(678 days)
For in vitro diagnostic use. For use on the Biocartis Idylla™ System only.
The Idylla™ MSI Test, for use on the Idylla™ System, uses formalin-fixed, paraffin-embedded (FFPE) tissue sections of human CRC tumor, from which nucleic acids are liberated, then analyzed using PCR amplification of seven monomorphic biomarkers (ACVR2A, BTBD7, DID01, MRE11, RYR3, SEC31A and SULF2) and subsequent melt-curve analysis. The Idylla™MSI Test reports results as either microsatellite stable (MSS), or microsatellite instability high (MSI-H) or invalid.
Idylla™ MSI Test is indicated for use by healthcare professionals for the qualitative identification of microsatellite instability (MSI) in colorectal cancer (CRC) tumors, indicative of mismatch repair deficiency, as an and in the identification of potential Lynch syndrome to help identify patients that would benefit from additional genetic testing to diagnose Lynch syndrome.
The results from the Idylla™ MSI Test should be interpreted by healthcare professionals in conjunction with other clinical findings, family history, and other laboratory data. The Idyla™ MSI Test should not be used for diagnosis of CRC. The clinical performance of this device to guide treatment decision for MSI high patients has not been established.
The Biocartis Idylla™ System covers the entire process from sample to result with fully integrated sample preparation followed by PCR amplification and high-resolution melting detection of the targeted sequences. The Idylla™ System consists of the Idylla™ Console connected to one or more Idylla™ Instruments (up to eight instruments). Idylla™ Cartridges, designed for specific applications, can be processed by the Idylla System using test specific software (Test Type Package, MSI TTP). The Idylla™ MSI Test procedure and data analysis are validated for FFPE tissue sections.
The Idylla™ MSI Test detects a novel panel of seven monomorphic biomarkers.
The Idylla™ MSI Test Cartridges are ready-for-use and contain the necessary reagents to perform sample preparation, PCR amplification and high-resolution detection, starting from insertion of FFPE tissue sections. The MSI TTP directs the processing of the sample within the cartridge.
The process steps in the Idylla™ MSI Test are:
- FFPE liquefaction and cell lysis: After insertion of the FFPE tissue section into the cartridge, a combination of chemical reagents, enzymes, heat, and High Frequency Ultrasound (HIFU) induces deparaffinization, disruption of the tissue and lysis of the cells. The nucleic acids are liberated for subsequent PCR amplification.
- PCR using biomarker-specific primers: All necessary PCR reagents are present in a stable formulation and are used to amplify seven biomarkers indicative for MSI status.
- Detection and analysis: Detection of these specific targets is performed using fluorescently labeled molecular beacons after PCR amplification. These beacons differentially melt from the wild type or mutated amplicons with increasing temperature. The fluorescence differences at melting temperatures are further analyzed by the MSI TTP and translated into genetic calls on biomarker level and MSI status on sample level.
- Reporting: At the end of the run, the result, reporting the MSI status, the number of mutated biomarkers, and an MSI score range in the analyzed sample is displayed on the console screen.
Here's a breakdown of the acceptance criteria and study details for the Idylla™ MSI Test based on the provided FDA 510(k) summary:
1. Table of Acceptance Criteria & Reported Device Performance
The document doesn't explicitly state "acceptance criteria" as a separate section with numerical targets. Instead, it demonstrates performance through various analytical and clinical studies, showing high concordance and hit rates. Below is a summary of the performance metrics reported, which implicitly serve as the evidence for meeting acceptance for substantial equivalence.
