The OSDI HER-2/neu Assay is an in vitro, diagnostic device intended for use in the quantitative determination of serum HER-2/neu in women with metastatic breast cancer who have an initial value of 15 ng/ml or greater. HER-2/neu values obtained may be used in the follow-up and monitoring of patients with metastatic breast cancer. HER-2/neu values should be used in conjunction with information available from clinical and other diagnostic procedures in the management of breast cancer. The clinical utility of the serum measurement of HER-2/neu as a prognostic indicator for early recurrence and in the management of patients on immunotherapy regiments has not been fully established.

Device Description

The HER-2/neu ELISA is a sandwich enzyme immunoassay, which utilizes two monoclonal antibodies to quantitate the extracellular domain (ECD) of the HER-2/neu protein in serum. The capture antibody (NB-3) has been immobilized on the interior surface of the microtiter plate wells. To perform the assay, an appropriate volume of serum is incubated in the coated well to allow binding of the antigen by the capture antibody. The captured HER-2/neu ECD is then reacted with a different anti-HER-2/neu antibody designated TA-1. The TA-1 antibody is biotinylated. The detection of the ECD of HER-2/neu is with a streptavidin horseradish peroxidase conjugate, which then catalyzes the conversion of the chromogenic substrate o-phenylenediamine into a colored product. The colored reaction product is quantitated by spectrophotometry (read absorbance at 490 mm) and is related to the amount of the HER-2/neu ECD in the serum sample. Six prepared HER-2/neu standards (0, 2.5, 7.5, 15, 25 and 35 ng/ml) allow construction of a standard curve for subsequent quantification of HER-2/neu in serum samples. Also available from OSDI is a set of HER-2/neu controls (10-CVX) designed to be used in conjunction with the ELISA for quality monitoring of assay performance. This control set consists of three (3) control levels at 2.9, 9.1 and 24.0 ng/mL HER-2/neu protein.

AI/ML Overview

The Oncogene Science Diagnostics, Inc. (OSDI) Manual HER-2/neu Microtiter ELISA is an in vitro diagnostic assay intended for the quantitative measurement of the extracellular domain of HER-2/neu protein in human serum. It is used in the follow-up and monitoring of patients with metastatic breast cancer whose initial serum level of HER-2/neu is 15 ng/mL or greater, in conjunction with other clinical and diagnostic procedures.

Here's an analysis of the acceptance criteria and the study proving the device meets those criteria:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are not explicitly stated in a quantitative table within the document but can be inferred from the performance metrics described for various aspects of the assay. The "reported device performance" are the results from the non-clinical and clinical studies.

