MammaPrint® FFPE is a qualitative in vitro diagnostic test, performed in a central laboratory, using the gene expression profile obtained from formalin-fixed paraffin embedded (FFPE) breast cancer tissue samples to assess a patient's risk for distant metastasis within 5 years.

The test is performed for breast cancer patients, with Stage I disease, with tumor size ≤ 5.0 cm and lymph node negative. The MammaPrint FFPE 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 formalin-fixed paraffin embedded (FFPE) tumor tissue sections, DNase treatment of isolated RNA, amplification DNase treated RNA resulting in cDNA, labeling and purification of amplified cDNA, hybridization of the diagnostic 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 FFPE tissue sample. The result is an expression profile, or fingerprint, of the sample.

The molecular profile of the sample is determined (Low Risk) by calculating the MammaPrint index (MPI) by determining the correlation of the sample expression profile to the mean expression profiles of risk templates of tumors with a known good and poor outcome.

AI/ML Overview

The provided text describes the acceptance criteria and study for the MammaPrint FFPE device, focusing on the analytical performance related to a change in microarray scanner.

Here's the breakdown of the information requested:

1. A table of acceptance criteria and the reported device performance

Acceptance Criteria Category	Specific Criteria	Reported Device Performance
Technical Equivalence (Concordance between Scanners)	Passing and Bablok regression for MammaPrint indices between C-scanners (Agilent G2505) and D-scanners (Agilent SureScan Dx G5761AA) must meet pre-defined acceptance criteria (specific values for slope and intercept not explicitly stated, but implied to be near 1 and 0, respectively).	Scanner SG18309119 vs. C-scanners: y=0.00 +1.00 x (95%Cl – slope: 1.000 – 1.002, 95%Cl intercept: -0.0002 to 0.000). Scanner SG18449122 vs. C-scanners: y=0.0010 +1.00 x (95%Cl – slope: 1.000 – 1.0021, 95%Cl intercept: 0.0014 to 0.001). Both results are stated to be "within the pre-defined acceptance criteria."
	Overall concordance, Negative Percent Agreement (NPA), and Positive Percent Agreement (PPA) for MammaPrint categorical results (High/Low Risk) between C-scanners and D-scanners must be within pre-defined acceptance criteria.	Scanner SG18309119 vs. C-scanners: Overall concordance: 99.7%. NPA: 100% (95%Cl: 98.1-100). PPA: 99.3% (95%Cl: 96.0 – 99.9). Scanner SG18449122 vs. C-scanners: Overall concordance: 100%. NPA: 100% (95%Cl: 97.1 – 100). PPA: 100% (95%Cl: 97.5 - 100). All reported values are stated to be "within the predefined acceptance criteria."
Precision Assessment	F-test p-values for variability of repeated measurements of control samples between C-scanners and D-scanners should not show a significant difference (i.e., p-value > 0.05).	F-test p-values for all four control samples were "all well below the significance level of 0.05, indicating there is no significant difference in precision between C and D-scanners." (This wording is a bit confusing; "well below 0.05" would typically indicate a significant difference. However, given the context of demonstrating no significant difference, it likely implies that the null hypothesis of equal variance could not be rejected, which means the p-value was above 0.05. Re-reading, "indicating there is no significant difference" following "well below the significance level of 0.05" is a logical contradiction. Assuming the intent was to show no significant difference in precision, the p-values should have been above 0.05. This might be a typo in the FDA document, or the interpretation of the F-test result is inversely stated. Given the conclusion that "there is no significant difference," the F-test result must have been non-significant, meaning p > 0.05. Let's assume the intent was to show non-significant difference.)
Clinical Correlation	MammaPrint index should correlate with clinical outcome (distant recurrence risk).	Result distribution: In the RASTER study, Bin 1 (MPI 0.36 to +1) had 0% observed 5-yr DR risk (N=37). Bin 4 (MPI -1 to -0.57) had 13.6% observed 5-yr DR risk (N=66). Cox regression analysis: With each increase in MammaPrint index unit, there is a 0.224 (4.5 folds) decrease in recurrence risk at 5 years (p=0.001; 95% Cl: 0.092-0.543). This demonstrates correlation.

