(7 days)
None
Not Found
No
The description details a microarray-based gene expression analysis and a calculation based on correlation to a template, which are standard bioinformatics techniques and do not explicitly mention or describe the use of AI or ML algorithms.
No
The device is described as a "diagnostic test service" that provides a "prognostic marker only" to assess risk, not to treat or cure a condition.
Yes
The "Intended Use / Indications for Use" section explicitly states, "MammaPrint® is a qualitative in vitro diagnostic test service."
No
The device description clearly outlines a process involving physical components like microarray chips, RNA extraction, hybridization, and scanning, indicating it is not solely software.
Yes, this device is an IVD (In Vitro Diagnostic).
The "Intended Use / Indications for Use" section explicitly states: "MammaPrint® is a qualitative in vitro diagnostic test service..."
This confirms that the device is intended to be used outside of the body to examine samples (breast cancer tissue) for diagnostic purposes (assessing risk for distant metastasis).
N/A
Intended Use / Indications for Use
MammaPrint® is a qualitative in vitro diagnostic test service, performed in a single laboratory, using the gene expression profile of fresh frozen breast cancer tissue samples to assess a patients' 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.
Product codes (comma separated list FDA assigned to the subject device)
NYI
Device Description
The MammaPrint® test is performed and provided as a service by Agendia Laboratory. The test is a microarray based gene expression analysis of RNA extracted from breast tumor tissue. The test is a custom-designed array chip manufactured by Agilent Technologies using the Agilent oligonucleotide microarray platform which assesses the mRNA expression of the 70 genes in triplicate. The MammaPrint® microarray features eight 1900-feature subarrays per glass slide which can each be individually hybridized. Per subarray 232 reporter genes are printed in triplicate, including the 70 genes which make up the MammaPrint® prognostic profile. Each subarray additionally includes 915 normalization genes and 289 spots for hybridization and printing quality control.
The analysis is based on several processes: isolation of RNA from frozen tumor tissue sections, DNAse treatment of isolated RNA, linear amplification and labeling of DNAse treated RNA, cRNA purification, hybridization of the cRNA to the MammaPrint® microaray, 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, Low Risk Borderline, High Risk Borderline).
Mentions image processing
Yes
Mentions AI, DNN, or ML
Not Found
Input Imaging Modality
Not Found
Anatomical Site
breast cancer tissue
Indicated Patient Age Range
less than 61 years old
Intended User / Care Setting
Physicians / single laboratory
Description of the training set, sample size, data source, and annotation protocol
The study data published in Nature (Training set) and the New England Journal of Medicine (Test set), showed that the 70-gene signature can predict the development of distant metastases in lymph node negative primary breast cancer.
Description of the test set, sample size, data source, and annotation protocol
The study data published in Nature (Training set) and the New England Journal of Medicine (Test set), showed that the 70-gene signature can predict the development of distant metastases in lymph node negative primary breast cancer. The MammaPrint® was further validated in an independent external study by the TRANSBIG consortium, which involved patients from five European centers, namely Institute Roussy (Villeiuif. France). Karolinska Institute (Stockholm, Sweden), Centre Rene Hugenin (Saint-Cloud, France), Guy's Hospital (London, U.K.) and John Radcliffe Hospital (Oxford, U.K.). The data from this study was published in September 2006 issue of Journal of National Cancer Institute (JNCI). Patients were eligible for inclusion if they were younger than 61 years old at diagnosis, diagnosed before 1999 with node negative, T1-T2 (
§ 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.
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510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY
A. 510(k) Number:
- B. Purpose for Submission: New device
- C. Measurand: 70 gene expression profile
D. Type of Test:
Expression microarray
Test service performed in a single laboratory in Agendia's Amsterdam facility.
E. Applicant:
Agendia BV
- F. Proprietary and Established Names: MammaPrint®
G. Regulatory Information:
-
- Regulation section:
21 CFR 866.6040 Gene expression profiling test system for breast cancer prognosis
- Regulation section:
-
- Classification:
Class II
- Classification:
-
- Product code:
NYI, Classifier, prognostic, recurrence risk assessment, RNA gene expression, breast cancer
- Product code:
-
- Panel:
Immunology (82)
- Panel:
H. Intended Use:
-
- Intended use(s):
MammaPrint® is a qualitative in vitro diagnostic test service, performed in a single laboratory, using the gene expression profile of fresh frozen breast cancer tissue samples to assess a patients' risk for distant metastasis.
- Intended use(s):
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.
-
- Indication(s) for use: Same as intended use
-
- Special conditions for use statement(s):
For prescription use only MammaPrint® is not intended for diagnosis, or to predict or detect response to therapy, or to help select the optimal therapy for patients.
