K Number

Device Name

THE CA 15-3 ASSAY FOR THE TECHNICON IMMUNO 1 SYSTEM (IN VITRO DIAGNOSTIC SYSTEM)

Manufacturer

BAYER CORP.

Date Cleared

1997-12-01

(374 days)

Product Code

MOI

Regulation Number

866.6010

Intended Use

The Bayer Immuno 1™ CA 15-3™ Assay is an in vitro, diagnostic, solid phase immunoassay intended to quantitatively measure CA 15-3™ Assay values in human serum on the Bayer Immuno 1 system. When used in conjunction with other clinical and diagnostic procedures, serial testing with the CA 15-3™ Assay is useful for monitoring the course of disease and therapy in metastatic breast cancer patients, and for detection of recurrence in previously treated Stage II, with greater than 2 positive lymph nodes, or Stage III breast cancer patients.

Device Description

The Bayer Immuno IIM CA 15-3™ Assay is a sandwich immunoassay in which one monoclonal antibody (115D8) is conjugated to fluorescein (R1) and a second monoclonal antibody (DF3) is conjugated to alkaline phosphatase (R2). Immuno 1 magnetic particles coated with antibody; the R1 conjugate, and patient sample, calibrator, or control are mixed simultaneously and incubated at 37℃ on the system. The R2 conjugate is then added, which binds to the immobilized CA 15-3™ to form a sandwich immunocomplex on the solid phase. The magnetic particles complexed with the immunological sandwich are then washed to separate unbound molecules, and a colorimetric substrate is added. The rate of conversion of substrate to a compound with absorbance at 405 and 450 nm is measured; the measured rate is proportional to the concentration of CA 15-3™ assay values in the sample. A cubic-through-zero curve fitting algorithm is used to generate standard curves.

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Predicate Devices

Not Found

Reference Devices

Centocor CA 15-3™ RIA

AI/ML (Artificial Intelligence / Machine Learning)

No
The device description mentions a "cubic-through-zero curve fitting algorithm" which is a standard mathematical method for generating standard curves in immunoassays, not an AI/ML technique. There are no other mentions of AI, ML, or related concepts in the provided text.

Therapeutic

No
This device is an in vitro diagnostic immunoassay that measures CA 15-3 values to monitor disease course and detect recurrence, rather than directly treating a condition.

Diagnostic

Yes
The "Intended Use / Indications for Use" section explicitly states that the device is "an in vitro, diagnostic, solid phase immunoassay." It also describes its use in monitoring disease course and therapy, and detecting recurrence, which are all diagnostic applications.

SaMD (Software as a Medical Device)

The device description clearly outlines a physical immunoassay process involving reagents, magnetic particles, incubation, washing, and colorimetric measurement, indicating it is a hardware-based in vitro diagnostic device, not software-only.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

The "Intended Use / Indications for Use" section explicitly states: "The Bayer Immuno 1™ CA 15-3™ Assay is an in vitro, diagnostic, solid phase immunoassay intended to quantitatively measure CA 15-3™ Assay values in human serum on the Bayer Immuno 1 system."

This statement clearly identifies the device as an in vitro diagnostic product.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

Product codes

MOI, 82

Device Description

The Bayer Immuno 1™ CA 15-3™ Assay is an in vitro device indicated for the quantitative measurement of CA 15-3™ assay values in human serum. When used in conjunction with other clinical and diagnostic procedures, serial testing with the CA 15-3™ Assay is useful for monitoring the course of disease and therapy in metastatic breast cancer patients, and for detection of recurrence in previously treated Stage II, with greater than two positive Tymph nodes, or Stage III breast cancer patients. The assay is designed to run on the Bayer Immuno 1™ Immunoassay System, a fully automated random-access analyzer which performs both homogeneous and heterogeneous immunoassays.

The assay has a range of 0.2 to 200 UlmL. The assay uses six calibrators with CA 15-3™ concentrations of 0, 12.5, 25, 50, 100, and 200 U/mL. A typical standard curve for the assay is presented in Figure 2.

Mentions image processing

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Not Found

Indicated Patient Age Range

Not Found

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Not Found

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

Not Found

Summary of Performance Studies

Nonclinical studies were performed to evaluate assay specificity and interfering substances, minimum detectable concentration, imprecision, linear range, hook effect, parallelism, and spiked recovery. In addition, accuracy was evaluated by comparison of the Immuno 1 CA 15-3™ Assay with the FDA-approved Biomira TRUQUANT® BR™ RIA, and Centocor CA 15-3™ RIA in a method concordance evaluation. Using a panel of 501 serum samples, this concordance analysis evaluated the relationship between CA 15-3™ assay values determined by the Immuno 1 CA 15-3™ Assay and the Biomira TRUQUANT® BR™ RIA and Centocor CA 15-3™ RIA.

Nonclinical Studies:

Characterization of the Antigen: The antigen used in Immuno 1 CA 15-3™ assay calibrators is the DF3 reactive determinant isolated from ZR75-1 Anchorage Dependent Cells, supplied by Centocor, Inc. Electrophoretic analysis confirmed consistency with previous descriptions of CA 15-3™ glycoprotein.
Immunoreactivity of the Antibodies: Monoclonal antibodies DF3 and 115D8 (from Centocor, Inc.) were characterized. Both are murine IgG1 and IgG2b subclasses, respectively. They bound to CA 15-3™ antigen quantitatively and specifically, exhibiting expected biophysical properties.
Specificity And Interfering Substances:
- Endogenous Interferants: Tested triglycerides, immunoglobulin, hemoglobin, heparin, bilirubin, or albumin. None showed significant interfering effects on CA 15-3™ assay recovery. (Table 1: Triglycerides (900 mg/dL) -0.2%, Immunoglobulin (5.3 g/dL) 0.5%, Hemoglobin (1.0 g/dL) 1.6%, Heparin (0.15 mg/mL) 1.3%, Bilirubin (25 mg/dL) 1.3%, Albumin (6.5 g/dL) -1.5%).
- Anti-neoplastic Drug Interferants: Tested chemotherapeutic drugs. Drug Pool #1 (Cyclophosphamide, Doxorubicin, 5-Fluorouracil, Methotrexate, Mitomycin C) inhibited CA 15-3™ recovery by -21.3%. Individually, only Methotrexate significantly interfered, with 300 ug/mL inhibiting recovery by 65%. (Table 2 & 3). The observed methotrexate effect is not likely to result in patient mismanagement.
- Common "Over the Counter" Drugs and Vitamin Supplements: Tested various vitamins, OTC drugs, caffeine, and codeine. None demonstrated significant interfering effects on CA 15-3™ assay recovery. (Table 4).
- Heterophilic Antibodies: Ten samples with high rheumatoid factor titers and six samples with high human anti-mouse antibody titers were assayed. Linear recovery of CA 15-3™, when diluted, indicated no significant heterophilic interference.
Linearity: Three clinical sample pools diluted with normal serum demonstrated linearity over the entire calibration range (recoveries between 98.2% and 105.2%).
Hook Effect: Tested up to 28.5 kU/mL of CA 15-3™ antigen; no hook effect was observed.
Spiked Recovery: Antigen spiked into seven patient samples at 30 and 120 U/mL showed recoveries ranging from 92.9% to 122.9%, demonstrating accurate quantitation.
Parallelism: Four patient serum sample pools diluted with Level 1 calibrator showed no deviation from linearity, with recoveries ranging from 88.2-116.9%.
Minimum Detectable Concentration: MDC was determined to be 0.13 U/mL, based on 1336 determinations, exceeding the method sheet claim of 0.2 U/mL.
Imprecision: Evaluated intra- and interassay reproducibility using commercial controls, calibrators, and an internal human serum control. (Table 5). Within-run CVs ranged from 1.3% to 3.4%; total CVs ranged from 3.0%. Excellent precision was observed.