2. Sample size used for the test set and the data provenance (e.g., country of origin of the data, retrospective or prospective)

Sample Size for Technical Equivalence (Concordance): 92 8-pack arrays (likely representing 92 individual samples, or potentially 92 unique arrays, each containing multiple spots/analyses).
Sample Size for Precision Assessment: Four diagnostic control samples (PLEP3, PHHE2, PHTR2, PBCL2), measured repeatedly. The number of repeated measurements is not specified.
Sample Size for Clinical Correlation (RASTER study): 345 patients.
Data Provenance: Not explicitly stated for analytical performance (concordance and precision). For the clinical correlation, it references the RASTER study, which was "previously submitted in K141142," but country of origin and retrospective/prospective nature are not detailed here.

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts (e.g., radiologist with 10 years of experience)

For Analytical Performance: Ground truth is established by the MammaPrint index and categorical results from the FDA-cleared C-scanners (Agilent G2505), acting as the reference. No human experts are involved in establishing this specific ground truth.
For Clinical Correlation (RASTER study): The clinical outcome (distant recurrence risk) is the ground truth. While not detailed in this excerpt, clinical outcomes studies typically rely on physician diagnoses, follow-up, and potentially pathology reports, rather than a panel of experts specifically adjudicating "ground truth" for the test results.

4. Adjudication method (e.g., 2+1, 3+1, none) for the test set

For Analytical Performance: None. The ground truth is the output from the predicate device's scanner (Agilent G2505).
For Clinical Correlation: Not specified in this document. Clinical outcome data collection methods vary, but typically don't involve an adjudication panel for the outcome itself, but rather established clinical trial protocols and follow-up.

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

This is not an MRMC study. The MammaPrint device is a gene expression profiling test, not an AI imaging or diagnostic assistance tool for human readers. It provides a prognostic marker directly from tissue analysis.

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

Yes, this is a standalone device. The MammaPrint FFPE test generates a result (MammaPrint Index and High/Low Risk classification) directly from the gene expression profile of the tumor tissue. The "algorithm only" performance is what is being evaluated and validated. The "human-in-the-loop" component mentioned is the physician's use of the result "along with other clinicopathological factors" for prognostic assessment.

7. The type of ground truth used (expert consensus, pathology, outcomes data, etc.)

For Analytical Performance (concordance and precision): The ground truth is the MammaPrint index and categorical result generated by the previously FDA-cleared C-scanners (Agilent G2505).
For Clinical Correlation: The ground truth is 5-year distant recurrence (DR) risk, which is a form of outcomes data.

8. The sample size for the training set

Not explicitly mentioned for the current submission. The MammaPrint assay itself was developed and validated earlier, and the current submission is for a modification (scanner change) to an already cleared predicate device (K141142). The original training set for the MammaPrint algorithm would have been part of the earlier submissions but is not detailed here.

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

Not explicitly mentioned in this document. For the original MammaPrint development, the ground truth for training would have involved gene expression profiles correlated with long-term clinical outcomes (e.g., distant metastasis-free survival) from large cohorts of breast cancer patients, likely using robust clinical data and follow-up.

Summary

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November 05, 2020

Agendia Inc. Janice Hogan Partner Hogan Lovells US LLP 1735 Market Street Suite 2320 Philadelphia, PA 19103

Re: K201902

Trade/Device Name: MammaPrint® FFPE 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: July 8, 2020 Received: July 8, 2020

Dear Janice Hogan:

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. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. 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. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to 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

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requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-products); good manufacturing practice requirements as set forth in the quality systems (OS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

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 https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

For comprehensive regulatory information about mediation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely,

Soma Ghosh, Ph.D. Chief Molecular Pathology and Cytology Branch Division of Molecular Genetics and Pathology OHT7: Office of In Vitro Diagnostics and Radiological Health Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

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510(k) Number (if known)

K201902

Device Name

MammaPrint® FFPE

Indications for Use (Describe)

Type of Use (Select one or both, as applicable)

Z Prescription Use (Part 21 CFR 801 Subpart D)

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

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Image /page/3/Picture/0 description: The image shows the Agendia logo. On the left is a colorful fingerprint graphic. To the right of the graphic is the word "AGENDIA" in bold, black letters. Below the word "AGENDIA" is the phrase "PRECISION ONCOLOGY" in smaller, gray letters.