Acceptance Criteria (Inferred)	Reported Device Performance
Specificity: Interference	"None of the potential endogenous or exogenous interferents demonstrated any significant interfering effects on HER-2/neu recovery." (Page 6)
Specificity: Cross-Reactivity	"The maximum effect seen with [Human Epidermal Growth Factor Receptor (HER-1)] cross-reactant was not significant (≤1%)... no cross-reactivity with HER-3." (Page 7)
Specificity: Heterophilic Antibodies and HAMA Interference	"HAMA and rheumatoid factors do not cause false negative or false positive results in the present assay format" when detector diluent contained Normal Mouse Serum (NMS). (Page 7)
Linearity	"When observed HER-2/neu concentration was plotted versus expected concentration, the slope and correlation coefficient of each data set were close to the optimum of 1, indicating linearity within the dynamic range of the assay." (Page 7)
Spike and Recovery (Matrix Effect)	"The total average percent recovery of all spiked serum samples in all three kit lots was 103%, indicating no effect of matrix on the ability of the ELISA to accurately measure HER-2/neu protein in serum." (Page 8)
Hook Effect (Antigen Excess)	"Separation of sample and reagent addition by washes prevents high-dose hook effect in this device." (Page 8)
Parallelism (Dilution Studies)	"Recoveries were evaluated at each of the dilution points for mean and standard deviation and were comparable for all three ELISA kit lots. Serum samples diluted in kit Sample Diluent result in accurate recovery of analyte at a range of concentrations in human samples." (Page 8)
End-to-End Variation (Robustness to timing)	"The expected variance in timing during normal use by skilled individuals should fall well short of these extended times tested and recoveries should be unaffected." (Page 9 - small increase/decrease (15%) observed only when times extended well beyond normal usage)
Plate Coating Variations	"Serum sample recovery, and therefore antibody coating, is consistent throughout plates and kit lots with 4.7-7.6 % CV." (Page 9)
Reproducibility (Intra- and Inter-assay)	OSDI Site, All Microtiter ELISA Lots Combined (Table 4.3-1): - Control 1 (24.2 ng/mL): Within Run %CV 3.8, Total %CV 7.8 - Control 2 (9.5 ng/mL): Within Run %CV 4.7, Total %CV 9.1 - Control 3 (2.9 ng/mL): Within Run %CV 7.8, Total %CV 10.6 - Control 4 (9.2 ng/mL): Within Run %CV 4.5, Total %CV 8.3 Three Sites and Three Kit Lots Combined (Table 4.3-2): - Control 1 (24.5 ng/mL): Within Run %CV 6.0, Total %CV 10.7 - Control 2 (9.8 ng/mL): Within Run %CV 7.0, Total %CV 10.4 - Control 3 (3.3 ng/mL): Within Run %CV 10.2, Total %CV 17.7 - Control 4 (9.5 ng/mL): Within Run %CV 6.9, Total %CV 10.8 "Within run CV's of 6-7% and total CV's of 10-11% for clinically relevant controls... well within acceptable limits for an assay of this type and for its intended use." (Page 9)
Sensitivity (Detection Limit)	"An MDD of 1.5 ng/mL was observed when assaying 232 replicates of Sample Diluent alone in three HER-2/neu ELISA kit lots. This MDD is acceptable for the intended use of this assay, where the lowest normal patient was 4.23 ng/ml and the cutoff determined in this study for elevated HER-2/neu values was 1.5 ng/ml." (Page 10)
Clinical Utility (Correspondence between HER-2/neu changes and clinical course)	Table 4.4-1: Correspondence of Metastatic Breast Cancer Patient Disease Status with Changes in Serum HER-2/neu: Visit-by-Visit - Change in HER-2/neu ≥20% ↑: 52 cases of Progression, 33 cases of No Progression (Total 85) - Change in HER-2/neu <20% ↑: 55 cases of Progression, 96 cases of No Progression (Total 151) Overall: "Data collected from this study show that changes in serum HER-2/neu concentrations over time in initially elevated metastatic breast cancer patients reflect changes in clinical status such as response to therapy or progression of disease." (Page 13-14)
Clinical Sensitivity and Specificity for Elevated HER-2/neu Cutoff	"The Upper Limit of Normals in this study with the ELISA was 15 ng/ml, which was confirmed as an appropriate cutoff by ROC analysis to give 95% specificity." (Page 12)

2. Sample size used for the test set and the data provenance

Test Set Sample Size:
- Non-clinical studies: The exact number of samples for each non-clinical test (interference, cross-reactivity, linearity, spike and recovery, hook effect, parallelism, end-to-end variation, plate coating variations, reproducibility, sensitivity) is not always explicitly stated as a single total number. For example:
  - Reproducibility: 130-131 runs and 385-390 replicates combined across three sites and three kit lots for 4 controls (Table 4.3-2).
  - Sensitivity (MDD): 232 replicates of Sample Diluent alone.
  - Interference: Not specified, but involved numerous compounds tested.
  - Linearity: Two controls combined to produce five samples.
- Clinical studies:
  - Longitudinal monitoring: 56 clinically evaluable metastatic breast cancer patients with initial elevated HER-2/neu (≥15 ng/mL). There were 236 visit-by-visit comparisons (Table 4.4-1).
  - Upper Limit of Normals determination: 38 healthy women (for longitudinal variability of 20% criterion).
  - Clinical sensitivity/specificity to establish 15 ng/ml cutoff: "patients with breast cancer," "patients with benign breast diseases," "other non-malignant diseases," and "normal healthy individuals." Specific numbers for each group are not provided for this specific analysis.
Data Provenance: Retrospective (Page 10, 12).
Country of Origin: The clinical evaluations were conducted at "two US clinical trial sites" (Page 2, 3), and studies were performed at OSDI, Cambridge, Massachusetts (Page 3).

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts

Number of Experts: Not explicitly stated as a specific number.
Qualifications of Experts: For the clinical studies, the "Clinical course or status was determined by the physician" (Page 10). It is implied these are qualified medical professionals, but specific qualifications (e.g., "radiologist with 10 years of experience") are not detailed.