Method Comparison Studies:

Introduction: Examined concordance of Immuno 1 CA 15-3™ Assay with Biomira TRUQUANT® BR™ RIA and Centocor CA 15-3™ RIA.
Samples: 501 human serum samples from BioClinical Partners, Inc. and Bayer Corporation (in-house specimen collection) were analyzed. (Table 6: 199 Normal samples (100 Premenopausal, 100 Postmenopausal), 150 Breast Cancer, 35 Lung Cancer, 35 Ovarian Cancer, 35 Colorectal Cancer, 46 Benign Breast Disease).
Normal Range: Determined for 199 normal healthy females. Mean serum values were 17.1 U/mL (Immuno 1), 17.6 U/mL (Biomira), and 18.6 U/mL (Centocor). Upper limits of normal (mean + 1.96 x SD) were 32.9 U/mL (Immuno 1), 30 U/mL (Biomira), and 33.5 U/mL (Centocor). 97.5th percentile cut-offs were 35.9 U/mL (Immuno 1), 35.6 U/mL (Biomira), and 31.3 U/mL (Centocor). No significant differences between assays. (Table 7 and Figure 4).
Method Concordance: Correlation study using 501 female serum samples (various clinical classifications). Ordinary least squares linear regression was used. (Table 8).
- Immuno 1 vs. Biomira: Slope 0.852, Intercept 3.48, N 498, R 0.93, Sy.x 11.40.
- Immuno 1 vs. Centocor: Slope 0.791, Intercept 3.23, N 499, R 0.96, Sy.x 9.26.
- Results demonstrate concordance and equivalence.

Reagent Stability Testing:

Shelf-Life Stability Testing: Four lots of reagents were tested for 18 months, indicating product stability within specifications.
On-System Stability Testing: On-system stability of 30 days was supported by testing four lots.
Shipping Stability Testing: Three lots subjected to heat/chill cycles supported refrigerated (2-8°C) shipping.

Calibrator Stability Testing:

Shelf-Life Stability Testing: Three lots of calibrators were stable for 12 months when stored at 2-10°C or below.
Open Vial Stability Testing: Two lots demonstrated open vial stability for 30 days when stored at 2-8°C.
Shipping Studies: Three lots subjected to freeze/thaw cycles supported stability for up to three cycles.

Multi-Site Clinical Studies:

Objectives:
1. Evaluate Immuno 1 CA 15-3™ Assay for monitoring disease course and therapy in metastatic breast cancer patients.
2. Evaluate Immuno 1 CA 15-3™ Assay for early detection of recurrence in previously treated Stage II or Stage III breast cancer patients.
3. Estimate upper limit of normal in healthy females.
4. Estimate maximum serial variability in normal healthy females.
5. Estimate clinical sensitivity in Stages 1-4 breast cancer patients.
6. Estimate clinical specificity in patients with benign breast diseases, other non-malignant diseases, non-breast cancers, pregnant women, and healthy individuals.
Investigators/Sites: Dr. Roy Beveridge (Fairfax Prince William Hematology Oncology Associates), Dr. Daniel W. Chan (Johns Hopkins Hospital), Dr. Herbert A. Fritsche (M.D. Anderson Cancer Center), Dr. Morton K. Schwartz (Memorial Sloan-Kettering Cancer Center), Dr. Alan H. B. Wu (Hartford Hospital).
Management Value for Monitoring Metastatic Breast Cancer: 158 metastatic breast cancer patients. Serial testing showed 64% sensitivity for detecting changes in clinical status. (Table 9).
Management Value for Early Detection of Recurrence: 182 Stage II and III breast cancer patients (17 showed recurrence).
- Immuno 1 (≥ 25% increase): Longitudinal sensitivity 53%, specificity 96%. (Table 10).
- Biomira (≥ 45% increase): Longitudinal sensitivity 53%, specificity 91%. (Table 11).
- Immuno 1 showed improved longitudinal specificity compared to Biomira.
Distribution of Immuno 1 CA 15-3™ Assay Results and Concordance with Clinical Condition: Concentrations across 216 premenopausal and 202 postmenopausal healthy women, 81 pregnant women, 114 benign breast disease, 180 other benign diseases, 318 breast cancer (various stages), and 210 non-breast cancer patients. (Table 12).
- Reference interval (95th percentile) for healthy women was 34.8 U/mL; an upper limit of normal of 35 U/mL is consistent with literature and predicate device.

Key Metrics

Method Comparison (Linear Regression):
- Immuno 1 vs. Biomira: R = 0.93
- Immuno 1 vs. Centocor: R = 0.96
Minimum Detectable Concentration (MDC): 0.13 U/mL
Imprecision (CV):
- Within-run CVs: 1.3% to 3.4%
- Total CVs: 3.0% (for Level 6 calibrator) up to 4.0%
Management of Metastatic Breast Cancer Patients:
- Sensitivity: 64% (98/152) for detecting changes in clinical status based on serial testing.
- Positive Predictive Value (PPV) of a ≥ 25% change in serial assay values: 95% (98/103).
Early Detection of Recurrence (Stage II or III Breast Cancer Patients):
- Immuno 1 (≥ 25% increase confirmed):
  - Sensitivity: 53%
  - Specificity: 96%
  - Predictive Value of Positive: 64%
  - Predictive Value of Negative: 93%
- Biomira (≥ 45% increase confirmed):
  - Sensitivity: 53%
  - Specificity: 91%
  - Predictive Value of Positive: 47%
  - Predictive Value of Negative: 93%
Normal Range: 95th percentile for healthy women = 34.8 U/mL; upper limit of normal = 35 U/mL.

Predicate Device(s)

Biomira TRUQUANT® BR™ RIA

Reference Device(s)

Centocor CA 15-3™ RIA

Predetermined Change Control Plan (PCCP) - All Relevant Information

Not Found

Regulation Number and Section

§ 866.6010 Tumor-associated antigen immunological test system.

(a)
Identification. A tumor-associated antigen immunological test system is a device that consists of reagents used to qualitatively or quantitatively measure, by immunochemical techniques, tumor-associated antigens in serum, plasma, urine, or other body fluids. This device is intended as an aid in monitoring patients for disease progress or response to therapy or for the detection of recurrent or residual disease.(b)
Classification. Class II (special controls). Tumor markers must comply with the following special controls: (1) A guidance document entitled “Guidance Document for the Submission of Tumor Associated Antigen Premarket Notifications (510(k)s) to FDA,” and (2) voluntary assay performance standards issued by the National Committee on Clinical Laboratory Standards.

Summary

1997

Holori

SUMMARY OF SAFETY AND EFFECTIVENESS For the Bayer Immuno 17M CA 15-37M Assay

This premarket notification is to add the quantitative measurement of CA 15-3™ assay values in human serum to the intended use of the Bayer Immuno 1™ Immunoassay System. The performance characteristics of the Bayer Immuno 1™ CA 15-3™ Assay and comparison of this assay to a predicate device, the Biomira TRUQUANT® BRTM RIA has been established in accordance with Section VI. (A) of the "Guidance Document For Submission of Turnor Associated Antigen Premarket Notifications, 510(k), to the FDA." Clinical evaluations of the Bayer Immuno 1™ CA 15-3™ Assay at four US clinical trial sites demonstrated clinical safety and effectiveness and substantial equivalence to the predicate device in accordance with Section VI.(B) of the Guidance Document. The information presented in this Summary of Safety and Effectiveness was derived from nonclinical performance and clinical evaluation studies comparing the performance of the Immuno 1 CA 15-37M Assay with that of the Biomira RJA. Clinical studies were conducted at four clinical trial sites with a suitable sampling of patients to support the diagnostic claims for this device.