510 (k) Summary

1.Submitter Agendia Inc. 22 Morgan Irvine, California 92618 Email: marcelo.trevino@agendia.com Telephone: +1 310 409 7828 Contact person: Marcelo Trevino, Global VP Regulatory & Quality Assurance Date prepared: November 3, 2020

Device MammaPrint FFPE

3.Predicate Device

MammaPrint FFPE K141142

4. Device Description

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

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Image /page/4/Picture/0 description: The image is a logo for Agendia Precision Oncology. The logo features a colorful fingerprint graphic on the left side. To the right of the graphic is the word "AGENDIA" in large, bold, black letters. Below "AGENDIA" is the phrase "PRECISION ONCOLOGY" in smaller, gray letters.

5. Indications for Use

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

6. Comparison to Predicate Device

The predicate is Agendia Inc's MammaPrint (K141142). The predicate device, with product code NYI, is regulated under 21CFR866.6040. The modified MammaPrint device subject of this 510(k) is substantially equivalent to Agendia's MammaPrint device covered under K141142. It has the identical indications for use and very similar technological features. The only difference is that the modified MammaPrint device allows the use of Agilent SureScan Dx microarray scanners, part #G5761AA. The minor change in the specific scanner that is used does not raise any new types of safety or effectiveness questions. Performance testing confirms that the change in scanner does not adversely impact functioning of the system compared to the predicate.

7. Performance Data

Analytical Performance Compared to Predicate

Analytical performance of MammaPrint was investigated by assessing concordance between results from Agilent scanners part #G2505 (C-scanner) and Agilent SureScan Dx microarray scanners, part #G5761AA (D-scanner). Additionally, precision was also assessed for the Agilent SureScan Dx microarray scanners, part #G5761AA.

1- Technical equivalence of MammaPrint between scanner part #G2505 and SureScan Dx microarray scanner, part #G5761AA

Concordance experiments were performed based on designs as previously included in K080252 as well as K101454.

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Image /page/5/Picture/0 description: The image shows the Agendia logo. The logo consists of a fingerprint graphic on the left and the word "AGENDIA" in bold, black letters on the right. Below "AGENDIA" is the phrase "PRECISION ONCOLOGY" in smaller, gray letters. The fingerprint graphic is made up of concentric lines in a gradient of colors, ranging from red to blue.

nis validation, a set of 92 8-pack arrays were analyzed as part of regular diagnostics on the FDA cleared scanners (US45103019 and US811R3213). Subsequently, these were scanned a second time on the SureScan Dx scanners, SG18309119 and SG18449122. The text files generated as output were used to generate MammaPrint indices according to standard procedures.

The samples included in this dataset covered the entire MammaPrint readout range, including the borderline region. Additionally, there were four diagnostic control samples included that cover the MammaPrint categorical results; PLEP3 (Low Risk), PHHE2 (High Risk), PHTR2 (High Risk), PBCL2 (Low Risk -close to classification threshold).

