MammaPrint is a qualitative in vitro diagnostic test service, performed in a central laboratory, using the gene expression profile of fresh breast cancer tissue samples to assess a patient's risk for distant metastasis (up to 10 years for patients less than 61 years old, up to 5 years for patients' ≥ 61 years).

The test is performed for breast cancer patients with Stage I or Stage II disease, with tumor size <= 5.0 cm and who are lymph node negative. The MammaPrint result is indicated for use by physicians as a prognostic marker only, along with other clinicopathological factors.

Device Description

The MammaPrint service is a microarray based gene expression analysis of a tumor. The analysis is based on several processes: isolation of RNA from frozen tumor tissue sections. DNA'se treatment of isolated RNA, linear amplification and labeling of DNA'se treated RNA, cRNA purification, hybridization of the cRNA to the MammaPrint microarray, scanning the MammaPrint microarray and data acquisition (feature extraction), calculation and determination of the risk of recurrence in breast cancer patients.

The MammaPrint analysis is designed to determine the gene activity of specific genes in a tissue sample compared to a reference standard. The result is an expression profile, or fingerprint, of the sample.

The correlation of the sample expression profile to a template (the mean expression profile of 44 tumors with a known good clinical outcome) is calculated and the molecular profile of the sample is determined (Low Risk, High Risk).

AI/ML Overview

The MammaPrint device is a qualitative in vitro diagnostic test service that assesses a patient's risk for distant metastasis in breast cancer. The study presented focuses on the analytical performance and reproducibility of the device, particularly when new scanners and bio-analyzers are used, and when the service is performed in different central laboratory sites.

Here's a breakdown of the requested information:

1. Acceptance Criteria and Reported Device Performance

The provided document describes analytical performance evaluation rather than a clinical study with specific diagnostic accuracy metrics (like sensitivity, specificity, AUC). The acceptance criteria are based on statistical comparisons to the predicate device's accepted variance and predetermined validation acceptance criteria.

Acceptance Criteria Category	Specific Acceptance Criteria	Reported Device Performance
Micro Array Scanners	Difference between mean, median, and standard deviation of MammaPrint Indices (between new and predicate scanners) falls within 1.96 * 0.030 (accepted variance of predicate device).	Differences fall within the accepted variance of the predicate device (1.96 * 0.030).
Bio Analyzers (RIN Measurements)	No significant difference in RNA Integrity Number (RIN) measurements between FDA-cleared and new Bio-analyzers (p-value for statistical test should indicate no significant difference).	Wilcoxon signed ranks test showed no significant difference in RIN measurements between FDA cleared and New Bioanalyzers (p=0.46 and p=0.47 respectively).
Central Laboratory Site Comparison (RNA Quality/RIN)	No significant difference in RNA quality (RIN measurement) between Amsterdam (L1) and US lab (L2). All results must comply with predefined validation acceptance criteria.	No significant difference in RNA quality of RIN measurement between Amsterdam (L1) and US lab (L2). All results comply with predefined validation acceptance criteria.
Central Laboratory Site Comparison (MammaPrint Index/Outcome)	No significant difference in MammaPrint Indices and outcome between European/Dutch (L1) and US/California (L2) lab. All results must comply with predefined validation acceptance criteria.	No significant difference in MammaPrint Indices between European/Dutch (L1) and US/California (L2) lab. All results comply with predefined validation acceptance criteria.

2. Sample Size and Data Provenance

The study focused on analytical validation rather than a clinical diagnostic accuracy study.