INDICATIONS FOR USE

The Bayer Immuno 1™ CA 15-3™ Assay is an in vitro, diagnostic, solid phase immunoassay intended to quantitatively measure CA 15-37M Assay values in human serum on the Bayer Immuno 1 system. When used in conjunction with other clinical and diagnostic procedures, serial testing with the CA 15-3™ Assay is useful for monitoring the course of disease and therapy in metastatic breast cancer patients, and for detection of recurrence in previously treated Stage II, with greater than 2 positive lymph nodes, or Stage III breast cancer patients.

Background

ﺴﻨﺔ

The Nature of the CA 15-3 ™ Determinant. The tumor marker of choice for breast cancer is the CA 15-37M assay[1]. The Immuno 1 CA 15-374 assay measures the serum level of a mucin, the high molecular weight glycoproteinaceous product of the MUC-1 gene [2,3]. Mucins are synthesized in two forms, secreted and membrane bound. Secreted mucins lubricate and protect the lumenal surfaces of the digestive, respiratory and reproductive epithelia. The structure of the membrane mucins have been deduced from nucleotide sequences of the MUC-J gene [2, 3]. Mucins contain a peptide backbone with very high serine and threonine content which serves as a scaffold for the attachment of a high density of O-linked oligosacharride side chains [4-6]. The N-terminus of the MUC-1 mucin (known as episialin, polymorphic epithelial mucin, or epithelial membrane antigen) is followed by a mucinous domain containing a large number of repetitive sequences, tandem repeats, each of which contains 20 amino acids with a large number of serine. threonine, and proline residues. The number of tandem repeats varies from 30 to 60 due to genetic polymorphism. Homology in the tandem repeat domains between various membrane-associated mucins is low suggesting that these regions serve solely as a scaffold for the attachment of O-linked oligosaccharide. Following the tandem repeat domain is a transmembrane segment and an intracellular domain. Immediately following synthesis in the endoplasmic reticulum, the MUC-1 gene product is cleaved such that the extracellular mucinous domain is separated from the transmembrane and intracellular domains [7]. The fragments remain associated, however, through non-covalent interactions.

The function of the membrane associated mucins is not well established, but since they inhibit cellular aggregation, they may play a role as antagonists of cellular adhesion [8]. The large size of episialin suggests that it extends approximately 500 nm above the cell surface, greatly in excess of the 10 nm extent of the glycocalyx [2]. This anti-adhesion function may maintain lumenal structure by inhibiting the adhesion of the lumenal and apical surfaces. The adhesion modulating properties of membrane bound mucins may also promote tumor cell metastasis [9].

The structure of membrane bound mucins also varies between normal and tumor cells. The number of O-linked residues and the length of the oligosaccharide chains are reduced in tumor cells compared with mucins from normal tissues. This has led to the development of monoclonal antibodies (MAbs) which show differential binding to tumor and normal cells. The MAb DF3 was derived from mice immunized with a membrane enriched fraction of a metastatic human breast carcinoma, and has been shown to bind to the heptapeptide sequence TRPAPGS [10, 11]. Binding of the DF3 MAb is reduced by pretreatment of episialin with neuraminidase [12], suggesting that the oligosaccharide side chain is critical for the binding of the DF3 antibody.

The DF3 MAb has been paired with an antibody of similar specificity, 115D8, in the tumor marker assay CA 1 5-3™. Both antibodies react with different epitopes which are expressed on certain high molecular weight mucinous glycoproteins. It has been reported that the breast cancer associated antigen contains epitopes for both DF3 and 115D8, thus permitting the detection of antigen by double antibody determinant assays [2-4, 10, 11].

CA 15-37M Serum Levels in Breast Cancer. Measurement of episialin in serum using the CA 15-37M Assay has shown considerable promise in the management of breast cancer patients. The overall sensitivity of serum CA 15-37M measurement is low for Stage I and II breast cancer, but increases to approximately 60-80% in late stage patients with metastatic disease [13-15]. Accordingly, the CA 15-374 assay has not been found useful for early detection of breast cancer in the asymptomatic population, but has shown considerable utility in management of patients with breast cancer diagnosed by other means. For example, longitudinal elevations of serum CA 15-3™ in breast cancer patients with no clinical evidence of disease has been shown to coincide with cancer recurrence in 37-77% of these patients [15-20]. In one study, serial increases in serum CA 15-37M values were shown to occur prior to clinical detection in 67% of patients with breast cancer recurrence, with a lead time of 4-48 months [20]. In a similar study, CA 15-3™ was found to detect breast cancer recurrence with a median lead time of 6 months [21]. It has also been shown that changes in serum concentrations of episialin correlate with changes in the clinical course of disease in patients with active breast cancer [16,17,18,20]. In fact, in one particular study, serum CA 15-37M values correlated with clinical status in 21/22 (95%) of breast cancer

patients [15]. In another study, CA 15-3™ values increased in 19/21 (90%) of patients with progressive cancer, and decreased in 7/9 (78%) of patients with disease regression [20]. Taken together, these results indicate that longitudinal measurement of CA 15-3™ in serum may be helpful in monitoring breast cancer patients, particularly those with metastatic disease; and may also be of value in monitoring for the detection of cancer recurrence in previously treated breast cancer patients with no clinical evidence of disease.

CA 15-3 ™ Serum Levels in Non-Breast Malignancies. Some non-breast malignant diseases are associated with elevated levels of CA 15-374 assay values [1, 22, 23]. Studies examining this issue have demonstrated that the occurrence of elevated circulating levels of CA 15-3™ assay values is not specific for breast cancer. Elevated serum CA 15-37M assay values have been demonstrated in patients with advanced ovarian, cervical, and endometrial cancer [1, 22-24].

CA 15-37M Serum Levels in Healthy Controls and Benign Disease. Recent studies have defined the specificity of the CA 15-37M assay. Patients with benign breast lesions had CA 15-37M levels that were not significantly different from those of healthy controls [10, 13, 15, 17]. While third trimester pregnancy is often associated with serum CA 15-3™ concentrations of up to 50 UlmL, lactation had no detectable effect on CA 15-37M levels [1]. The upper limit of normal values of CA 15-37M assay values, as determined in different studies employing different assay systems, ranged from 15 to 50 U/mL.

Elevated serum CA 15-3™ levels have been reported to be associated with several non-malignant conditions. Like CEA, inflammatory liver disease and liver cirrhosis result in low, but significant elevations in certain patients [1]. Additionally, several studies have demonstrated the detection of slightly elevated CA 15-3TM assay values in the circulation of individuals with generalized autoimmune disease [1].

DEVICE DESCRIPTION

Indicated Use. The Bayer Immuno 1™ CA 15-3™ Assay is an in vitro device indicated for the quantitative measurement of CA 15-3™ assay values in human serum. When used in conjunction with other clinical and diagnostic procedures, serial testing with the CA 15-37M Assay is useful for monitoring the course of disease and therapy in metastatic breast cancer patients, and for detection of recurrence in previously treated Stage II, with greater than two positive Tymph nodes, or Stage III breast cancer patients. The assay is designed to run on the Bayer Immuno 17ª Immunoassay System, a fully automated random-access analyzer which performs both homogeneous and heterogeneous immunoassays.

