MammaPrint is a qualitative in vitro diagnostic test service, performed in a single laboratory, using the gene expression profile of fresh breast cancer tissue samples to assess a patient's risk for distant metastasis.

The test is performed for breast cancer patients who are less than 61 years old, 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 turnor 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

Here's a breakdown of the acceptance criteria and the study information for the MammaPrint® device, based on the provided text:

MammaPrint® Device Performance Study Analysis

1. Acceptance Criteria and Reported Device Performance

The provided document primarily focuses on demonstrating substantial equivalence to a predicate device (Agendia BV's MammaPrint (K070675)) rather than setting explicit, quantifiable clinical acceptance criteria for de novo approval. However, it does present analytical performance targets relative to the predicate and clinical outcomes from supporting studies.

Here's a table summarizing the implicit acceptance criteria (based on predicate performance) and the reported device performance from the analytical studies:

Acceptance Criteria / Performance Metric	Target (derived from predicate or equivalency)	Reported Device Performance (New MammaPrint HD)
Analytical Performance
Repeatability and Precision (MammaPrint index Standard Deviation)	At least as good as predicate (≤ 0.030)	At least as good as predicate (0.030)
Repeatability and Precision (MammaPrint index Variance)	At least as good as predicate (≤ 0.001)	At least as good as predicate (0.001)
Concordance in MammaPrint outcome (HD vs. LD microarray)	Within 97.7% technical accuracy of predicate	98.9%
MammaPrint Index difference between scanners (mean, median, standard deviation)	Within predicate's accepted variance (1.96 * 0.030)	Falls within accepted variance
Accuracy of classifying as High Risk or Low Risk	Better than predicate (97.7% accuracy, 1.1% false negative)	98.9% (0.5% false negative classification)
Percentage of "Borderline Samples"	Not explicitly stated as a target, but reported for context	Less than 5%
"Borderline Samples" Classification Accuracy	Not explicitly stated as a target, but reported for context	Approximately 90% (10% chance of false classification)

2. Sample Size and Data Provenance for Test Set (Analytical Performance)

Precision Evaluation (Method Validation):
- Sample Size: 3 samples with different outcome levels (high, low, borderline) were run repeatedly over 20 days. Two replicates per sample level per day. Also included High Risk Control (HRC) and Low Risk Control (LRC). Total 120 (3 samples x 2 replicates x 20 days), plus controls.
- Data Provenance: Not explicitly stated, but likely internal laboratory data from Agendia BV. Retrospective, as these were established samples.
LD and HD Microarray Comparison:
- Sample Size: 98 historical MammaPrint service samples.
- Data Provenance: Samples from the period 2004 through 2007. Origin country is not explicitly stated in this section, but the company is based in the Netherlands. This is retrospective data.
Scanner Performance Comparison:
- Sample Size: 26 newly hybridized slides (representing 104 samples). This included three samples with varying risk profiles, repeated, and LRC/HRC controls.
- Data Provenance: Likely internal laboratory data from Agendia BV. Retrospective.

For Clinical Performance, the document refers to four published studies. These studies serve as the clinical evidence for the device's utility, rather than a single "test set" in the context of analytical validation. The details provided are for each study:

Nature Paper (1): 78 patients
NEJM Paper (2): 151 patients
MammaPrint Paper (3): Not a clinical study for patient outcomes, focuses on reproducibility of (1) and (2).
Transbig Paper (4): 302 patients

The provenance of these clinical studies would generally be international (e.g., European for Transbig). All are retrospective analyses of patient cohorts.

3. Number of Experts and Qualifications for Ground Truth (Test Set)

For the analytical performance studies, "ground truth" refers to the known characteristics of the samples (e.g., whether they were designed to be high, low, or borderline risk, or previously classified by the predicate device). No human experts are used to establish ground truth in these analytical comparisons; instead, it relies on the pre-determined characteristics of the control samples or the results from the predicate device.

For the extensive clinical studies referenced (Nature, NEJM, Transbig), the ground truth for patient outcomes (e.g., metastasis-free survival) would have been established through long-term clinical follow-up by medical professionals, including oncologists and pathologists. The number and specific qualifications of these experts are not detailed in this 510(k) summary, as it refers to published peer-reviewed literature. Typically, these studies involve panels of clinicians (e.g., pathologists, oncologists) determining initial diagnosis and patient outcomes.

4. Adjudication Method for the Test Set

Analytical Performance: No adjudication method as human interpretation is not involved in comparing array results or scanner outputs. The comparisons are quantitative.
Clinical Performance (Referenced Studies): The 510(k) summary does not detail the specific adjudication methods used in the referenced clinical studies (e.g., for outcome endpoints like metastasis). Such details would typically be found within the full publications.

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

No MRMC study is mentioned or implied. MammaPrint is a gene expression profiling test, which is an automated, quantitative assay. Human readers do not directly interpret the "cases" (gene expression profiles) in the same way they would an image. The device provides a quantitative risk score (Low Risk, High Risk) that physicians use to make decisions, but the device itself does not involve human interpretation for its output. Therefore, a study demonstrating how human readers improve with AI vs. without AI assistance is not applicable here.

