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510(k) Data Aggregation
(526 days)
Agendia Inc.
The MammaPrint FFPE NGS kit is a qualitative in vitro diagnostic test for use by clinical laboratories using target enrichment Next Generation Sequencing (NGS) technology for gene expression profiling of the 70-gene MammaPrint Breast Cancer signature on formalin-fixed, paraffin-embedded (FFPE) breast cancer tissue samples. The test is used to assess a patient's risk to develop distant metastasis within 5 years and up to 10 years after diagnosis.
The MammaPrint FFPE NGS kit is performed for breast cancer patients with Stage I or Stage II disease, with tumor size ≤ 5.0 cm and lymph node negative. The test result is indicated for use by physicians as a prognostic marker only, along with other clinicopathological factors.
The MammaPrint FFPE NGS kit is a sequencing-based gene expression analysis of a tumor. The analysis is based on several processes: isolation of RNA from FFPE breast cancer tissue sections; library preparation of RNA resulting in cDNA adapter-ligated sequences; enrichment of the 70 genes (capture step); sequencing of the enriched library in the flow cell and data acquisition; MammaPrint Index calculation of the risk classification in breast cancer patients.
Data analysis is performed according to the MammaPrint FFPE NGS algorithm (resulting in MammaPrint Index or MPI). This algorithm was designed and programmed by Agendia and incorporated into a proprietary software program, which loads the FASTQ data file. The software loads file, performs quality control checks and determines the molecular profile of the sample by calculating the MammaPrint index by determining the correlation of the sample's 70 gene expression profile to the mean expression profiles of tumors with a known good and poor outcome.
The provided text describes the acceptance criteria and the study that proves the MammaPrint FFPE NGS kit meets these criteria, primarily by demonstrating substantial equivalence to its predicate device (MammaPrint FFPE microarray).
Here's a breakdown of the requested information:
1. Table of Acceptance Criteria and Reported Device Performance
The document doesn't explicitly outline a formal "acceptance criteria" table with pre-defined thresholds for performance metrics. Instead, it demonstrates performance through concordance studies and reproducibility assessments against the predicate device, or by showing high agreement for controls. The clinical performance is demonstrated by comparing the survival outcomes of the new device to those of the predicate device on the same patient cohort from the RASTER study.
Acceptance Criteria (Implied by the study design and results presented):
| Performance Metric | Implied Acceptance Criteria (Achieved by the device) | Reported Device Performance |
|---|---|---|
| Method Comparison (Study-1): | ||
| OPA (High vs. Low) | High concordance with predicate device. | 97.42% (95% Cl: 93.55, 98.99) |
| NPA (High vs. Low) | High concordance with predicate device. | 93.85% (95% C1: 85.22, 97.58) |
| PPA (High vs. Low) | High concordance with predicate device. | 100.00% (95% CI: 95.91, 100.00) |
| Method Comparison (Study-2): | ||
| OPA (ranging across sites) | High concordance with predicate device. | 91.09% to 92.41% |
| PPA (ranging across sites) | High concordance with predicate device. | 91.39% to 99.34% |
| NPA (ranging across sites) | High concordance with predicate device. | Lower: 82.89% to 93.42% (noted as impacted by borderline samples) |
| Repeatability of RNA Isolation (Categorical Results): | 100% agreement between repeat isolations. | 100% agreement (95%Cl: High Risk: 86.2-100.0, Low Risk: 81.6-100.0, Borderline: 34.2 –100.0) |
| Reproducibility of Controls (Categorical Results): | 100% agreement for known controls across sites/operators/lots. | 100% agreement for both CTRL-HR and CTRL-LR (Table 1: 94.2%-100% CI for both). |
| Precision/Reproducibility (Categorical Results: Study-1): | High agreement for different risk categories. | PREC-BD (Borderline): 81% (72.0, 88.5) (Table 3) |
| PREC-HR (High Risk): 100% (96.9, 100) (Table 3) | ||
| PREC-LR (Low Risk): 100% (96.9, 100) (Table 3) | ||
| Precision/Reproducibility (Categorical Results: Study-2): | High agreement, especially for samples not near threshold. | Range from 58% to 100% (Table 5). Samples 2 (Low Risk) and 5 (High Risk) had 83% and 58% agreement respectively. |
| Detection Limit (Valid Rates): | Acceptable valid rates at specified RNA input and quality. | DV200 "poor" (35-49%) with ≥100 ng RNA: 80% to 100% valid rates. |
| DV200 "Standard" (35-80%) with ≥100 ng RNA: 80% to 100% valid rates. | ||
| Clinical Performance (DRFI): | Similar survival outcomes to predicate device. | Kaplan-Meier plots suggest significant difference in survival curves among different risk groups for MammaPrint FFPE NGS kit, p=0.001. |
| 5-year DRFI: Low Risk 98.1%, Borderline 92.6%, High Risk 88.2% (Table 6). | ||
| 10-year DRFI: Low Risk 95.2%, Borderline 92.6%, High Risk 82.0% (Table 6). | ||
| The results "indicated that both devices show similar clinical performance" based on RASTER study follow-up. |
2. Sample Sizes and Data Provenance
- Method Comparison Study-1: 155 samples used. Data acquired retrospectively as these samples were "previously processed on MammaPrint FFPE microarray as part of routine diagnostics."
