of 86 -97%.

Sensitivity: Resolved sensitivity relative to ER SBA/DCC at a cut off of > 11% was 98.6% (71/72), with a 95% confidence interval of 92.5 - 100%.

Specificity: Resolved specificity relative to ER SBA/DCC at a cut off of > 11% was 78.6% (22/28), with a 95% confidence interval of 59.1 - 91.7%. One possible reason for the low specificity in relation to the other values, was the few number of SBA/DCC negative cases in the randomly selected pool (n=28).

3. Prognosis and prediction: Allred, et. al. also investigated the prognostic value of ER, using formalin fixed, paraffin embedded tissue and clone 6F11. In a group of 820 patients receiving no adjuvant therapy, he found the difference in disease free survival (DFS) at five years follow up to be ten per cent. In a subset of 800 patients receiving endocrine therapy, however, ER positivity by IHC was associated with a 25% improvement in DFS over 5 years' duration'.

Conclusions: Ventana claims that ER Primary Antibody (clone 6F11) is substantially equivalent to Abbott ER-ICA Monoclonal, subject of P880026, which was downclassified from class III to Class II as a result of 21 CFR 864.1860, Immunohistochemistry Reagents and Kits. Performance characteristics of the Ventana ER Primary Antibody were validated in a comparison with an FDA approved chemical cytosol receptor assay for ER, serum binding assay/dextran coated charcoal (SBA/DCC). Demonstrated performance characteristics were: 98.6% relative sensitivity, 78.6% relative specificity, 93% concordance. Ventana further claims that ER Primary Antibody (clone 6F11) is a useful aid in assessing prognosis and prediction of therapy outcome for the breast cancer patient.

## Bibliography:

- 1. Allred, D. C. et. al. Prognostic and predicitve facotrs in breast cancer by immunohistochemical analysis. Mod. Pathol., 11: 155-168, 1998.
- 2. Bevitt, D. J. et. al. New monoclonal antibodies to oestrogen and progesterone receptors effective for paraffin section immunohistochemistry. J. Pathol. 183: 228-232, 1997.
- 3. Abbott ER-ICA Monoclonal Package Insert. Abbott Laboratories Diagnostics Division. Customer Support Center (USA) 1-800-323-9100.

Ventana Medical Systems, Inc.

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Image /page/5/Picture/1 description: The image shows the seal of the Department of Health & Human Services (HHS). The seal features an abstract image of an eagle with three stripes forming its body and head. The words "DEPARTMENT OF HEALTH & HUMAN SERVICES • USA" are arranged in a circular pattern around the top of the seal. The seal is black and white.

AUG 12 1999

Food and Drug Administration 2098 Gaither Road Rockville MD 20850

Ms. Judith Frederick, MS, CCRA Senior Clinical Coordinator Ventana Medical Systems, Inc. 3865 North Business Center Drive Tucson, Arizona 85705

Re: K984567

Trade Name: Ventana ER Primary Antibody (Clone 6F11) Regulatory Class: II Product Code: MYA Dated: July 22, 1999 Received: July 26, 1999

Dear Ms. Frederick:

We have reviewed your Section 510(k) notification of intent to market the device referenced above and we have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration.

If your device is classified (see above) into either class II (Special Controls) or class III (Premarket Approval), it may be subject to such additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 895. A substantially equivalent determination assumes compliance with the Current Good Manufacturing Practice requirements, as set forth in the Quality System Regulation (QS) for Medical Devices: General regulation (21 CFR Part 820) and that, through periodic QS inspections, the Food and Drug Administration (FDA) will verify such assumptions. Failure to comply with the GMP regulation may result in regulatory action. In addition, FDA may publish further announcements concerning your device in the Federal Register. Please note: this response to your premarket notification submission does not affect any obligation you might have under sections 531 through 542 of the Act for devices under the Electronic Product Radiation Control provisions, or other Federal laws or regulations.

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Page 2

Under the Clinical Laboratory Improvement Amendments of 1988 (CLIA-88), this device may require a CLIA complexity categorization. To determine if it does, you should contact the Centers for Disease Control and Prevention (CDC) at [phone].

This letter will allow you to begin marketing your device as described in your 510(k) premarket notification. The FDA finding of substantial equivalence of your device to a legally marketed predicate device results in a classification for your device and thus, permits your device to proceed to the market.

If you desire specific advice for your device on our labeling regulation (21 CFR Part 801 and additionally 809.10 for in vitro diagnostic devices), please contact the Office of Compliance at [phone]. Additionally, for questions on the promotion and advertising of your device, please contact the Office of Compliance at [phone]. Also, please note the regulation entitled, "Misbranding by reference to premarket notification"(21 CFR 807.97). Other general information on your responsibilities under the Act may be obtained from the Division of Small Manufacturers Assistance at its toll-free number [phone] or [phone], or at its internet address "http://www.fda.gov/cdrh/dsma/dsmamain.html".

Sincerely yours.

Steven Sutman

Steven I. Gutman, M.D, M.B.A. Director Division of Clinical Laboratory Devices Office of Device Evaluation Center for Devices and Radiological Health

Enclosure

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510(k) Number (if known): K984567

Device Name: ER Primary Antibody (Clone 6F11)

# Indications For Use:

Ventana Medical Systems' ER Primary Antibody (Clone 6F11) may be used in immunohistochemical methods to aid in the identification of estrogen receptor (ER) antigen in normal and neoplastic cells as an aid in the diagnosis of ER positive tumors. The anti-ER (Clone 6F11) is intended for laboratory use to qualitatively stain ER antigen in sections of formalin fixed, paraffin embedded tissue on a Ventana automated slide staining device. It is further intended as an aid in the assessment of prognosis in the evaluation of breast cancer. Detection chemistries used by Ventana to detect ER are DAB, AEC, Alkaline phosphatase red and blue. Light microscopy is used to detect the staining of cell components. It is indicated for use in the management, prognosis and prediction of therapy outcome of breast cancer.

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Concurrence of CDRH, Office of Device Evaluation (ODE)

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Division of Clinical Laboratory Devices
510(k) Number K984567

**Prescription Use**
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**Over-The-Counter Use**

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