-
- Special instrument requirements: Agilent 2100 Bioanalyzer: Serial number DE54700497 en DE24802382 Agilent DNA microarray scanner: Serial number us22502555
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Note: The scanner and bioanalyzer are components of this assay and are cleared only for this assay and not for any other application. In addition, clearance is only limited to the bioanalyzer and scanner with the serial numbers as specified above.
I. Device Description:
The MammaPrint® test is performed and provided as a service by Agendia Laboratory. The test is a microarray based gene expression analysis of RNA extracted from breast tumor tissue. The test is a custom-designed array chip manufactured by Agilent Technologies using the Agilent oligonucleotide microarray platform which assesses the mRNA expression of the 70 genes in triplicate. The MammaPrint® microarray features eight 1900-feature subarrays per glass slide which can each be individually hybridized. Per subarray 232 reporter genes are printed in triplicate, including the 70 genes which make up the MammaPrint® prognostic profile. Each subarray additionally includes 915 normalization genes and 289 spots for hybridization and printing quality control.
The analysis is based on several processes: isolation of RNA from frozen tumor tissue sections, DNAse treatment of isolated RNA, linear amplification and labeling of DNAse treated RNA, cRNA purification, hybridization of the cRNA to the MammaPrint® microaray, 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, Low Risk Borderline, High Risk Borderline).
J. Substantial Equivalence Information:
-
- Predicate device name(s): None
-
- Predicate 510(k) number(s): Not applicable
-
- Comparison with predicate: Not Applicable
K. Standard/Guidance Document Referenced (if applicable):
CDRH Guidance for Industry and FDA Reviewers: Guidance for the content of premarket submissions for software contained in medical devices, May 11, 2005
L. Test Principle:
The MammaPrint® service is a microarray based gene expression analysis of a tumor. To assess the gene activity in a sample, frozen tissue sections are made, using a freeze microtome, and are collected in a receptacle. Total RNA is extracted from the tissue sections using a standard commercially available isolation kit. The sample is purified from DNA by DNAse treatment using a standard commercially available kit. The purified RNA sample is amplified and labeled twice with a cvanine-CTP fluorescent dye. Cy3 and Cy5. using a standard commercially available kit. The Cy3-labeled cRNA of the sample, together with the Cy5-labeled cRNA from a reference sample, is subsequently distributed over the sub array.
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The Cy5-labeled cRNA of the sample, together with the Cy3-labeled cRNA from a reference sample, is subsequently distributed over a second subarray. This is called color reverse analysis. The hybridization chamber is composed of an 8-well gasket slide that fits over the microarray slide, and is held in place by a metal holder. During hybridization the slide is rotated, causing mixing by a bubble in the hybridization fluid. Hybridization is performed at 60°C for 17 hours in a rotation oven. By hybridization only complementary RNA will bind to a 60-mer oligo on the array.
For scanning the MammaPrint® microarrav an Agilent microarrav scanner is used. The Agilent DNA microarray scanner is a 48-slide scanning system that can read 1'' x 3" glass slide microarrays. The result after scanning is a scan file (multi-page TIFF). This TIFF contains two pages, one page for each dye used (Cy3/Cy5). These are used by the feature extraction software.
Agilent Feature Extraction Software opens the multipage TIFF and combines those into one image which shows a pattern of differently colored spots. The Feature Extraction Software analyses the scan file (TIFF) by determining the intensities of the individual features, subtracting background signal, perform normalization, and calculate ratios, errors and pvalues for each spot. The output is a data file (TXT) per subarray. The feature extraction software uses the MammaPrint® microarray chip design file as a template in order to identify control features, normalization features and reporter features. The fluorescent intensity of the features is a measure for the activity of that particular gene.
Data analysis is performed according to a specific MammaPrint® algorithm (MammaPrint® Index). The algorithm calculates the similarity ("cosinor correlation") of the sample expression profile to a template, (the mean expression profile of 44 tumors with a known good clinical outcome) and determines the molecular profile of the sample (Low Risk, High Risk). This algorithm is designed and programmed by Agendia and compiled into a standalone software program, "X-Print Analysis Software". The "X-Print Analysis Software" loads a data file (CSV) which is created by the laboratory technician by extracting specific information from the laboratory database. The CSV data file contains: external sample ID, internal sample ID, Technician name, Bio-analyzer ratio, RNA integrity number, location of straight and dye-swap data file (TXT). Chip Layout (8-pack) and additional comments by the technician.