4. Adjudication method for the test set

The document implies that "Clinical course or status was determined by the physician" (Page 10). This suggests individual physician assessment rather than a detailed, multi-reader, adjudicated ground truth process for each patient case, at least as explicitly described for the clinical status.
For the visit-by-visit analysis (Table 4.4-1), changes greater than or equal to 20% in HER-2/neu values were considered reflective of disease progression, while less than 20% increase reflected lack of progression. This 20% criterion was internally derived from the longitudinal variability in normal patients (six serial samples from 38 healthy women), suggesting an objective, pre-defined threshold rather than expert adjudication for each change.

5. If a multi reader multi case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance

No, a multi-reader multi-case (MRMC) comparative effectiveness study was not done. This device is an immunoassay (ELISA), not an AI-powered image analysis tool that would typically involve human readers.

6. If a standalone (i.e. algorithm only without human-in-the-loop performance) was done

Yes, the device was evaluated in a standalone manner. The ELISA assay itself provides quantitative values for HER-2/neu. The non-clinical studies (e.g., interference, linearity, reproducibility, sensitivity) directly assess the performance of the assay independent of human interpretation or intervention beyond performing the laboratory procedure correctly. The clinical studies then correlated the values generated by the ELISA with the clinical course of the disease, but the device itself is a measurement tool, not an interpretive algorithm that a human would then refine.

7. The type of ground truth used

Non-clinical studies:
- Defined Antigen: The standard antigen (p105) itself is a characterization of the "ground truth" for what the assay is supposed to detect (Page 3).
- Spiked Samples/Known Concentrations: For analytical performance like linearity, spike and recovery, and hook effect, samples with known, defined concentrations of HER-2/neu were used as ground truth.
- Clinical Studies:
  - Clinical Course/Status Determined by Physician: For the metastatic patient serial monitoring, the ground truth for disease progression or lack thereof (stable/responding disease) was the "clinical course or status determined by the physician" (Page 10). This would be based on a combination of medical assessments and other diagnostic procedures.
  - Retrospective Sample Storage: The ground truth relied on patient samples collected and stored in specimen banks, with the assumption that HER-2/neu concentration remained stable during storage (Page 10).
  - Statistical Threshold: The 20% change criterion for correlating HER-2/neu values with clinical status was established based on longitudinal variability in normal patients, acting as a statistical ground truth for what constitutes a "significant" change (Page 12).
  - ROC Analysis: Used to confirm 15 ng/ml as an appropriate cutoff for 95% specificity, likely against a diagnosed patient population as ground truth (Page 12).

8. The sample size for the training set

The document does not explicitly describe a separate "training set" in the context of typical machine learning models. Instead, the non-clinical studies (which involve extensive characterization of the assay's performance) and the selection of assay parameters (like antibody choice or standard curve concentrations) can be thought of as the "development" phase that would precede a formal clinical validation. The standard development of an ELISA kit does not typically involve a "training set" in the same way an AI/ML model does.

9. How the ground truth for the training set was established

As a traditional immunoassay, there isn't a "training set" in the machine learning sense. The assay's "ground truth" during its development would have been established through:
- Characterization of the Antigen and Antibodies: (Pages 3-4) Understanding the specific extracellular domain of HER-2/neu (p105) and developing highly specific monoclonal antibodies (NB-3 and TA-1) that bind to it. This involves biological and biochemical validation.
- Standard Curve Development: Using prepared HER-2/neu standards of known concentrations (0, 2.5, 7.5, 15, 25, and 35 ng/ml) to establish accurate quantification (Page 2).
- Analytical Validation: Extensive non-clinical studies to demonstrate performance characteristics (specificity, linearity, sensitivity, reproducibility) using samples with known properties (e.g., spiked samples, controls derived from cell lines or serum pools). (Pages 3-9)

Summary

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SEP 2 9 2000 510(k) SUMMARY FOR THE ONCOGENE SCIENCE DIAGNOSTICS, INC. MANUAL HER-2/NEU MICROTITER ELISA

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

The assigned 510(k) number is: K994112 - 11 - 11 - 1

During the period since the original submission, Oncogene Science Diagnostics, Inc. was acquired by Bayer Diagnostics, Tarrytown, NY (12/1/99) and became Oncogene Science, a part of Bayer Diagnostics. Throughout this document, Oncogene Science will be referred to as Oncogene Science Diagnostics,

GENERAL INFORMATION 1.