Description of the Assay. The Bayer Immuno IIM CA 15-3™ Assay is a sandwich immunoassay in which one monoclonal antibody (115D8) is conjugated to fluorescein (R1) and a second monoclonal antibody (DF3) is conjugated to alkaline phosphatase (R2). Immuno 1 magnetic particles coated with antibody; the R1 conjugate, and patient sample, calibrator, or control are mixed simultaneously and incubated at 37℃ on the system. The R2 conjugate is then added, which binds to the immobilized CA 15-3™ to form a sandwich immunocomplex on the solid phase. The magnetic particles complexed with the immunological sandwich are then washed to separate unbound molecules, and a colorimetric substrate is added. The rate of conversion of substrate to a compound with absorbance at 405 and 450 nm is measured; the measured rate is proportional to the concentration of CA 15-3™ assay values in the sample. A cubic-through-zero curve fitting algorithm is used to generate standard curves. A schematic representation of the magnetic separation sandwich assay technique is presented in Figure 1 below.

POTENTIAL ADVERSE EFFECTS OF THE DEVICE ON HEALTH

।

The Immuno 1 CA 15-3™ Assay is intended for in vitro diagnostic use only. There are no known potential adverse effects on the health of clinically managed patients when this device is used as indicated. It is imperative that the physician use the Immuno 1 CA 15-37M test results in conjunction with the patient's overall clinical assessment and other diagnostic tests. False test results could affect physician decisions regarding treatment. If falsely low, treatment may be delayed in cases of recurring breast cancer. If falsely high, new therapy may be instituted These false positive and false negative values should not lead to patient unnecessarily. mismanagement as it is indicated that CA 15-3™ assay values be used in conjunction with the results of the patient's overall clinical assessment.

Image /page/6/Figure/1 description: The image shows a diagram of a multi-step process involving reagents, antibodies, and a sample analyte. The process includes steps labeled "INCUBATE" and "WASH", indicating a sequence of incubation and washing stages. The diagram also identifies components such as "MP REAGENT (MAGNETIC PARTICLES ANTIFLUORESCEN)", "REAGENT 2 (ENZYME LABELED MONOCLONAL ANTIBODY)", "SAMPLE ANALYTE", and "REAGENT 1 (FLUORESCENCE LABELED MONOCLONAL ANTIBODY)". The diagram illustrates the interactions between these components, likely representing a biochemical assay or detection method.

Figure 1. Schematic Representation of the Magnetic Separation Sandwich Immunoassay of the Bayer Immuno 1 m System.

Image /page/6/Figure/3 description: This image shows a graph of absorbance units (AU) per minute versus U/mL. The graph shows a linear relationship between the two variables. The data points are (0, 0.0073), (12.5, 0.0925), (25, 0.1700), (50, 0.3198), (100, 0.6031), and (200, 1.1002).

Figure 2. Standard Curve for the Bayer Immuno 1 ™ CA 15-3™ Assay.

PRECAUTIONS AND WARNINGS

This device is not indicated for breast cancer screening, as a predictor of stage of disease, or as a sole diagnostic tool to confirm the presence of malignant breast disease. CA 15-37M assay values should be used for the management of breast cancer patients in conjunction with the information from a complete clinical evaluation including physical exam and other diagnostic tests.

Confirmed breast carcinoma patients, in particular patients with Stage I or II disease, can have CA 15-37M assay values that are frequently in the same range as healthy individuals [13-15]. Additionally, patients with certain non-malignant conditions (inflammatory liver disease, liver cirrhosis, generalized autoimmune disease, and third trimester pregnancy), and patients with certain non-breast malignancies (advanced ovarian, cervical, or endometrial carcinoma) can exhibit elevations in CA 15-37M assay levels [1, 22-24]. As such, serum CA 15-37M assay levels should not be interpreted as absolute evidence of the presence of malignant disease.

The CA 15-37M concentration in a given specimen determined with assays from different manufacturers can vary due to differences in assay methodology and reagent specificity. The results reported by the laboratory to the physician must include the identity of the CA 15-37M assay used. Values obtained with different CA 15-3™ assays cannot be used interchangeably. If in the course of monitoring the patient, the assay method used for determining serial CA 15-37M levels is changed, additional sequential testing should be carried out to confirm baseline values.

SUMMARY OF STUDIES

Nonclinical studies were performed to evaluate assay specificity and interfering substances, minimum detectable concentration, imprecision, linear range, hook effect, parallelism, and spiked recovery. In addition, accuracy was evaluated by comparison of the Immuno 1 CA 15-37M Assay with the FDA-approved Biomira TRUQUANT® BR™ RIA, and Centocor CA 15-3™ RIA in a method concordance evaluation. Using a panel of 501 serum samples, this concordance analysis

evaluated the relationship between CA 15-37M assay values determined by the Immuno 1 CA 15-3™ Assay and the Biomira TRUQUANT® BR™ RIA and Centocor CA 15-3™ RIA .

NONCLINICAL STUDIES

Characterization of the Antigen. The antigen used in the Immuno 1 CA 15-3™ assay calibrators is the DF3 reactive determinant (designated DF3 antigen) isolated from the supernatant fluid of an in vitro culture of ZR75-1 Anchorage Dependent Cells. The antigen is manufactured by Centocor, Inc., and is supplied to Bayer in a partially purified form. Antigenic preparations, analyzed by non-reducing SDS-PAGE, and immunoblotted with alkaline phosphatase conjugated DF3 MAb revealed two high molecular weight bands of 290 and 490 kDa. This electrophoretic analysis demonstrated that the antigenic calibrator preparation used in the assay is consistent with previous descriptions of the CA 15-37M glycoprotein.

Immunoreactivity of the Antibodies. Monoclonal antibody preparations DF3 and 115D8 are used in the Immuno 1 CA 15-37M Assay. Both antibodies are manufactured by Centocor, Inc., and are supplied to Bayer in partially purified form.

The DF3 and 115D8 antibodies were characterized in a series of experiments. Isotype analysis demonstrated that the MAbs DF3 and 115D8 were of the murine IgG1 and IgG2b subclasses respectively. Relative affinity analysis revealed that both antibodies bound to CA 15-3™ antigen in a similar and saturable manner, and bound to "native" CA 15-37" antigen with an apparent higher relative affinity than to desialyated CA 15-374 antigen. Additionally, the binding of MAb 115D8 to native antigen was found to be more sensitive to changes in pH than was MAb DF3. Biochemical analysis of MAb preparations, under conditions of reducing or non-reducing SDS-PAGE and isoelectric focusing revealed bands characteristic of murine immunoglobulin molecules of the IgG isotype. These results demonstrate that the DF3 and 115D8 MAbs bind to the CA 15-3™ antigen quantitatively and specifically, and display biophysical properties expected of mouse monoclonal antibodies.

Specificity And Interfering Substances. The recovery of CA 15-37M assay values from the Medical Decision Pool, an internal serum-based control containing a CA 15-3™ concentration of approximately 31 UlmL, was studied before and after spiking with the potentially interfering substances listed below. Each potential interferant was tested in duplicate, at five equally spaced concentration levels, on one system, using one lot of assay reagent.

Endogenous Interferants. The Immuno 1 CA 15-37M Assay was performed on the Medical Decision Pool to which was added various concentrations of either triglycerides, immunoglobulin, hemoglobin, heparin, bilirubin, or albumin. The highest concentration of each potential interferant used was greater than that normally observed during routine clinical testing. The highest concentration of each potential endogenous interferant tested and the maximum effect on the observed CA 15-374 recovery are summarized in Table 1. None of the potential endogenous interferants demonstrated any significant interfering effects on CA 15-3TM assay recovery.