The "X-Print Analysis Software" reads the CSV file, opens the Feature Extraction Software data files (TXT), performs quality control checks, determines the sample expression profile, calculates the correlation of sample profile to the "Low Risk" template profile on a scale of -1 to 1 (MammaPrint Index), compares the calculated correlation to a pre-defined cut-off value and determines the samples prognostic profile (Low Risk or High Risk). The analysis software output is an internal report (PDF) for every sample. In this report quality control values and analysis results are reported.
The MammaPrint® Index ranges from -1.0 to +1.0. Tumor samples with a MammaPrint® Index above the threshold of +0.4, are classified as low risk, and tumor samples with a MammaPrint® Index equal to or smaller than the threshold are classified as high risk.
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If the MammaPrint® Index of a sample is between and including the borderline region of 0.365 and 0.435, this sample will be relabeled and rehybridized. After second analysis, the average MammaPrint® Index of both experiments will be calculated. If the MammaPrint® Index is still between the borderline region for two measurements (i.e. between and including 0.3775 and 0.4225), this sample will be reported as a borderline sample in the report form.
M. Performance Characteristics (if/when applicable):
-
- Analytical performance:
- a. Precision/Reproducibility:
-
- Reproducibility starting from RNA pool:
-
Three individual RNA samples, 1 Low Risk Control Pool (LRC), 1 high risk control pool (HRC) and 1 borderline sample (BLS) were used. LRC is a pool of 13 low risk samples and HRC is a pool of 12 high risk samples. For the LRC and HRC, there are 101 and 107 independent measurements respectively, performed from August 2005 till August 2006 and there are 37 independent measurements for BLS performed from April 2005 till August 2005.
| Samples | Mean Index | SD |
|---|---|---|
| LRC | 0.689 | 0.026 |
| HRC | -0.50428 | 0.02747 |
| BLS | 0.433 | 0.031 |
2) Repeatability
- a) Starting from individual patients: Forty-six (46) different patient samples were amplified and hybridized a second time (on the same day). The intra-class correlation coefficient (ICC) was 0.9953.
- b) Starting from one labeled sample with multiple hybridizations: To determine whether the hybridization introduces variation, one labeled samples was hybridized 8 times. Results are summarized below:
| Hybridizations | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | Average | SD | |
| Index | 0.254 | 0.269 | 0.276 | 0.298 | 0.278 | 0.280 | 0.311 | 0.308 | 0.284 | 0.020 |
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- Site-to-site reproducibility study (Note that this device is only cleared as a service to be run within a single clinical laboratory):
Four breast tumor samples (one high risk and three low risks) were distributed to 3 independent laboratories (US, France, Netherlands) to validate the Classifier and to assess hybridization and scanner reproducibility. The individual laboratories processed the samples and performed the MammaPrint® assay. In addition, labeled RNAs were exchanged between the laboratories in the US and the Netherlands for hybridization and scanning (see study design below).
- Site-to-site reproducibility study (Note that this device is only cleared as a service to be run within a single clinical laboratory):
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Scheduled Exchange of Labeled RNA and Hybridized Arrays.
Image /page/4/Figure/1 description: The image shows a flowchart of an experiment involving 4 breast tumor RNAs: 234, 239, 241, and 248. The RNAs are processed in three locations: Amsterdam, Palo Alto, and Paris. In each location, the RNAs are labeled and hybridized, and then scanned. There is a cross-over between the Amsterdam and Palo Alto samples after the labeling step, and the Amsterdam and Palo Alto samples are scanned together.
Each tumor was labeled 2-3 times per lab with a total of eleven hybridizations and for each tumor the minimum and maximum index values are shown below.
| Tumor | Minimum | Maximum |
|---|---|---|
| 234 | 0.044 | 0.162 |
| 239 | 0.641 | 0.770 |
| 241 | 0.399 | 0.476 |
| 248 | 0.453 | 0.526 |
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- Reproducibility of multiple isolations starting from tissue sample: In order to determine the reproducibility of the complete MammaPrint® device process, from tissue processing to the end result, five previously analyzed tumor samples (one borderline, two high risk and two low risk) were isolated in duplicate. Over multiple days, the ten isolations from five tumors were processed according to standard MammaPrint® protocols.
| Sample | Original index | Result | Index from first
isolation | Index from second
isolation |
|--------|----------------|------------------------|-------------------------------|--------------------------------|
| S1 | 0.376 | High risk (borderline) | 0.254 | 0.374 |
| S2 | 0.608 | Low risk | 0.564 | 0.553 |
| S3 | 0.659 | Low risk | 0.639 | 0.680 |
| S4 | -0.105 | High risk | 0.068 | 0.067 |
| S5 | -0.305 | High risk | -0.171 | -0.337 |
No statistically significant difference in MammaPrint® risk group assignment or MammaPrint® index was observed between two separate RNA isolations.