Trade Name:

Oncogene Science Diagnostics, Inc. (OSDI) Manual HER-2/neu Microtiter ELISA

Classification Name: Tumor-Associated Antigen Immunological Test Systems

Walter P. Carney, Ph.D. President 80 Rogers Street Cambridge, MA 02142 Phone # 617-492-7289 Fax # 617-492-8438

Date

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0000003

In this 510(k) promarket application and notification, the performance and clinical safety and effectiveness of the Oncogcne Science Diagnostics Inc. (OSDI) HER-2/nou ELISA for the management and monitoring of metastatic breast cancer patients with initial elevated HER-2/neu has been established by extcrnal clinical studies in the target population of longitudinal metastatic breast cancer patients and by comparison to accepted diagnostic procedures in accordance with the "Guidance Document For Submission of Tumor Associated Antigen Premarket Notifications, 510(k), to the FDA". Clinical evaluations of the HER-2/neu ELISA at two US clinical trial sites demonstrated clinical safety and cffectiveness. Non-clinical studies indicate this assay is a stable, reproducible, highly specific and sensitive assay in which serum components and therapeutic agents do not interfere. Together these studies validated clinical performance characteristics and the comparison to accepted diagnostic procedures.

The HER-2/neu growth factor receptor encodes a full length HER-2/neu protcin (p185) that is overexpressed in breast cancer cells. The extracellular domain (ECD) of this receptor (p97-115kDa) is shed into the serum of healthy women and is elevated above normal levels in women with metastatic breast cancer. Many reports indicate that monitoring the HER-2/neu serum levels has clinical utility in the management of women with metastatic breast cancer. The OSDI HER-2/neu Manual Microtiter ELISA accurately measures HER-2/neu ECD in serum.

2. INDICATIONS FOR USE

The OSDI HER-2/nou ELISA is an in vitro diagnostic assay intended to quantitalively measure the extraccllular domain of HER-2/nou protein in human serum. HER-2/ncu values obtained may be used in the follow-up and monitoring of patients with metastatic breast cancer whose initial serum level of HER-2/neu is 15 ng/ml or greater. HF/R-2/neu values should be used in conjunction with information available from clinical and other

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510(k) Notification Safety and Effectiveness

diagnostic proccdures in the management of breast cancer. The clinical utility of serum measurement of HER-2/neu as a prognostic indicator for early detection of recurrence and in the management of patients on immunotherapy regimens has not been fully established.

DEVICE DESCRIPTION 3.

Description of the Method: The HER-2/neu ELISA is a sandwich cnzyme immunoassay. which utilizes two monoclonal antibodies to quantitate the extraccllular domain (ECD) of the HER-2/neu protein in scrum. The initial report describing the assay was published in 1991 (Carney, W. P., et al., Journal of Tumor Marker Oncology, 6(2): p. 53-72). Both the capture and detector anti-HER-2/neu monoclonal antibodies specifically bind the ECD of the HER-2/neu protein. The capture antibody (NB-3) has been immobilized on the interior surface of the microtiter plate wells. To perform the assay, an appropriate volume of serum is incubated in the coated well to allow binding of the antigen by the capture antibody. The captured HER-2/neu ECD is then reacted with a different anti-HER-2/neu antibody designated TA-1. The TA-1 antibody is biotinylated. The detection of the ECD of HER-2/neu is with a streptavidin horseradish peroxidase conjugate, which then catalyzes the conversion of the chromogenic substrate o-phenylenediamine into a colored product. The colored reaction product is quantitated by spectrophotometry (read absorbance at 490 mm) and is related to the amount of the HER-2/neu ECD in the serum sample. Six propared HER-2/neu standards (0, 2.5, 7.5, 15, 25 and 35 ng/ml) allow construction of a standard curve for subsequent quantification of HER-2/neu in servin samples. Also available from OSDI is a sct of HER-2/neu controls (10-CVX) designed to be used in conjunction with the ELISA for quality monitoring of assay performance. This control set consists of three (3) control levels at 2.9, 9.1 and 24.0 ng/mL HER-2/ncu protein.

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SUMMARY OF STUDIES 4.

Non-clinical studies were carried out to validate the performance of the mcthod according to the protocol cntitled "Protocols for Non-Clinical Performance Evaluation of the OSI)I HER-2/neu Microtitor ELISA". Protocols were performed at OSDI, Cambridge, Massachusetts. These studies included evaluation of interfering substances, crossreactivity, hctcrophilic antibodies, HAMA interference, Herceptin® interference, sample linearity, parallelism (sample dilution), hook cffect, reproducibility, spike and recovery, end-10-end variability, plate coating variability and reagent lot-10-lot variation.