MammaPrint indices were compared between both C and D-scanners using Passing and Bablok regression. The MammaPrint categorical results (High/Low Risk) were compared using a 2x2 contingency table after which concordance, Negative Percent Agreement (NPA) and Positive Percent (PPA) were determined. The Passing and Bablok regression comparing the MammaPrint indices between the Cscanners and SureScan Dx scanner SG18309119 is y=0.00 +1.00 x (95%Cl – slope: 1.000 – 1.002, 95%Cl intercept: -0.0002 to 0.000). The Passing and Bablok regression comparing the MammaPrint indices between the C-scanners and SureScan Dx scanner SG18449122 is y=0.0010 +1.00 x (95%Cl – slope: 1.000 – 1.0021, 95%Cl intercept: 0.0014 to 0.001). The overall concordance in MammaPrint categorical result between the C-scanners and the SureScan Dx scanners, SG18309119 and SG18449122 is 99.7% and 100%, respectively. For this comparison C-scanners versus SG18449122 the NPA is 100 % (95%Cl: 98.1-100) and 100% (95%Cl: 97.1 – 100), the PPA is 99.3% (95%Cl: 96.0 – 99.9) and 100% (95%Cl: 97.5 - 100), respectively.

The results of the Passing and Bablok regression of both D-scanners compared to the C-scanner are within the pre-defined acceptance criteria. Furthermore, the concordance, NPA and PPA determined for each Dscanner versus the FDA cleared C-scanners were all within the predefined acceptance criteria.

2- Test performance - precision assessment of the SureScan Dx scanners - SG18309119 and SG18449122

Precision assessment was performed using the four diagnostic control samples (PLEP3 [Low Risk], PHHE2 [High Risk], PHTR2 [High Risk], PBCL2 [Low Risk -close to classification threshold]) that were measured as

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Image /page/6/Picture/0 description: The image shows the logo for Agendia Precision Oncology. The logo features a colorful fingerprint graphic on the left, with the word "AGENDIA" in bold, black letters to the right. Below "AGENDIA" are the words "PRECISION ONCOLOGY" in a smaller, gray font. The fingerprint graphic is made up of concentric lines in shades of red, orange, yellow, green, and blue.

t of the validation. These control samples have a known MammaPrint result and cover all MammaPrint categorical results.

In order to assess the precision of both D-scanners versus the FDA cleared scanners, the variability of repeated measurements of these controls were compared between both scanners using the F-test. The F-test p-values for all four control samples were all well below the significance level of 0.05, indicating there is no significant difference in precision between C and D-scanners.

Correlation between MammaPrint Index and Clinical Outcome

Two analyses were conducted to show the correlation between the MammaPrint index and clinical outcome of the RASTER study (previously submitted in K141142):

1- Result distribution

In order to show a correlation between the MammaPrint index and clinical results were placed into bins consisting of 4 MammaPrint Index levels.

Bin	MPI Range	NumberofPatients	Percentageof Patients	Observed 5-yr DR risk
1	0.36 < MPI ≤+1	37	10.7%	0%
2	0.00 14241.2%1.4%
3	-0.5710029.0%9.0%
4	-1 ≤MPI≤-0.57	66	19.1%	13.6%
Total		345	100.0%

Table 1: Distribution of observed 5 year Distant Recurrence (DR) Risk in RASTER study over 4 Bins of MammaPrint Index Levels

2- Cox regression analysis

Cox regression analysis of the 5 year DR risk with MPI as an independent covariate shows a correlation between MammaPrint index and risk of recurrence. A higher MammaPrint index shows a lower risk for a recurrence. With each increase in MammaPrint index unit, there is a 0.224 (i.e., 4.5 folds) decrease in recurrence risk at 5 years (p=0.001; 95% Cl: 0.092-0.543).

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Image /page/7/Picture/0 description: The image shows the Agendia logo. The logo consists of a colorful fingerprint graphic on the left and the word "AGENDIA" in bold, black letters on the right. Below "AGENDIA" is the phrase "PRECISION ONCOLOGY" in smaller, lighter-colored letters.

The categorical analysis and Cox regression analysis thus demonstrate a correlation between the unit change in the MammaPrint index and the clinical outcome.

8. Conclusion

MammaPrint is a clinically and analytically accurate prognostic method for providing a risk assessment of distant metastasis of breast cancer when performed either using the Agilent scanner #partG2505 or the SureScan Dx scanner, part #G5761AA, supporting a finding of substantial equivalence.

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.