Micro Array Scanners: Used a selection of 25 slides from which MammaPrint Indices were generated. These slides consisted of approximately 100 samples and 20 control samples (LRC and HRC). The samples were analyzed during regular diagnostics. The provenance (country of origin) is not explicitly stated for these specific samples, but the company is based in the Netherlands with a US lab mentioned later. The samples were likely retrospective as they were previously run for diagnostic purposes.
Bio Analyzers: A selection of about 60 samples covering the complete RIN measuring range. Data provenance is not explicitly stated.
Central Laboratory Sites (RNA Isolation): 36 samples selected based on sufficient tissue material. The samples had previously shown acceptable RNA quality at the Amsterdam lab.
Central Laboratory Sites (Amplification/Labeling and Hybridization): 99 samples were used. All samples had been previously subjected to a diagnostic MammaPrint test at the Amsterdam Lab (Lab 1). The result distribution was 54 high risk, 38 low risk, and 7 borderline.
- Data Provenance: The central laboratories include Amsterdam (Netherlands) and a US laboratory (California). The samples used for comparison were run in both labs. This implies a retrospective analysis of samples previously processed.

3. Number of Experts and Qualifications for Ground Truth

The document does not describe a process where experts established a ground truth for a test set in the traditional sense of diagnostic accuracy studies (e.g., radiologists reviewing images). The ground truth for the analytical validation appears to be the results obtained from the predicate device or a previously established reference standard within Agendia's own validated processes.

4. Adjudication Method

Not applicable for this type of analytical validation study. There was no expert adjudication process for establishing a "ground truth" for a test set. The comparisons were statistical analyses of measurements (MammaPrint Indices, RIN values) between different instrumentations or lab sites.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

No, this document does not describe a MRMC comparative effectiveness study involving human readers. This is an analytical validation of the device's technical performance and reproducibility, not a study on how human interpretation improves with AI assistance.

6. Standalone (Algorithm Only) Performance

Yes, the study describes the standalone performance of the MammaPrint algorithm, focusing on the analytical performance characteristics like precision and reproducibility across different hardware and lab sites. It evaluates the consistency of the algorithm's output (MammaPrint Index, RIN) under various conditions.

7. Type of Ground Truth Used

The "ground truth" in this context refers to the established and accepted performance of the predicate device and the existing, validated MammaPrint procedures. For instance:

For scanner validation, the MammaPrint Indices generated by the FDA-cleared predicate scanners served as the reference.
For bio-analyzer validation, the RIN measurements from FDA-cleared bio-analyzers served as the reference.
For central lab site comparison, the results from the Amsterdam lab (L1), where samples had been previously subjected to diagnostic MammaPrint, served as the reference for comparison with the US lab (L2).

This is an internal reference standard/predicate device comparison.

8. Sample Size for the Training Set

The document does not describe a training set for a machine learning algorithm. MammaPrint is a gene expression profiling test, which typically relies on a fixed algorithm/signature derived from earlier research, not an adaptive machine learning model that is "trained" in an ongoing manner. The document focuses on the analytical validation of the test's execution and reproducibility.

9. How the Ground Truth for the Training Set Was Established

Not applicable, as there is no mention of a training set for a machine learning algorithm in the provided text. The MammaPrint test itself is based on a specific gene expression profile correlated to clinical outcome. The methods for establishing this original correlation (which forms the basis of the "template" mentioned in the device description) would be found in the foundational clinical studies for MammaPrint, not in this analytical validation document. The "template" (mean expression profile of 44 tumors with a known good clinical outcome) explicitly mentioned in the device description is effectively the core of their model, derived from prior clinical data.

Summary

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Section 5: 510(k) Summary

1. Assigned 510(k) number
The assigned 510(k) number is

JAN 2 8 2011

Company

Agendia BV Science Park 406 1098XH Amsterdam The Netherlands Telephone : 31 20 462 1500

Contact

Guido Brink, Senior Director Regulatory Affairs and Quality Assurance

Date Prepared

May 21, 2010

Proprietary Name

MammaPrint®

6. Classification Name

Gene expression profiling test system, for breast cancer prognosis.

7. Common Name

Multivariate device for cancer prognosis

8. Classification

Class II, regulated under 21 CFR 866.6040, product code NYI

9. Predicate Device

Agendia BV's MammaPrint (K081092)

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10. Device Description

11. Intended Use

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12. Performance Data (non-clinical)

Analytical performance

MammaPrint analytical (i.e., non-clinical) performance characteristics investigated comprise Precision and Reproducibility compared to the predicate device.