Anti-neoplastic Drug Interferants. Because of the possibility that serum CA 15-37M measurements might be performed while patients are undergoing a regimen of chemotherapy, CA 15-37M assay values were measured in the Medical Decision Pool after spiking the pool with an individual drug or a cocktail of drugs commonly used to treat The identities of the individual chemotherapeutic drugs and their final cancer. concentrations in the drug cocktail are presented in Table 2.

| Interferant | Highest Concentration
Tested | Maximum Effect |
|----------------|---------------------------------|----------------|
| Triglycerides | 900 mg/dL | -0.2% |
| Immunoglobulin | 5.3 g/dL | 0.5% |
| Hemoglobin | 1.0 g/dL | 1.6% |
| Heparin | 0.15 mg/mL | 1.3% |
| Bilirubin | 25 mg/dL | 1.3% |
| Albumin | 6.5 g/dL | -1.5% |

Table 1. Endogenous Interference

| Pool Number | Drug | Highest Concentration
Tested (1X) |
|---------------|------------------------------|--------------------------------------|
| Drug Pool #1 | Cyclophosphamide (Cytoxan) | 700 ug/mL |
| | Doxorubicin (Adriamycin) | 51.8 ug/mL |
| | 5-Fluorouracil | 346 ug/mL |
| | Methotrexate | 30 ug/mL |
| | Mitomycin C | 13.8 ug/mL |
| Drug Pool # 2 | Diethylstelbestrol | 23 ug/mL |
| | Tamoxifen Free Base | 60 ug/mL |
| | Tamoxifen Citrate (Novadex) | 60 ug/mL |
| | Vincristine | 1.4 ug/mL |
| Drug Pool # 3 | Aminoglutethemide (Cytadren) | 398 ug/mL |
| | Cis-Platin | 173 ug/mL |
| | Megace | 243 ug/mL |

Table 2. Chemotherapeutic Drugs Used For Interference Testing

Inhibition of CA 15-3™ recovery (-21.3%) by the chemotherapeutic Drug Pool # 1 was noted (Table 3). The five individual drugs which comprised Drug Pool #1 were evaluated individually. Only methotrexate exhibited any change in percent recovery with increasing drug concentration. Methotrexate, at a concentration of 300 ug/mL inhibited CA 15-37M recovery by approximately 65% as shown in Figure 3.

High dose methotrexate chemotherapy with autologous stem cell rescue is presently used in the treatment of primary breast carcinoma [25]. The use of high doses of methotrexate leading to plasma concentrations of 104 to 103 M (45.4-454 ug/mL) is sufficient to allow passive entry of methotrexate into tumor cells, overcome drug resistance, and prolong

drug action so that more tumor cells can be exposed to the drug during DNA synthesis (26). Methotrexate exhibits a "triphasic" pattern of clearance from the circulation, with the highest serum levels of the drug (104 to 103 M), exhibiting a terminal clearance halflife of 8-15 hours (average 10.4 hours) [26,27,28]. Therefore, a patient with a high therapeutic serum level of methotrexate would clear the drug from the circulation (assuming the slowest rate of elimination) to a level which will not significantly effect Immuno 1 CA 15-37M Assay quantitation after four days.

The observed methotrexate effect is not likely to result in patient mismanagement for several reasons. First. Stage II or Stage III patients, clinically free of disease, and being monitored for the early detection of breast cancer recurrence, are not likely to be receiving methotrexate therapy at the time of testing. Secondly, for patients who may be receiving methotrexate treatment, testbleeds for serum tumor marker immunoassay analysis would commonly be drawn between cycles of chemotherapy, just prior to the initiation of a new round of treatment. Given standard chemotherapeutic regimens, these serum specimens would be collected well after the last dose of drug was received, and after sufficient time to allow the clearance of methotrexate to a level which would not significantly effect the assay.

Interferant	Maximum Effect
Therapeutic Drug
Pool 1	-21.3%
Therapeutic Drug
Pool 2	0%
Therapeutic Drug
Pool 3	2.0%

Table 3. Drug Pool Interference

000210

Image /page/12/Figure/1 description: This image is a plot of the percent recovery versus methotrexate concentration. The x-axis is labeled "Methotrexate (ug/mL)" and ranges from 0 to 300. The y-axis is labeled "% Recovery" and ranges from 0 to 120. The plot shows a curve that decreases as the concentration of methotrexate increases, starting at approximately 100% recovery at 0 ug/mL methotrexate and decreasing to approximately 33% recovery at 300 ug/mL methotrexate.

Figure 3. Methotrexate Interference

Common "Over the Counter" Drugs and Vitamin Supplements. The potential interference of common vitamin supplements, "Over the Counter" (OTC) drugs, caffeine, and codeine in the Immuno 1 CA 15-37M Assay was investigated. Each of the substances were added individually to a patient sample serum pool with CA 15-37M assay concentration of approximately 100 U/mL. The concentrations of drugs added were greater than that normally observed during routine clinical testing. The concentration of each potential interferant tested and the maximum effect on the observed Immuno 1 CA 15-3™ Assay recovery are summarized in Table 4. None of the vitamins, OTC drugs, caffeine, or codeine demonstrated any significant interfering effects on CA 15-37M assay recovery.

000211

| Interferant | Highest Concentration
Tested | Maximum Effect |
|-----------------|---------------------------------|----------------|
| Vitamin A | 10 IU/mL | 0.2% |
| Thiamin (B1) | 3.0 µg/mL | 0.3% |
| Riboflavin (B2) | 3.4 µg/mL | 0.7% |
| Vitamin B6 | 4.0 µg/mL | 0.0% |
| Vitamin B12 | 0.012 µg/mL | -2.7% |
| Vitamin C | 30 µg/mL | -1.5% |
| Vitamin D2 | 0.8 IU/mL | -0.3% |
| Vitamin E | 0.6 IU/mL | 1.9% |
| Niacin | 0.4 mg/mL | 2.1% |
| Folic Acid | 0.8 µg/mL | -3.5% |
| Caffeine | 100 µg/mL | 1.1% |
| Codeine | 240 µg/mL | -2.1% |
| Acetaminophen | 200 µg/mL | -2.7% |
| Ibuprofen | 400 µg/mL | -1.5% |
| Aspirin | 500 µg/mL | 0.6% |

Table 4. Common vitamins, OTC Drugs and Codeine Interference

Heterophilic Antibodies. To investigate the effectiveness of the assay's reagent formulation in minimizing heterophilic antibody interferences, ten samples with high rheumatoid factor titers and six samples with high human anti-mouse antibody titers were assayed with two lots of reagents. All samples were tested both undiluted and at a 50% dilution using the Level 1 Immuno 1 CA 15-3™ calibrator (containing no CA 15-37M antigen). All samples recovered CA 15-3™ assay values linearly when diluted 50% with the Level 1 CA 15-37M calibrator. The percent recovery for all samples ranged from 83.9% to 92.6%. This observed linear CA 15-3™ recovery indicates a lack of significant heterophilic interference in the assay and demonstrates the effectiveness of the reagent formulation in minimizing these interferences.

To determine if CA 15-3TM assay recoveries are linear over the entire calibration Linearity. range, three clinical sample pools containing a high concentration of CA 15-37M assay values were diluted with normal serum (low CA 15-37M assay values) to final concentrations of 100%

(undiluted) 75%, 50%, 25%, and 0% (low CA 15-3™ serum only). Each pool was assayed with two lots of Immuno 1 CA 15-3TM reagent.

The three pools of sera with CA 15-37M assay values of approximately >180 U/mL diluted linearly. Recoveries of the intermediate dilutions were all between 98.2% and 105.2% of the expected value. These results clearly demonstrate the linearity of CA 15-3™ assay recoveries over the entire calibration range.