The OSDI HER-2/neu EIJSA was cvaluated as an aid in the management of metastatic breast cancer patients with elevated scrum HER-2/neu values during the course of disease and therapy. This clinical cvaluation was conducted at two US clinical trial sites.

Characterization of the Antigen, 4.1

The standard antigen used in the OSDI HER-2/neu ELISA is the extracellular domain (ECD) of HRR-2/nou, p105. It is derived from a recombinant NIII 3T3 mouse ccll line designated 3-30. HER-2/neu p105 antigen is harvested from the cell culture serum-free conditioned medium and concentrated 10-fold. Western blot analysis shows a glycoprotein with a molccular wcight range between 97 and l 15 kd.

4.2 Characterization of the Antibodies

The NB-3 monoclonal antibody is used to capture the ECD and is therefore coated onto the surface of the microtiter well. The TA-1 monoclonal anti-HER-2/neu is used as the reagent to detect the HER-2/neu ECD. NB-3 is purified from tissue culture supernatant, produced in bioreactors, using Protein G+ affinity chromatography. The TA-1 antibody is purified from mouse ascites using Protein

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A + affinity chromatography. Production and purification of antibodies utilize standard protocols and the antibodies are manufactured in Cambridge, MA. I All lots arc isotyped and characterized by SDS-PAGE, isoelectric focusing and Coomassie Blue staining prior to functional testing.

Assay Performance 4.3

Specificity: Interference 4.3.1

The recovery of HER-2/neu from patient samples was studied before and after spiking the serum samples with the potentially interfering substance, Each potential interforent was tested at concentrations that were higher than the recommended dietary or therapeutic doses. Endogenous compounds (common serum constituents) were tested at concentrations greater than those normally observed during routine clinical testing.

The OSDI HER-2/neu ELISA was performed on serum samples or pools of serum to which were added various concentrations of either triglycerides, hemoglobin, immunoglobulin, bilirubin, albumin, heparin or cholesterol. HER-2/neu values were also measured in serum samples after spiking with either an individual chemotherapeutic drug, "Over the Counter" (OTC) drug, vitamin or IIERCEPTIN® (Trastuzumab), trademark of Genentech BioOncology, South San Francisco, CA. Nonc of the potential endogenous or exogenous interferents demonstrated any significant interfering effects on HER-2/neu recovery.

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510(k) Notification 0000007 Safety and Effectiveness

Cross-Reactivity 4.3.2

Possible cross-reactions in the OSDI HER-2/ncu EI.JSA wcrc studied by assaying purified Human Epidermal Growth Factor Reccptor (HER-1) spiked into Sample Diluent . The maximum effect seen with this cross-reactant was not significant (≤1%). Assays with cell line lysatcs have indicated that there is also no cross-reactivity with HER-3.

Heterophilic Antibodies and HAMA Interference 4.3.3

Hetcrophile antibodies possess a broad range of non-specific reactivities with other species of immunoglobulins. They include human anti-mousc antibodies (HAMA) and rheumatoid factors (RF). Thesc antibodies may interfere with immunoassays and cause false positive or negative results. The OSDI HER-2/ncu ELISA uses two mouse monoclonal antibodics, for capture and detection, but has Normal Mouse Serum (NMS) in the detector reagent to reducc this interference. A number of IJAMA and rhoumatoid factor positive serum samples were assayed in the HER-2/neu ELISA with significant interference for a small percentage of specimens without NMS in the detector diluent, but none when detector diluent contained NMS. HAMA and rhcumatoid factors do not cause false negative or false positive results in the present assay format.

Linearity 4.3.4

To evaluate the linearity of recovered values, two controls containing different levels of HER-2/neu were combined in discreet proportions to produce five samples of defined concentrations for assay. When observed HER-2/neu concentration was plotted versus expected concentration, the slope and correlation coefficient of each data set were close to the optimum of 1, indicating lincarity within the dynamic range of the assay.

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Spike and Recovery 4.3.5

In order to determine the cffect of sample matrix on the ability of the HER-2/neu ELISA to detect HER-2/neu, recovered dosages of spiked analyte in Sample Diluent (ideal matrix) were compared to recovered dosages of spiked analyte into five different patient sera. The total average percent recovery of all spiked serum samples in all three kit lots was 103%, indicating no effect of matrix on the ability of the ELISA to accurately measure HER-2/ncu protein in serum.