1 - Micro Array Scanners

In concordance with experiments performed for an additional scanner clearance in K080252, experiments were performed.

A selection of 25 slides performed as dual hybridizations from which MammaPrint Indices of the 1ª scan was generated using the FDA cleared scanners (serial US45103019 and US22502555) were used, consisted of approximately 100 samples and 20 times the control samples LRC and HRC.

The samples and controls were analyzed during regular diagnostics. For the scanner validation these same 25 slides were scanned a second time using the new scanners (US810R3210 and US811R3213). Txt files generated as output were used to generate MammaPrint Indices following standard procedures. Subsequently MammaPrint Indices of both scans were compared. The hybridized samples included: three samples with either high, low, borderline results with repeated results were generated per sample. Additionally, control samples LRC and HRC were included. MammaPrint indices were compared between both scans using Passing and Bablok regression analysis and a comparison of the variance per scanner.

The difference between the mean, median and standard deviation for all samples levels between both scanners fall within the accepted variance of the predicate device of 1.96*0.030.

2 – Bio Analyzers

A selection of about 60 samples that cover the complete RIN measuring range was analyzed on the FDA cleared Bio-analyzers (Serial nr DE24802382 and DE54700497), as well as the new Bio-Analyzers (Serial nr DE72901757 and DE72902838).

Depending on the distribution of the data a statistical test was performed to determine if there is a significant difference in RIN measurements between both Bio-analyzers.

The RIN measurements of the samples on both Bio-Analyzers were collected. Subsequently the D'Agostino-Pearson test on the RIN differences of both analyzers showed that there a normal distribution (p<0.0001).

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Therefore a Wilcoxon signed ranks test was used which showed that there was no significant difference in RIN measurements between the FDA cleared and New Bioanalyzers (p=0.46 and p=0.47 respectively).

3 - Central Laboratory sites Europe and U.S.

Validation of MammaPrint at the European and U.S. central laboratories was performed in two parts. All experiments were performed using FDA cleared equipment and in compliance with FDA cleared MammaPrint procedures.

PART 1: RNA ISOLATION

Samples were selected based on sufficient tissue material available for sectioning and isolation at the US laboratory (Lab 2). These samples have previously shown to generate acceptable quality of RNA at the Amsterdam laboratory (Lab 1). After isolation the concentration and RNA quality (RNA integrity number, RIN) was assessed using the Bioanalyzers; all values have to meet the standard quality controls for MammaPrint (RIN>7). Isolations were performed on three different days, twelve samples each day, in total 36 samples.

PART 2: AMPLIFICATION/LABELING AND HYBRIDIZATION

For validation of the labeling, amplification and hybridization steps of MammaPrint at the US lab (Lab 2). RNA from 99 samples was used. All samples have been previously subjected to a diagnostic MammaPrint test at the Amsterdam Lab (Lab 1). Based on the Amsterdam result the following result distribution was selected:

Hiah risk: n=54 (54.5%) -
। Low risk: n=38 (38.3%)
-Borderline: n=7 (7.1%)

RNA was amplified, labeled and hybridized according to standard MammaPrint protocols on FDA cleared MammaPrint Low (HD) 8-pack array.

The 99 samples the standard control samples (Low Risk Control and High Risk Control) were analyzed. To show MammaPrint stability over time and to determine variation in MammaPrint Index, LRC and HRC were analyzed on each labeling day and on additional days resulting in 20 data points per control sample.

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Statistical analysis that have been performed on the data are:

. Passing and Bablok regression analysis
. Bland & Altman analysis
McNEMARS TEST
. Analysis on Control Pools: LRC AND HRC

The studies show that there is no significant difference in RNA quality of RIN measurement between Amsterdam (L1) and US lab (L2). All results comply with the predefined validation acceptance criteria as described in the validation plan.

Moreover when comparing of MammaPrint Index and Outcome, it is concluded that there is no significant difference in MammaPrint Indices between European / Dutch (L1) and US / California (L2) lab. All results comply with the predefined validation acceptance criteria as described in the validation plan.