Hook Effect. Extremely high concentrations of CA 15-374 assay values seen in some malignant conditions may cause a "hook effect" in an assay. An excess of analyte saturates both label and capture antibody and causes the reported concentration to "hook" back into the assay range rather than be flagged as above range. CA 15-37M antigen, isolated from human ascites and purchased from BioDesign (Kennebunk, ME), was value-assigned by testing on the Immuno 1, and diluted in Level 1 calibrator at concentrations of 28500, 14250, 7125, 3562, 1781, 890, 445, 223, 111, 56, 28, 14, and 7 U/ml. This collection of samples was tested in the assay using two lots of reagents. The assay response, measured as the observed reaction rate, did not "hook" back into the assay range, even at the highest concentration of antigen tested (28.5 kU/mL). These results demonstrate the lack of a hook effect in the Immuno 1 CA 15-3™ Assay at CA 15-3TM assay values ≤ 28,500 U/mL.

Spiked Recovery. To determine how well CA 15-37M antigen, when spiked into a patient sample, is recovered by the Immuno 1 CA 15-37M Assay, antigen was spiked into seven patients at two different CA 15-3™ concentrations (30 and 120 U/mL). Each sample was assayed in quadruplicate with one lot of Immuno 1 CA 15-3™ reagent.

Recoveries of assay values for all samples ranged from 92.9 to 122.9%. No significant deviation was noted with regard to expected versus observed assay values. These results demonstrate the accurate quantitation of spiked and recovered CA 15-3™ assay values using the Immuno 1 assay.

Parallelism. To confirm the use of the Immuno 1 CA 15-3™ Level 1 calibrator (0.0 U/mL) as a sample diluent and to further verify assay linearity, four patient serum sample pools containing a high level of CA 15-3™ values were diluted with Level 1 calibrator to final concentrations of 100% (undiluted), 75%, 50%, 25%, and 0% (Level 1 calibrator only). Each dilution of each sample pool was assayed with two lots of reagents. Linear regression analysis for the determination of deviations from linearity for each of these clinical samples showed no deviation from linearity. The recovery of CA 15-37M assay values ranged from 88.2-116.9%. The accurate recovery of CA 15-3™ assay values in diluted patient samples further illustrates the linearity of the Immuno 1 CA 15-3™ Assay throughout the entire calibration range and confirms the use of the Immuno 1 CA 15-3™ Level 1 calibrator as a sample diluent.

)

Minimum Detectable Concentration. Analytical sensitivity of the Immuno 1 CA 15-3™ assay was evaluated by determination of the Minimum Detectable Concentration (MDC). The MDC is defined as the minimum concentration of CA 15-37M which can be statistically distinguished from the concentration of the lowest standard as calculated from a typical standard curve. Specifically, the MDC of the Immuno 1 CA 15-3™ assay was determined as the CA 15-3™ concentration corresponding to an absorbance value equal to two within-run standard deviations above the mean absorbance value of the zero calibrator.

An MDC of 0.13 UlmL was observed, based on 1336 determinations of the Level 1 calibrator (0 U/mL), at four clinical trial sites, using three lots of calibrators and three lots of reagents. This level of analytical sensitivity is acceptable for an assay of this type with an upper limit of normal at approximately 35 UlmL. This level of sensitivity exceeds the Immuno 1 CA 15-3™ Method Sheet claim of 0.2 U/mL.

Intra- and interassay reproducibility were evaluated for two levels of Imprecision. commercial controls, three lots of Technicon SETpoint™ CA 15-3™ calibrators, and an internal human serum control material at the medical decision level. These materials were assayed with three lots of reagent over a period of 10 days in two runs per day at four

clinical trial sites. Imprecision estimates pooled across sites, reagent lots, and calibrator lots are shown in Table 5.

Table 5

lmmuno 1 CA 15-3™

Pooled Across Clinical Trial Sites, Reagent and Calibrator Lots
				Within-Run		Total
	Product
/Levels	Mean
(U/mL)	Number of
Observations	SD
(U/mL)	CV
(%)	SD
(U/mL)	CV
(%)
	CONTROLS: BioRad Lot 44010, Immuno 1 CA 15-3 Medical Decision Pool Lot
	BioRad 1	13.5	445	0.46	3.4	0.54	4.0
	BioRad 2	35.0	445	0.84	2.4	1.09	3.1
	MDP	33.2	446	0.53	1.6	1.09	3.3
	Technicon SETpoint CA 15-3 Calibrators: Pooled Across Lots
	Level 1	0.1	774	0.05	-	0.06	-
	Level 2	13.3	1325	0.23	1.8	0.47	3.5
	Level 3	26.0	1338	0.55	2.1	0.96	3.7
	Level 4	50.7	1340	1.22	2.4	1.85	3.7
	Level 5	102.3	1334	1.89	1.9	3.52	3.4
	Level 6	190.9	301	2.47	1.3	5.73	3.0

Within-run CVs ranged from 1.3% to 3.4%, while total CVs ranged from 3.0%. Excellent precision was obtained at all analyte concentrations tested covering the range of the assay (0.0 to 200 UlmL). These results show that the recovery of Immuno 1 CA 15-374 assay values are highly reproducible over time, using different lots of reagent, when tested in different laboratories.

METHOD COMPARISON STUDIES

Introduction. The objective of the method comparison studies was to examine the concordance of sample assay values obtained using the Bayer Immuno 1™ CA 15-3™ Assay with those obtained using the Biomira TRUQUANT® BR™ RIA and the Centocor CA 15-3™ RIA. Patient sample CA 15-3™ or BR 27.29 values generated by the three methods were compared by correlation analysis and a determination of normal range cutoff.

ﻤﺴﺴﺴﺴ

Human serum samples from approximately 501 patients obtained from BioClinical Partners, Inc. (Sharon, MA), and our in-house specimen collection (Bayer Corporation, Business Group Diagnostics; Tarrytown, NY), were analyzed using the Immuno 1 CA 15-3™ Assay, the Biomira TRUQUANT® BR™ RIA and the Centocor CA 15-3™ RIA. The samples were analyzed at two investigational sites. The Immuno 1 testing and Centocor RIA analysis was performed in the Research and Development Laboratories at Bayer Corporation, Tarrytown, N.Y. The Biomira RIA analysis on all patient samples was performed by Dianon Systems, Inc. in their laboratories in Stratford, CT. Samples above the upper limit of the Immuno 1, Biomira, or Centocor standard curves were diluted and re-assayed. The number, source, and clinical classification of the patient samples used in this concordance study are summarized in Table 6.

| CLINICAL

CLASSIFICATION	SOURCE	NUMBER OF SAMPLES
Normal (Premenopausal)	BioClinical Partners	100
Normal (Postmenopausal)	BioClinical Partners	100
Breast Cancer (Single Point)	Bayer Diagnostics	150
Lung Cancer	BioClinical Partners	35
Ovarian Cancer	BioClinical Partners	35
Colorectal Cancer	BioClinical Partners	35
Benign Breast Disease	BioClinical Partners	46
Total		501

Table 6. Summary of Patient Samples Used In The Clinical Concordance Evaluation.

Normal Range. The range of values for normal specimens was determined for all methods by calculation of the mean, median, standard deviation, range, mean + 1.96 x SD, and 97.5th percentile of CA 15-3TM or BR 27.29 assay values in 199 normal healthy females (100 premenopausal and 99 post-menopausal).