4.3.6 Hook Effect (Antigen Excess)

Extremely high concentrations of HER-2/neu seen in some malignant conditions may cause a "hook cffcct" in certain assay formats. 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. A 526 ng/ml stock solution of p105 HER-2/neu was serially diluted six times at 1:2 and assayed. A reportable concentration was obtained only when the expected concentration of the diluted sample approaches the range of the standard curve (0-35 ng/ml). Separation of sample and reagent addition by washes prevents high-dose hook effect in this device.

Parallelism (Dilution Studies) 4.3.7

Five scparate clinical samples containing elevated levels of HER-2/ncu, and two Boving Serum-based controls containing spiked HFR-2/neu were serially diluted two-fold for four dilutions. Recoveries were evaluated at cach of the dilution points for mean and standard deviation and were comparable for all three ELISA kit lots. Scrum samples diluted in kit Sample Dilucnt result in accurate recovery of analyte at a range of concentrations in human samples.

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510(k) Notification Safety and Effectiveness

4.3.8 End-to-End Variation

The effect on HER-2/neu recovery when incubation times of critical reagent steps were varied from sample to sample within a single assay run was evaluated. When times extended well beyond normal usage, some increase or decrease in recovery was observed (15%). However, the expected variance in timing during normal use by skilled individuals should fall well short of these extended times tested and recoveries should be unaffected.

4.3.9 Plate Coating Variations

In order to evaluate antibody coaling variations, a human serum sample was assayed over entire plates. Serum sample recovery, and therefore antibody coating, is consistent throughout plates and kit lots with 4.7-7.6 % CV.

4.3.10 Reproducibility

Intra- and inter-assay reproducibility were evaluated at OSDI and at two clinical trial sites. Three controls and a human scrum pool were assayed for reproducibility and results are shown in Tables 4.3-1 and 4.3-2. Imprecision data pooled across HER-2/neu ELISA kit lots and across assay sites showed within run CV's of 6-7% and total CV's of 10-11% for clinically relevant controls. A low control (3 ng/ml), below the lowest normal patient sample, gave higher within run and total % CV's of 10.2 and 17.7, respectively. This is well within acceptable limits for an assay of this type and for its intended use.

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Oncogene Science Diagnostics, Inc. Manual HER-2/neu Microtiter ELISA

510(k) Notification Safety and Effectiveness

	Number ofRuns	Number ofReplicates	Mean(ng/mL)	Within Run		Total
				STD DEV	%CV	STD DEV	%CV
Control 1	45	135	24.2	0.91	3.8	1.90	7.8
Control 2	45	135	9.5	0.45	4.7	0.86	9.1
Control 3	45	134	2.9	0.22	7.8	0.30	10.6
Control 4	45	135	9.2	0.42	4.5	0.77	8.3

Table 4 3-J Junecision for OSDI Site, All Microtiter ELISA Lots Combined

Table 4.3-2 Imprecision for Three Sites and Three Kit Lots Combined

	Number ofRuns	Number ofReplicates	Mean(ng/mL)	Within Run		Total
				STD DEV	%CV	STD DEV	%CV
Control 1	130	385	24.5	1.46	6.0	2.62	10.7
Control 2	131	390	9.8	0.69	7.0	1.03	10.4
Control 3	131	386	3.3	0.34	10.2	0.59	17.7
Control 4	130	385	9.5	0.65	6.9	1.02	10.8

4.3.11 Sensitivity (Detection Limit)

Sensitivity of the HER-2/neu Microtiter ELISA was evaluated at OSDI and at two clinical trial sites by determining the Minimum Detectable Dose (MDD). An MDI> of 1.5 ng/mL was observed when assaying 232 replicates of Sample Diluent alone in three HER-2/new ELISA kit lots. This MDD is acceptable for the intended use of this assay, where the lowest normal pationt was 4.23 ng/ml and the cutoff determined in this study for elevated HER-2/neu values was 1.5 ng/ml.

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4.4 CLINICAL STUDIES

4.4.1 Introduction

To assess the safety and effectivencss of the OSDI HER-2/neu Microtiter ELISA, clinical studies were performed at two investigational sites on pationt samples from four different locations. All patients were studied retrospectively. Assay values were determined for surplus scrum samples which had been collected and stored (-70° C) in specimen banks prior to the study. Studies performed at OSDI have shown no evidence of changes in HER-2/neu concentration in patient samples stored at -80° C for 5 % years.