- Method Comparison Study-2: 303 samples used. Data acquired retrospectively from "previously collected patient samples."
- Repeatability of RNA Isolation: Not explicitly stated, but implies multiple FFPE tumor blocks, each sectioned twice.
- Reproducibility of Controls: 2 control samples (CTRL-HR, CTRL-LR), each processed 50 times across 4 external sites, 2 operators per site, 6 NGS runs, and 3 lot numbers. Total of 100 measurements for controls.
- Precision/Reproducibility Assessment-Study-1: 3 samples (High Risk, Borderline, Low Risk). Each processed 96 times (4 external sites x 2 operators x 6 NGS runs x 2 duplicates per run).
- Precision/Reproducibility Assessment-Study-2: 8 samples. Each processed repeatedly (from RNA isolation to sequencing) by 2 operators at 3 sites, with 4 runs per site and 2 replicates per run. This results in 16-24 replicates per sample (table indicates N of 16 or 24).
- Detection Limit: 8 FFPE breast cancer samples with "poor" DV200 quality were evaluated, with various dilutions. Additional unstated number of "Standard" DV200 samples.
- Clinical Validation (RASTER Study):
- Original RASTER enrollment: 427 patients.
- Subset of FFPE samples from RASTER study used for NGS kit validation: 345 samples.
- Samples successfully processed for analysis: 316 samples.
- Data provenance: Multicenter observational study conducted in the Netherlands between 2004 and 2006 (for initial RASTER study). The current analysis uses updated 5- and 10-year follow-up data. This is prospective observational data with the device evaluated retrospectively on archived samples.
3. Number of Experts and Qualifications for Ground Truth
The document does not mention the use of experts to establish ground truth for the analytical or reproducibility test sets. The ground truth for these studies is based on comparisons against the predicate device's results or known control sample values.
For the clinical validation, the ground truth for distant recurrence-free interval (DRFI) and breast cancer specific survival (BCSS) is outcomes data from the RASTER study, defined as distant breast cancer recurrence or death from breast cancer. The establishment of these clinical outcomes likely involved clinical experts (e.g., oncologists, pathologists, and medical records review) over the 10-year follow-up period, but the specific number and qualifications of these experts are not detailed in this submission.
4. Adjudication Method for Test Set
The document does not describe an adjudication method (such as 2+1 or 3+1) for the test sets. For analytical studies, agreement is based on direct comparison of results between NGS and microarray platforms or within the NGS platform itself (repeatability/reproducibility). For the clinical study, outcomes (DRFI, BCSS) are established from patient follow-up data.
5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study
No MRMC comparative effectiveness study was performed as this device is a gene expression profiling test system, not an imaging device requiring human reader interpretation. The comparison is between the new NGS-based assay and the predicate microarray-based assay.
6. Standalone Performance
The "standalone" performance shown is the analytical performance of the MammaPrint FFPE NGS kit itself (repeatability, reproducibility, detection limit) and its clinical prognostic ability, which is an algorithm-only output (MammaPrint Index). The clinical validation study (RASTER) assesses the algorithm's ability to stratify patients by risk based on actual clinical outcomes. The device performance (result classification: High Risk, Borderline, Low Risk) is reported directly from the algorithm without a human-in-the-loop component for classification.
7. Type of Ground Truth Used
- Analytical Performance (Method Comparison, Repeatability, Reproducibility): The ground truth is effectively the predicate device's results or the known values of control samples. For repeatability/reproducibility, the ground truth is consistency of the device's own output.
- Clinical Validation: The ground truth is outcomes data (Distant Recurrence-Free Interval - DRFI, and Breast Cancer Specific Survival - BCSS) from prospectively collected clinical follow-up in the RASTER study. This is directly observed patient outcomes over 5 and 10 years.
8. Sample Size for the Training Set
The document focuses on the performance of the MammaPrint FFPE NGS kit, which is stated to use an "unchanged" 70-gene signature and scoring algorithm from the predicate device. The original MammaPrint 70-gene signature was developed much earlier. The description states: "This algorithm was designed and programmed by Agendia and incorporated into a proprietary software program, which loads the FASTQ data file." The specifics of the training set for the original MammaPrint algorithm are not provided in this document. This submission is for a new platform (NGS) of an existing and cleared diagnostic system, demonstrating its equivalence, rather than a new algorithm development.
9. How the Ground Truth for the Training Set was Established
As noted above, the original training of the MammaPrint algorithm is not detailed here. The clinical validation in this document uses the RASTER study data, where the ground truth is clinical outcome data (DRFI, BCSS) established through follow-up. This RASTER data is used for validation of the new NGS platform, not for training a new algorithm.
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(120 days)
Agendia Inc.
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.
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.
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.
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