| Metric (Implicit Acceptance Criteria) | Reported Device Performance |
|---|---|
| Analytical Performance | |
| Analytical Specificity (LoB) | Correctly generated 'Invalid' when no sample. Correctly generated 'MSS' for wild-type samples. No cross-reactivity between mutant/wild-type targets or interference from known mutations. |
| Analytical Sensitivity (LoD) - Contrived | Estimated LoD was 30% allelic frequency. Correct MSI status calls with estimated allelic frequency LoD using contrived specimens, ranging from 15 to 30% for low input background in individual biomarkers. (91.7% at 10% AF for low input, 100% at 10% AF for high input, and 100% at 15% AF or higher for both inputs). |
| Analytical Sensitivity (LoD) - Clinical | 100% correct calls for 7 clinical samples (MSI-H and MSS) tested at ~33% neoplastic cell content and lowest sample input size (25 mm² per 10 µm section). This confirms LoD at 33% neoplastic cell content. |
| Reproducibility (Across sites, lots, days) | 100% identical and correct MSI status calls across three sites for all seven clinical samples tested (252 valid runs). No relevant effects of inter-instrument, inter-lot, or inter-day variability. Biomarker reproducibility >85% for most markers, with some exceptions noted for specific samples (e.g., Sample 2 BTBD7 at 77.78%, Sample 4 BTBD7 at 66.67%, Sample 7 ACVR2A at 88.89% - these are noted to be discordant from the clinical sample characterization). |
| Non-Interfering Substances | Hemoglobin (2 mg/mL), Triglycerides (37 mmol/L), and Paraffin (1 additional 10 µm section) showed 100% concordance with control results, indicating no impact on Idylla™ MSI Test. |
| Mucinous Content Interference | Samples with mucinous cell content up to 60% generated correct MSI status calls (96.5% of runs, 55/57), indicating no negative impact. |
| Necrotic Content Interference | Necrosis content up to 73% had no impact on the Idylla™ MSI Test, generating correct MSI status calls. |
| Clinical Performance (Vs. Predicate) | |
| PPA (Idylla vs. OncoMate) | 96.88% (95% CI: 83.78 - 99.92) across all samples (sequential + enrichment). For sequential cohort, 100% (78.2 - 100%). For enrichment cohort, 94.12% (71.31 - 99.85). |
| NPA (Idylla vs. OncoMate) | 99.07% (95% CI: 94.95 - 99.98) across all samples (sequential + enrichment). For sequential cohort, 99.07% (94.95 - 99.98%). For enrichment cohort, N/A (all were MSI-H). |
| OPA (Idylla vs. OncoMate) | 98.57% (95% CI: 94.93 – 99.83) across all samples. For sequential cohort, 99.19% (95.55 - 99.98). For enrichment cohort, 94.12% (71.31 - 99.85). |
| Clinical Performance (Vs. Germline NGS) | |
| PPA (Idylla vs. Germline NGS) | 92.00% (95% CI: 73.97 – 99.02) across all samples for identifying Lynch cases. For sequential cohort, 80% (28.36 - 99.49). For enrichment cohort, 95% (75.13 – 99.87). |
| NPA (Idylla vs. Germline NGS) | 89.81% (95% CI: 82.50 – 94.80) across all samples. For sequential cohort, 89.81% (82.51 - 94.80). For enrichment cohort, N/A. (Note: less informative as Lynch negative samples by NGS can still be MSI-H due to sporadic dMMR). |
| OPA (Idylla vs. Germline NGS) | 90.22% (95% CI: 83.99 – 94.20) across all samples. For sequential cohort, 89.38% (82.18 - 94.39). For enrichment cohort, N/A. |
2. Sample Size Used for the Test Set and Data Provenance
-
Analytical Studies (LoD, Reproducibility, Interference):
- LoD Estimation (Contrived): 24 replicates per allelic frequency level (Table 3), various levels (50%, 30%, 20%, 15%, 10%, 5%) for both high and low sample input.
- LoD Estimation (Clinical): 4 FFPE samples (titration experiments), with 6 replicates per concentration level.
- LoD Confirmation (Clinical): 7 FFPE clinical samples (MSI-H, MSS, borderline) tested in 10 replicates per lot (n=20) over 5 days on multiple instruments (total n=140 from all seven samples).
- Reproducibility: 7 individual FFPE clinical specimens (MSI-H and MSS) tested at 3 laboratory sites, with 12 replicates per sample per site (total 36 tests/sample, 252 valid runs across all sites).
- Interference (Hemoglobin, Triglycerides, Paraffin): 7 FFPE colorectal cancer clinical samples, tested in 5 replicates with each interfering substance.