The results of the normal range analysis are present in Table 7 and Figure 4. The mean serum CA 15-3™ or BR 27.29 values for all samples analyzed by the Immuno 1 CA 15-3™ Assay, the Biomira TRUQUANT® BR™ RIA and the Centocor CA 15-37M RIA were 17.1 U/mL, 17.6 U/mL and 18.6 U/mL respectively. The mean value + 1.96 x SD gave an upper range of 32.9

U/mL for the Immuno 1 CA 15-3™ Assay, 30 U/mL for the Biomira TRUQUANT® BR™ RIA and an upper range of 33.5 U/mL for the CA 15-3™ Centocor RIA. The 97.5th percentile normal range cut-off for the Immuno 1 was 35.9 U/mL for the Biomira RIA and 35.6 U/mL for the Centocor RIA. As shown in Table 7 and Figure 4, there were no significant differences between the three assays in the CA 15-37M or BR 27.29 mean, median, or cutoff ranges for the entire collection of specimens. Notably, all three methods denoted modest differences in the mean, median and cutoff ranges for the pre- versus the post-menopausal samples (Table 7). Overall, the means and ranges of the Biormira RIA assay values (Figure 4) were very similar to those generated by the Immuno 1 CA 15-3™ assay and, thereby demonstrate the concordance of the two methods in MUC -1 gene product quantitation.

	PRE-MENOPAUSAL			POST-MENOPAUSAL			TOTAL
	IMMUNO
1	CENTOCOR	BIOMIRA	IMMUNO
1	CENTOCOR	BIOMIRA	IMMUNO
1	CENTOCOR	BIOMIRA
n	100	100	100	99	99	99	199	199	199
Mean	14.1	15.5	15.7	20	21.7	19.6	17.1	18.6	17.6
Median	13.9	14.5	15	19.2	21	18.7	14.9	16.8	16.4
Range	4.2-32.4	4.4-31.7	6.6-34.7	5.9-43.6	8.1-43.2	8.9-36	4.2-43.6	4.4-43.2	6.6-36
Mean
+1.96 SD	25.1	25.7	25.9	37.8	38.2	32.3	32.9	33.5	30
97.5th
Percentile	27	27.1	25.7	38.9	41.5	33.2	35.9	35.6	31.3

Tuble 7. Normal Range Cut-off Analysis Results (U/mL)

Image /page/18/Figure/4 description: This image is a bar graph that shows the frequency of assay values for three different substances: Biomira, Centocor, and Immuno 1. The x-axis represents the assay values in U/mL, ranging from 5 to 45. The y-axis represents the frequency, ranging from 0 to 70. The graph shows that Centocor has the highest frequency at an assay value of 15, while Immuno 1 has a high frequency at an assay value of 10.

Figure 4. Distribution of Normals

Method Concordance. In order to compare the values obtained from serum samples analyzed by the Immuno 1 CA 15-3™ Assay with Biomira TRUQUANT® BR™ and Centocor CA 15-3™ RIA derived values, a correlation study using 501 female serum samples was performed. The 501 serum samples consisted of approximately 199 normal samples (pre- and post-menopausal), 150 single point breast cancer specimens, 35 lung cancer specimens, 35 ovarian cancer specimens, 35 colorectal cancer specimens, and 46 benign breast disease samples, and one unknown disease sample with elevated an CA 15-37M and BR 27.29 value. Ordinary least squares linear regression statistics were used to compare the assay values obtained.

The calculated statistics for sample results that were within the linear range (i.e. standard curve range) of the Immuno 1 Assay, Biomira RIA and the Centocor RIA are presented in Table 8. These results demonstrate that the quantitation of CA 15-3™ and CA 27.29 assay values by the methods is concordant and equivalent.

| | CA 15-3 TM CORRELATION
(STANDARD CURVE RANGE SAMPLES ONLY) | | | | |
|-----------------------|---------------------------------------------------------------------------|-----------|-----|------|-------|
| | ORDINARY LINEAR LEAST SQUARES
REGRESSION | | | | |
| | SLOPE | INTERCEPT | N | R | Sy.x |
| Immuno 1 vs. Biomira | 0.852 | 3.48 | 498 | 0.93 | 11.40 |
| Immuno 1 vs. Centocor | 0.791 | 3.23 | 499 | 0.96 | 9.26 |

Table 8. Correlation of All Methods (Standard Curve Range Samples Only)

Conclusions from Method Comparison Studies. The results of this comparative clinical analysis of serum assay values in a panel of healthy subjects, and patients with non-malignant and malignant diseases clearly demonstrates the concordance of the Immuno 1 CA 15-3™ Assay with the Biomira TRUQUANT® BR™ RIA results. Normal reference ranges were essentially equivalent, and correlation showed good concordance between the Immuno 1 and the predicate device.

REAGENT STABILITY TESTING

Shelf-Life Stability Testing. Reagents were subjected to temperature stress at 25°C, 30°C, and 40°C and tested at selected time points. Additional reagent stored at 2-8°C was tested in parallel with the stressed reagents at all time points tested. At each timepoint, the sensitivity of the calibrators, as well as the recovery of controls and the Medical Decision Pool (a serum based control material manufactured at Bayer Corporation and used internally for routine quality control) were monitored.

A shelf-life of 18 months is recommended for CA 15-3™ reagents. The data for the four lots (Experimental 1 & 2 ; Trials 1 & 2) indicated that the product will still be well within the product stability specifications at 18 months. In all cases, at the latest time point tested, calibrator sensitivity (2-8°C) and control recoveries were within acceptable limits.

On-System Stability Testing. Testing of the Experimental lots was performed over a 32 day period every 26 weeks for the entire shelf life of the reagent. Five packs of reagents were opened at the beginning of each 32 day study. One reagent pack was tested at each time point (days 0, 7, 14, 21, and 32) for each study performed at weeks 0, 26, 52, 78 and 104. During the testing of the Trial reagent lots, one reagent pack remained on the system for the duration of the 32 day study and was performance tested at the defined time points (Davs 0. 7. 14. 21. and 32), Additionally, a fresh reagent pack, maintained at 2-8℃ was run at each time point in parallel with the test reagent pack as a control. At each timepoint, the sensitivity of the calibrators, as well as the recovery of controls and the Medical Decision Pool were monitored against both the day 0 and individual time point calibration curves (Day 7, 14, 21, and 32). On-system stability studies on four lots of Immuno 1 CA 15-37M Assay reagents support an on-system stability recommendation of 30 days. The specifications for minimum calibrator sensitivity and control recovery were met throughout all studies.

Shipping Stabilty Testing. Three lots of reagents were subjected to three heat/chill cycles (three days at 40°C, then three days at 2-8°C). Following these cycles, the reagents were stored at 2-

8°C. and performance was evaluated at selected timepoints. Additional reagents were stored at 2-8°C and run alongside the stressed reagents at all time points. The sensitivity of the calibrators and the recovery of controls and the Medical Decision Pool were monitored at each timepoint.

No significant change in control recovery, as compared to the non-stressed control reagent, was noted throughout the duration of testing. Recoveries for the cycled reagent remained well within specification. Shipping, temperature stress, and temperature cycling stability data on three lots of Immuno 1 CA 15-37M assay reagents support the requirement of refrigerated (2-8°C) shipping of these reagents.

Reagent stability is summarized in the Immuno 1 CA 15-3™ method insert sheet. Labeling. Expiration dates are also indicated on the labels of each reagent kit.