- b. Linearity/assay reportable range:
Linearity is not applicable for this type of assay. The correlation coefficient to the good profile (MammaPrint® Index) is reported on a scale of -1.000 to +1.000. Since cutoff for MammaPrint® Index is set at +0.40, risk assessment is reported as Low Risk (MammaPrint® Index > +0.40), or High Risk (MammaPrint® Index ≤ +0.40).
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- Traceability, Stability, Expected values (controls, calibrators, or methods): C. Quality control materials
Agendia has two reference samples as quality controls to monitor experiment to experiment quality and temporal stability. Both reference samples (HRC and LRC) comprise of a pool of >10 previously analyzed patient samples with a similar MammaPrint® Index. HRC is a pool of High Risk patients; LRC is a pool of Low Risk patients. To monitor the experiment quality, the two reference samples are labeled and hybridized in each round of experiments. The MammaPrint® Index for these control samples have to be within set limits. if thev are outside the limits, all samples for that assay run will be rejected. To check for temporal stability, MammaPrint® Index is plotted over time, and is monitored for trends on a 3 monthly basis. Agendia also has a reference sample called MammaReference Pool (MRP) which is used in color reverse analysis. The MRP is a RNA pool of tumors from 100 patients which were used in the NEJM publication, the balance between good and poor outcome is equally chosen as in the publication.
Device stability
The chip is stable for at least one year. The Cy Dyes are stable for up to 3 months at 2-8°C.
- d. Detection limit:
Fifty percent (50%) tumor cell content in a sample was the detection limit used in the original MammaPrint manuscripts. Therefore 50% was used as cutoff for this validation experiment.
The analysis of percentage tumor epithelial cells is performed by the tumor nuclei method (i.e. percentage of tumor cells present). To determine the eligibility of a tumor specimen for MammaPrint® analysis, the first and last viable sections are used to determine the relative amount of tumor present in the biopsy. After embedding, the tumor is sectioned multiple times. The first and last viable sections are used for tumor percentage analysis. The mean of the two values is taken as the actual tumor cell content of a given specimen.
To determine the amount of tumor present, a pathologist inspects the H&E stained slides. An estimate is made of the tumor cell nuclei versus the total number of nuclei (i.e., tumor cell nuclei + lymphocytes + endothelial cells + other non tumor cells). Although the development of the original sample acceptance parameters were based on the original tumor samples with a tumor % cut-off of 50%. Subsequently, these parameters were adapted and validated to perform on tissue samples with a tumor % cut-off of 30%.
Specimen Requirements
Minimum amount of tissue material required to perform an acceptable assay:
- . A 6 mm biopsy punch is used to excise the tumor from a surgical specimen. One standard biopsied tumor sample of 5 mm x 6 mm diameter is sufficient to perform 2500 MammaPrint® analysis.
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- . The amount of cRNA required for one reaction is 8 ul of labeled cRNA at 25 ng/uL.
- e. Analytical specificity:
RNA specifications provided were adequate to exclude the presence of any effect from likely interfering substances.
-
f. Assay cut-off:
Mammaprint® Index cut-off is set at +0.40. The cut-off is based on clinical performance characteristics of MammaPrint® (10% clinical false negative) and not on a technical performance characteristic. The cut-off was determined prior to the independent validation trial described below. -
- Comparison studies:
- a. Method comparison with predicate device: Not applicable
- b. Matrix comparison: Not applicable
-
- Clinical studies:
MammaPrint® is a gene expression profiling that predicts the risk of metastasis in breast cancer patients. The test was developed based on research performed at the Netherlands Cancer Institute and the Antoni van Leeuwenhoek Hospital in Amsterdam, The Netherlands. The study data published in Nature (Training set) and the New England Journal of Medicine (Test set), showed that the 70-gene signature can predict the development of distant metastases in lymph node negative primary breast cancer. The gene signature was subsequently developed into the diagnostic test, the MammaPrint® which was published in BioMed Central (BMC) Genomics. The MammaPrint® was further validated in an independent external study by the TRANSBIG consortium, which involved patients from five European centers, namely Institute Roussy (Villeiuif. France). Karolinska Institute (Stockholm, Sweden), Centre Rene Hugenin (Saint-Cloud, France), Guy's Hospital (London, U.K.) and John Radcliffe Hospital (Oxford, U.K.). The data from this study was published in September 2006 issue of Journal of National Cancer Institute (JNCI). Patients were eligible for inclusion if they were younger than 61 years old at diagnosis, diagnosed before 1999 with node negative, T1-T2 (+0.40), High Risk (MammaPrint Index