6. Standalone Performance Study

Yes, the entire submission describes the standalone performance of the MammaPrint device (the algorithm/system itself) in classifying tissue samples as High Risk or Low Risk based on gene expression profiles. The analytical performance metrics (precision, reproducibility, classification accuracy) are all measures of the algorithm's performance in isolation from human interpretation.

7. Type of Ground Truth Used

Analytical Performance:
- Known outcome levels for control samples (high, low, borderline).
- Results from the predicate device (FDA cleared Low Density microarray).
Clinical Performance (Referenced Studies):
- Long-term clinical follow-up data: Metastasis-free survival.
- Clinicopathological factors (e.g., tumor size, lymph node status, age).

8. Sample Size for the Training Set

The document mentions: "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)." This "template" of 44 tumors appears to be a core component of the algorithm's development.

The broader clinical studies referenced (e.g., Nature Paper (1) with 78 patients) were likely instrumental in the initial development and refinement (training) of the 70-gene profile itself. The MammaPrint Paper (3) describes the "Development of MammaPrint" and "Converting a breast cancer microarray signature into a high-throughput diagnostic test," implying further training and optimization steps using data from earlier studies.

Therefore, the initial "training set" for defining the gene profile involved patient cohorts (e.g., the 78 patients in the Nature paper, or the 44 specific "good clinical outcome" tumors).

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

For the "44 tumors with a known good clinical outcome" that form the template, the "known good clinical outcome" would have been established through:

Long-term clinical follow-up: Patients whose tumors formed this template would have had a prolonged period without recurrence or distant metastasis.
Clinicopathological data: Standard clinical assessments (e.g., pathology reports, surgical findings) would have confirmed the characteristics of these tumors and the patients' disease stage.

For the broader training of the 70-gene profile, as implied by the referenced papers, the ground truth was established by:

Clinical outcome data: Patients were followed for several years (e.g., 5-10 years) to determine their metastasis-free survival or overall survival.
Pathology: Original biopsy/surgical pathology diagnoses, including tumor type, grade, and lymph node status, were used.
Expert Consensus: While not explicitly stated for individual training cases in this summary, the clinical outcomes and pathological assessments that defined "good clinical outcome" and "poor clinical outcome" would have been made by qualified medical professionals (pathologists, oncologists).

Summary

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Section 5: 510(k) Summary

Assigned 510(k) number The assigned 510(k) number is
Company

umber is K0800252

21 2008

Agendia BV Slotervaart Medical Center 9D Louwesweg 6, 1066EC Amsterdam The Netherlands Telephone : 31 20 512 9161 Facsimile : 31 20 5129162

Contact Guido Brink, Director Quality Management and Regulatory Affairs
Date Prepared January 21, 2008
Proprietary Name MammaPrint®
Classification Name Gene expression profiling test system, for breast cancer prognosis.
Common Name Multivariate device for cancer prognosis
Classification Class II, regulated under 21 CFR 866.6040, product code NYI
Predicate Device Agendia BV's MammaPrint (K070675)

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10. Device Description

11. Intended Use

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.

First, the technical validity of the high density microarray platform was established by method validation (i.e., precision evaluation) according the EP5-2A protocols of NCCLS This experimental design consisted of repeated runs over 20 days in which 3 samples with different outcome levels, high, low and borderline, were performed on MammaPrint index High Density 8-pack arrays. Per day one run is performed consisting of two replicates of each sample level. Additionally both control samples high and low risk

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control (HRC, LRC) as used for MammaPrint were also be taken along with one of the replicates.

The repeatability and precision of the MammaPrint index of all levels of sample outcomes, were at least as good as the performance of the predicate device (Standard Deviation and Variance of 0.030 and 0.001 respectively).

Second, a comparison was made between the FDA cleared Low Density (LD) microarray and, under QSR design controlled, High Density (HD) microarray. For the LD and HD comparison 98 samples will be selected for MammaPrint service from the period 2004 through 2007. All samples will be re-hybridized on 8-pack HD arrays. However, samples originating from 2004 through 2005 will be relabeled and hybridized for both LD and HD 8-pack arrays.

From 2006 onwards the original MammaPrint Index from previous LD hybridizations will be used. For these samples the labeled cRNA will be taken and re-hybridized on HD 8pack MammaPrint arrays. Subsequently, a comparison of the MammaPrint index of both array types were performed.

Results showed a 98.9% concordance in MammaPrint outcome between HD and LD microarray which falls with the 97.7% technical accuracy of the predicate device.

Third the High Density sample set from the precision evaluation was used to determine the performance between the Agilent DNA microarrays. A set of 26 newly hybridized slides (104 samples) were scanned first on the FDA cleared scanner (serial nr: US22502555, Agendia DPd Id: 002) and subsequently on the new scanner (serial nr: US45103019 Agendia DPd Id: 112). 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.