14. Conclusion

MammaPrint is a clinically and analytically accurate prognostic marker for providing a risk assessment of distant metastasis of breast cancer when performed in either Agendia's European or US central laboratory.

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Image /page/5/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" around the perimeter. Inside the circle is an emblem featuring a stylized human figure with outstretched arms, representing care and protection.

Public Health Service

Food and Drug Administration 10903 New Hampshire Avenue Silver Spring, MD 20993

Agendia BV c/o Mr. Guido Brink Director, Regulatory Affairs Science Park 406 1098 XH Amsterdam The Netherlands

JAN 2 8 2011

Re: K101454

Trade/Device Name: MammaPrint® Regulation Number: 21 CFR§866.6040 Regulation Name: expression profiling test system for breast cancer prognosis Regulatory Class: Class II Product Code: NYI Dated: December 23, 2010 Received: December 27, 2010

Dear Mr. Brink:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and 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, Drig, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). 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 class II (Special Controls), it may be subject to such additional controls. Existing major regulations affecting your device can be found in Title 21, Code of Federal Regulations (CFR), Parts 800 to 895. In addition, FDA may publish frinther announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must

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Page 2 – Mr. Guido Brink

comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Parts 801 and 809); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); and good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820). This letter will allow you to begin marketing your device as described in your Section 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.

ff you desire specific advice for your device on our labeling regulation (21 CFR Parts 801 and 809), please contact the Office of In Vitro Diagnostic Device Evaluation and Safety at (301) 796-5450. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.

You may obtain other general information on your responsibilities under the Act from the Division of Small Manufacturers, International and Consumer Assistance at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address http://www.fda.gov/cdrh/industry/support/index.html.

Sincerely yours,

M Chan

Maria M. Chan, Ph.D. Director · Division of Immunology and Hematology Devices Office of In Vitro Diagnostic Device Evaluation and Safety Center for Devices and Radiological Health

Enclosure

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Image /page/7/Picture/15 description: The image shows a logo with the word "agenda" in a stylized font. Above the word "agenda" is a graphic that resembles a fingerprint or a stylized representation of a brain. Below the word "agenda" is a smaller text, which is difficult to read due to the image quality.

ection 4: Indications for Use Statement

Indications for Use Form

K101454 510(k) Number (if known):

Device Name: MammaPrint®

Indications for Use:

MammaPrint is a qualitative in vitro diagnostic test service, performed in a central laboratory, using the gene expression profile of fresh breast cancer tissue samples to assess a patients' risk for distant metastasis (up to 10 years for patients less than 61 years old, up to 5 years for patients' ≥ 61 years).

The test is performed for breast cancer patients with Stage I or Stage II disease, with a tumor size of ≤ 5.0 cm and lymph node negative. The MammaPrint result is indicated for use by physicians as a prognostic marker only, along with a other clinicopathological factors.

Prescription Use XX (Part 21 CFR 801 Subpart D) AND/OR

Over-The-Counter Use (21 CFR 801 Subpart C)

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

Concurrence of CDRH, Office of Device Evaluation (ODE)

			Page 1 of 1
	Division Sign-Off

Office of In Vitro Diagnostic
Device Evaluation and Safety

	K101454
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Agendia BV - Amsterdam 2010-1061 Additional Scanner

Regulation Number and Section

§ 866.6040 Gene expression profiling test system for breast cancer prognosis.

(a)
Identification. A gene expression profiling test system for breast cancer prognosis is a device that measures the ribonucleic acid (RNA) expression level of multiple genes and combines this information to yield a signature (pattern or classifier or index) to aid in prognosis of previously diagnosed breast cancer.(b)
Classification. Class II (special controls). The special control is FDA's guidance document entitled “Class II Special Controls Guidance Document: Gene Expression Profiling Test System for Breast Cancer Prognosis.” See § 866.1(e) for the availability of this guidance document.