4.4.2 Metastatic Patient Serial Monitoring - HER-2/neu Management Value

The clinical utility of the OSDI HER-2/neu Microtiter E.I.ISA in monitoring metastatic breast cancer patients with initial elevated HER-2/neu was evaluated using retrospective scrum. Scrial serum HER-2/neu values were measured over a 6 to 12 month period in clinically evaluable patients with Stage IV breast cancer undergoing therapy. 33.7 % had initial clevated serum samples (HER-2/neu concentrations cqual to or greater than 15 ng/mL). These fifty-six patients were then evaluated for correspondence of changes in their serum HER-2/neu values with changes in their clinical course of disease.

A visit-to-visit analysis of the study results are presented in Table 4.4-1. The changes in serum HER-2/neu values from one visit to the next were calculated for cach patient. These changes were scparated into 2 groups; Group I had changes in serum HER-2/neu which paralleled the clinical course of discase and Group II had changes in scrum HER-2/nen values that did not parallel the clinical course of discase. Clinical course or status was determined by the physician. Correspondence of HER-2/neu changes

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to clinical status were determined as follows: An increase of 20% or greater from the previous visit was reflective of disease progression. If the change is less than a 20% increase from the previous visit, this was reflective of a lack of discase progression during therapy (including responding or stable discase). Stable and responding were consolidated since both reflect effectiveness of a current therapy. The 20% critcrion was derived from the longitudinal variability in normal patients (determined from the HER-2/neu values in six serial samples from cach of 38 healthy women).

Table 4.4-1: Correspondence of Metastatic Breast Cancer Patient Disease Status with Changes in Serum HER-2/neu: Visit-by-Visit

		Pro-gression	No Pro-gression	TOTAL
ChangeIn HER-2/neu	≥20% ↑	52	33	85
ChangeIn HER-2/neu	<20% ↑	55	96	151
ChangeIn HER-2/neu	TOTAL	107	129	236

Change in Clinical Status

The following figures show typical examples of changes in HER-2/neu, over time, for 2 of the 56 metastatic breast cancer patients from the clinical study. Figure 4.4-1 shows a patient whose discase is responding and Figure 4.4-2 shows a patient with progressive discase.

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510(k) Notification 0000013 Safety and Effectiveness

Figure 4.4-1: Monitoring of a 38-year-old Stage IV Breast Cancer Patient with the OSDI HER-2/neu ELISA. Longitudinal changes in OSDI HER-2/neu values correlate with changes in disease status.

Image /page/11/Figure/3 description: This image is a graph showing HER-2/neu levels in ng/mL over a period of 125 days from the first draw. The graph shows an initial HER-2/neu level of around 70, which increases slightly before decreasing significantly after 75 days. The graph also includes annotations indicating chest x-ray results and treatment changes, such as starting Adriamycin and Taxol Tx, and Miloxanthrone & Cyclophospharnide Tx.

Figure 4.4-2:Monitoring of a 74-year-old Stage IV Breast Cancer Patient with the OSDI HER-2/neu ELISA. Longitudinal changes in OSDI HER-2/neu values correlate with changes in disease status.

Image /page/11/Figure/5 description: This image is a line graph showing HER-2/neu levels in ng/mL over time in days from the first draw. The x-axis represents the days from the first draw, ranging from 0 to 200. The y-axis represents HER-2/neu levels, ranging from 0 to 2000 ng/mL. The graph shows an increase in HER-2/neu levels over time, with annotations indicating chest X-ray results and treatment changes, such as starting and stopping Arimidex Tx and starting Navelbine Tx.

These data support the clinical utility of the OSDI HER-2/neu Microtiter ELISA for use in management (monitoring) during the course of disease and therapy of metastatic breast cancer patients whose initial HER-2/neu levels are elevated above 15 ng/ml.

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Distribution of HER-2/neu Concentrations; Scositivity and Specificity 4.4.3

The HER-2/neu ELISA was used to estimate the clinical (cross-sectional) sensitivity in patients with breast cancer and characterize the frequency distribution of HER-2/neu ELISA values in a population of breast cancer patients by stage of disease. Specificity of the HER-2/neu ELISA was determined in patients with benign breast diseases, other non-malignant diseases, and in normal healthy individuals.

The Upper Limit of Normals in this study with the ELISA was 15 ng/ml, which was confirmed as an appropriate cutoff by ROC analysis to give 95% specificity. These two results validate the use of 15 ng/ml as the baseline for elevated HER-2/neu concentrations used in the metastatic patient serial monitoring portion of this study.

The longitudinal measurements for healthy women seen in this study showed a standard deviation of 10%. Ninety-five percent of the time, serial ELISA values should be within twice this SD. Therefore, the normal variability expected in longitudinal sample analysis would be 20%, and anything greater would be potentially indicative of changes duc to cancer.