Classification performance

Based on the analytical performance of MammaPrint using the High Density microarray platform, the accuracy of classifying a sample as High Risk or Low Risk, is 98,9%, (i.e., 0.5% false negative classification). This performance is better than the predicate device which has a classification accuracy of 97.7% (i.e., 1.1% false negative classification).

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agencia

Borderline Sample

As a result of the technical inaccuracy, analytical measurements (i.e., MammaPrint Index) can fall within a pre-defined area around the classification cut-off between the High Risk and Low Risk profile (i.e., "Borderline Sample").

Based on the results of independent MammaPrint analyses over a time period of over 2 years, it has been shown that less than 5% of the analyzed samples are considered to be "Borderline Samples".

"Borderline Samples" have approximately a 90% classification accuracy (i.e. 10% chance of false classification).

Looking at all generated data it is concluded that the MammaPrint analysis on a High Density microarray platform using the Agilent DNA microarray scanner G2505B with serial number US45103019 is considered to be substantially equivalent to the FDA cleared MammaPrint device with 510k number K070675.

13. Clinical Data

Clinical performance testing is based on the following studies:

	Purpose	Results	Comments
Nature Paper (1)	Development ofbreast cancerprognosis 70-geneprofile (LNO, <55)	2002, 78 patients,6.4% adjuvanttreatment	Within 5 yearmetastasis risk byprofile multivariateOR 18
NEJM Paper (2)	Validation of the70-gene profile inconsecutive seriesof breast cancerpatients (LNO,<53)	2002, 151 patients,5.2% adjuvanttreatment	Metastasis-freesurvival by profile at10 yrs: low riskprofile 87%, highrisk profile 44%(at 5 yrs: 93% and56% respectively)
MammaPrint Paper (3)	Development ofMammaPrint	2006, reproducibilityof (1) and (2) onMammaPrint	Highly reproducibleMammaPrint asdiagnostic tool
Transbig Paper (4)	IndependentEuropeanvalidation of 70-gene signature(LNO, <61)	2006, 302 patients,no adjuvanttreatment	Metastasis-freesurvival by profile at10 yrs: low riskprofile 88%, highrisk profile 71%(at 5 yrs: 96% and83% respectively)

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14. Conclusion

MammaPrint is a clinically and analytically accurate prognostic marker for providing a risk assessment of distant metastasis of breast cancer.

(1) Gene expression profiling predicts clinical outcome of breast cancer;

Laura J. Van 't Veer et al; Nature (2002) 415, p530-536.

(2) A Gene-Expression Signature as a Predictor of Survival in Breast Cancer;

Marc J. Van de Vijver et al;. New Engl J Med (2002) 347, p1999-2009.

(3) Converting a breast cancer microarray signature into a high-throughput diagnosiic test; Annuska M. Glas et al; BMC Genomics (2006) accepted.

(4) Validation and clinical utility of a 70-gene prognostic signature for women with node-negative breast cancer; Marc Buyse et al; J Natl Cancer Inst (2006), 98, p1183-1192.

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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 department's name around the perimeter. Inside the circle is a stylized representation of a human figure embracing a globe, symbolizing the department's mission to protect the health of all Americans.

Public Health Service

'JUL 2 1 2008

Food and Drug Administration 2098 Gaither Road Rockville MD 20850

Agendia BV c/o Mr. Guido Brink Director Quality Management and Regulatory Affairs Slotervaart Medical Center 9D Louwesweg 6, 1066EC Amsterdam The Netherlands

Re: K080252

Trade/Device Name: MammaPrint® Regulation Number: 21 CFR 866.6040 Regulation Name: Gene expression profiling test system for breast cancer prognosis Regulatory Class: Class II Product Code: NYI Dated: May 7, 2008 Received: May 9, 2008

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, Drug, 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 mishranding and adulteration.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), 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 898. In addition, FDA may publish further 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 comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801); 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

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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), please contact the Office of In Vitro Diagnostic Device Evaluation and Safety at (240) 276-0450. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding postmarket surveillance, please contact CDRH's Office of Surveillance and Biometric's (OSB's) Division of Postmarket Surveillance at 240-276-3474. For questions regarding the reporting of device adverse events (Medical Device Reporting (MDR)), please contact the Division of Surveillance Systems at 240-276-3464. 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 (240) 276-3150 or at its Internet address http://www.fda.gov/cdrh/industry/support/index.html.

Sincerely yours,

Maria M Clan

Maria M. Chan. Ph.D. Acting Division 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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510(k) SUBMISSION FOR MAMMAPRINT SERVICE IN THE U.S.

Section 4: Indications for Use Statement

Indications for Use Form

510(k) Number (if known):

K080252

Device Name: MammaPrint®

Indications for Use:

The test is performed for breast cancer patients who are less than 61 years old, Stage I or Stage II disease, with a tumor size of ≤ 5.0 cm and fymph 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)

m. Can

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ると

Jision Sign-Off

Office of In Vitro Diagnostic Device Evaluation and Safety

Kofo 252

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.