CALIBRATOR STABILITY TESTING

)

Shelf-Life Stability Testing. The calibrator U/mL
N (%) | > 100 to 200
U/mL
N (%) | > 200 U/mL
N (%) | Median
U/mL |
|-------------------------|-----|----------- | HEALTHY | | | Premenopausal | 216 | 216 (100%) | Postmenopausal | 202 | 182 (90.0%) | TOTAL HEALTHY | 418 | 398 (95.2%) | PREGNANT | 81 | 78 (96.3%) | BENIGN BREAST | 114 | 109 (95.6%) | NON-BREAST BENIGN | | | Autoimmune/Inflamm. | 55 | 45 (81.8%) | GI/Pancreas/Gallbladder | 51 | 51 (100%) | Gynecological | 13 | 12 (92.3%) | Urinary | 7 | 5 (71.4%) | Cardiopulmonary | 26 | 21 (80.8%) | Liver | 21 | 19 (90.5%) | Benign Other | 7 | 6 (85.7%) | TOTAL non-breast benign | 180 | 159 (88.3%) | NON-BREAST CANCER | | | Bladder/Urinary | 22 | 21 (95.5%) | Cervix | 18 | 15 (83.3%) | Colorectal/Stomach | 43 | 39 (90.7%) | Liver | 10 | 9 (90.0%) | Lung | 32 | 23 (71.9%) | Ovary | 45 | 27 (60.0%) | Pancreas | 18 | 12 (66.7%) | Uterus | 13 | 9 (69.2%) | Other Ca. | 9 | 7 (77.8%) | TOTAL non-breast cancer | 210 | 162 (77.1%) | BREAST CANCER | | | Stage 1 | 71 | 55 (77.5%) | Stage 2 | 79 | 65 (82.3%) | Stage 3 | 68 | 42 (61.8%) | Stage 4 | 98 | 29 (29.6%) lots were stressed at 2-8°C, 30°C and 40°C and tested at selected timepoints. Additional calibrators stored at 35 to 100
------------|------------------------------|-------------------------------|---------------------|----------------|
| | | | |
| 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 15.1 |
| 20 (10%) | 0 (0.0%) | 0 (0.0%) | 18.9 |
| 20 (4.8%) | 0 (0.0%) | 0 (0.0%) | 16.3 |
| 3 (3.7%) | 0 (0.0%) | 0 (0.0%) | 18.3 |
| 5 (4.4%) | 0 (0.0%) | 0 (0.0%) | 20.4 |
| | | | |
| 10 (18.2%) | 0 (0.0%) | 0 (0.0%) | 22.6 |
| 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 16.7 |
| 1 (7.7%) | 0 (0.0%) | 0 (0.0%) | 15.9 |
| 2 (28.6%) | 0 (0.0%) | 0 (0.0%) | 15.6 |
| 5 (19.2%) | 0 (0.0%) | 0 (0.0%) | 16.0 |
| 2 (9.5%) | 0 (0.0%) | 0 (0.0%) | 15.8 |
| 1 (14.3%) | 0 (0.0%) | 0 (0.0%) | 14.9 |
| 21 (11.7%) | 0 (0.0%) | 0 (0.0%) | -- |
| | | | |
| 0 (0.0%) | 0 (0.0%) | 1 (4.5%) | 21.1 |
| 3 (16.7%) | 0 (0.0%) | 0 (0.0%) | 18.1 |
| 4 (9.3%) | 0 (0.0%) | 0 (0.0%) | 19.5 |
| 1 (10.0%) | 0 (0.0%) | 0 (0.0%) | 19.7 |
| 6 (18.8%) | 0 (0.0%) | 3 (9.4%) | 25.2 |
| 12 (26.7%) | 3 (6.7%) | 3 (6.7%) | 29.8 |
| 2 (11.1%) | 4 (22.2%) | 0 (0.0%) | 22.0 |
| 3 (23.1%) | 0 (0.0%) | 1 (7.7%) | 24.7 |
| 2 (22.2%) | 0 (0.0%) | 0 (0.0%) | 24.9 |
| 33 (15.7%) | 7 (3.3%) | 8 (3.8%) | 23.3 |
| | | | |
| 11 (15.5%) | 3 (4.2%) | 2 (2.8%) | 23.0 |
| 8 (10.1%) | 0 (0.0%) | 6 (7.6%) | 23.2 |
| 18 (26.5%) | 4 (5.9%) | 4 (5.9%) | 26.6 |
| 33 (33.7%) | 17 (17.3%) | 19 (19.4%) | 56.7 |

CONCLUSIONS DRAWN FROM ALL THE STUDIES

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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Valid Scientific Evidence. The conclusions drawn from these studies are based upon valid scientific evidence. Data were gathered following a well designed protocol, in research laboratories operating under the principles of Good Laboratory Practices. The patient population was well characterized and patient histories were thoroughly documented.

Method Performance Immuno 1 CA 15-3™ Assay nonclinical performance, including analytical sensitivity (minimum detectable concentration), imprecision, parallelism, linear range, hook effect, and spiked recovery met accepted specifications for an assay of this type.

Safety and Effectiveness. The clinical studies demonstrate the safety and effectiveness of serial Immuno 1 CA 15-37M Assay values for monitoring the course of disease and therapy in metastatic breast cancer patients, and for early detection of recurrence in previously treated Stage II or Stage III breast cancer patients who are clinically free of disease. The correlations between Immuno 1 CA 15-37M Assay values and disease course demonstrate that this assay may be used in conjunction with other clinical indicators to monitor success or failure of therapy for metastatic disease and to signal possible recurrence to malignant disease.

Substantial Equivalence The method concordance studies confirm the substantial equivalence of the Immuno 1 CA 15-37M Assay with the Biomira TRUQUANT® BR™ RIA predicate device. There is a high degree of correlation between the Immuno 1 and Biomira TRUQUANT® BR™ specimen values. Multi-site clinical studies demonstrated longitudinal sensitivity and specificity for predicting recurrence of disease are equivalent for the two tests. Therefore, based upon the analytical and clinical concordance established in these studies, the Bayer Immuno 1 CA 15-3™ Assay and the Biomira TRUQUANT® RIA are equivalent with respect to method performance, clinical utility and device safety and effectiveness.

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ਤੇ ਤੋ

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Image /page/35/Picture/1 description: The image shows the logo for the U.S. Department of Health & Human Services. The logo consists of a stylized eagle with its wings spread, and the text "DEPARTMENT OF HEALTH & HUMAN SERVICES • USA" arranged in a circular fashion around the eagle. The logo is black and white.

Public Health Service

DEC - I 1997 Food and Drug Administration 2098 Gaither Road Rockville MD 20850

Gabriel J. Muraca, Jr. Manager, Requlatory Affairs BAYER CORPORATION 511 Benedict Avenue Tarrytown, NY 10591-5097

Re: K964703 Trade Name: Bayer Immuno 1 TM CA 15-3TM Assay Regulatory Class: II Product Code: MOI, 82 Dated: November 21, 1996 November 22, 1996 Received:

Dear Mr. Muraca:

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 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 requlations.

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 (770)488-7655.

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 (301) 594-4588. Additionally, for questions on the promotion and advertising of your device, please contact the Office of Compliance at (301) 594-4639. 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 (800) 638-2041 or at (301) 443-6597 or at its internet address "http://www.fda.gov/cdrh/dsmamain.html"

Sincerely yours,

Steven Butman

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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K964703 510(k) Number (if known):

Bayer Immuno 17M CA 15-37M Assay Device Name:

Indications For Use:

The Bayer Immuno 17M CA 15-37M Assay is an in vitro, diagnostic, solid phase immunoassay intended to quantitatively measure CA 15-3 Assay values in human serum on the Bayer Immuno 1 system. When used in conjunction with other clinical and diagnostic procedures, serial testing with the CA 15-3 Assay is useful for monitoring the course of disease and therapy in metastatic breast cancer patients, and for detection of recurrence in previously treated Stage II, with greater than 2 positive lymph nodes, or Stage III breast cancer patients.

(PLEASE DO NOT WRITE BELOW THIS LINE - CONTINUE ON ANOTHER PAGE IF NEEDED)

Concurrence of CDRH, Office of Device Evaluation (ODE)

Prescription Use (Per 21 CFR 801.109)

(Division Sign-Om
Division of Clinical Laboratory Devigee
510(kumber K964703 Over-The-Counter Use

Optional Format 1-2-96)