Conclusions from the Clinical Studies 4.4.4

The results of this retrospective clinical trial demonstrate that the OSDI Manual HER-2/neu Microtiter FILISA is reproducible, and is safe and effective for the management and follow-up of patients with metastatic breast cancer whose initial serum levels of HER-2/neu are 15 ng/ml or greater.

This study carefully examined the potential role of longitudinal serum IIER-2/neu concentrations in the management of patients with mctastatic breast cancer. The results are consistent with published reports showing HER-2/neu to be useful in the metastatic clinical setting. Data collected

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from this study show that changes in serum HER-2/neu concentrations over time in initially clevated metastatic breast cancer pations reflect changes in clinical status such as response to therapy or progression of discase.

The reproducibility CV's obtained by the two clinical laboratories in the OSDI HER-2/ncu ELISA fall within typical ranges for manual microtiter assays of <10% intra-assay and <15% interassay. The detection limit is acceptable for the intended use of this assay. This demonstrates that this assay should provide reliable and reproducible results when tested by different laboratories using different manufactured lots of reagents at different times.

5. CONCLUSIONS DRAWN FROM ALL THE STUDIES

Valid Scientific Evidence

The conclusions drawn from these studies are based upon valid scientific evidence. Data were gathered following a well-designed protocol, in a clinical research laboratory operating under the principles of Good Clinical Practices. Clinical data were gathered during well controlled investigations conducted by qualified experts. Patient case histories were well documented. The results of this study are comparable to literature reports of experiences with HER-2/neu assays.

Method Performance

OSDI HER-2/neu ELISA results are highly reproducible with a maximum interassay %CV pooled over reagent lots and clinical sites of 11% over the clinically useful range of the assay. The assay shows no interference by common serum components or by vitamins, over-the-counter drugs, chemotherapeutic agents or

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HERCEPTIN®. Other performance characteristics including analytical sensitivily and specificity, cross-reactivity, linearity, and antigen cxcess hook effect were excellent.

Safety and Effectiveness

These clinical studies confirm the safety and effectiveness of the OSDI HER-2/neu Microtiter ELISA as an aid in the follow-up and management of mctastatic breast cancer patients whose initial HER-2/neu concentration is 15 ng/ml or greater. The correspondence between HER-2/neu concentrations and the patients' clinical course of disease demonstrate that the OSDI HER-2/neu Microtiter ELISA may be used in conjunction with other clinical indicators to confirm disease progression and response to therapy in metastatic breast cancer pationts with initial elevated levels of HER-2/neu.

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DEPARTMENT OF HEALTH & HUMAN SERVICES

Image /page/15/Picture/1 description: The image shows the logo for the U.S. Department of Health & Human Services. The logo consists of a circular seal with the text "DEPARTMENT OF HEALTH & HUMAN SERVICES • USA" arranged around the perimeter. Inside the circle is an abstract symbol resembling an eagle or bird in flight, composed of three curved lines.

SEP 2 9 2000

Food and Drug Administration 2098 Gaither Road Rockville MD 20850

Walter P. Carney, Ph.D. Director Oncogene Science Bayer Corporation 80 Rogers Street Cambridge, Massachusetts 02142-1168

K994112 Re:

Trade Name: Oncogene Science Diagnostics, Manual HER-2/neu Microtiter ELISA Regulatory Class: II Product Code: NCW Dated: June 30, 2000 Received: July 3, 2000

Dear Dr. Carney:

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

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 noutleation. The PDF Intellig ssification 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 11 you desire spoolite an novitro diagnostic devices), please contact the Office of Compliance at additionally 007.10 for mi mise craguestions on the promotion and advertising of your device, (201) 591 1500: Traditionally) (1) 594-4639. Also, please note the regulation produced "Misbranding by reference to premarket notification" (21CFR 807.97). Other general information on your responsibilities under the Act may be obtained from the Division of Small mionifacturers Assistance at its toll-free number (800) 638-2041 or (301) 443-6597 or at its internet address "http://www.fda.gov/cdrh/dsma/dsmamain.html".

Sincerely yours,

Steven Dutman

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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Page 1 of 1

510 (k) Number (if known): K994112

Device Name: OSDI HER-2/neu Assay

Indications For Use:

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

Peter E. Mafrici

rence of CDRH, Office of Device Evaluation (ODE)

Prescription Use (per 21 CFR 801.109)

Over-the-counter Use

(Optional Format 1-2-96)

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.