- Interference (Mucin): 19 FFPE clinical samples (MSI-H and MSS) with varying mucinous content, tested in 3 replicates each (total 57 runs, 55 valid).
- Interference (Necrosis): 4 FFPE clinical samples with various necrosis levels, tested in triplicate with and without necrosis.
- Data Provenance: The document states "clinical samples" and "hospital biobanks", indicating human sample origin. The reference to "Colorectal Cancer Family Registry" for the enrichment cohort suggests these are existing, well-characterized clinical samples. It is retrospective in nature, using archived FFPE tissue sections. Origin country is not explicitly stated but implies multicenter (multiple sites for reproducibility) data.
-
Clinical Performance Study:
- Total samples: 143 samples.
- Sequential cohort: 123 samples (sequentially selected from two hospital biobanks).
- Enrichment cohort: 20 confirmed Lynch cases (obtained from the Colorectal Cancer Family Registry).
- Data Provenance: Retrospective, FFPE tissue sections of human CRC tumor.
3. Number of Experts Used to Establish Ground Truth for the Test Set and Their Qualifications
The document does not explicitly state the number of experts or their specific qualifications for establishing the ground truth for the test set as per a typical adjudication process for imaging or pathology.
The ground truth reference methods are:
- OncoMate™ MSI Dx Analysis System (Predicate Device): This is a legally marketed predicate device, implying its results are accepted as a reference standard.
- Germline NGS for MMR Genes: This is a molecular diagnostic method, considered a gold standard for identifying Lynch syndrome.
- Sample Characterization: For analytical studies, samples were characterized as MSI-H or MSS based on known properties (e.g., cell lines, clinical samples).
Therefore, the "experts" here are essentially the established and validated methods of the predicate device and germline NGS, rather than human expert readers adjudicating images.
4. Adjudication Method for the Test Set
No explicit adjudication method (like 2+1, 3+1) is mentioned or implied for the clinical performance study. The comparison is made directly against established reference methods: the OncoMate™ MSI Dx Analysis System and Germline NGS for MMR genes. These are direct comparisons between the device's output and the reference method's output.
5. If a Multi Reader Multi Case (MRMC) Comparative Effectiveness Study Was Done, and Effect Size of Human Readers Improvement with AI vs. Without AI Assistance
This device, the Idylla™ MSI Test, is an in vitro diagnostic (IVD) test that automatically reports results (MSS, MSI-H, or invalid) based on molecular analysis of tissue samples. It is not an AI-assisted diagnostic imaging device or a software that assists human readers in interpreting clinical data. Therefore, an MRMC comparative effectiveness study involving human readers with and without AI assistance is not applicable to this device. There is no human-in-the-loop component for result generation, so there's no "effect size of how much human readers improve with AI vs without AI assistance" to report.
6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) Was Done
Yes, the studies performed are inherently standalone performance of the Idylla™ MSI Test. The device provides an automated result (MSS, MSI-H, or Invalid) directly from the FFPE tissue sample without intermediate human intervention for interpretation of the molecular signals. The performance metrics (PPA, NPA, etc.) represent the algorithm's standalone accuracy against reference methods.
7. The Type of Ground Truth Used
The ground truth used for the clinical performance study was:
- Predicate Device Output: The results from the OncoMate™ MSI Dx Analysis System.
- Molecular Gold Standard: Germline Next Generation Sequencing (NGS) for DNA mismatch repair (MMR) genes, particularly for identifying Lynch cases.
For analytical studies, the ground truth was also based on well-characterized samples (e.g., cell lines with known allelic frequencies, clinical samples previously characterized as MSI-H or MSS).
8. The Sample Size for the Training Set
The document does not provide information about a separate "training set" or its sample size. This is typical for IVD submissions which validate a fixed algorithm or assay rather than continuously learning AI models. The development and optimization of such a test would involve internal R&D, but the submission focuses on the validation of the finalized product.
9. How the Ground Truth for the Training Set Was Established
As no specific training set is mentioned in the context of an AI/ML model, this question is not directly applicable. For the development of the Idylla™ MSI Test, the "ground truth" for optimizing the assay (e.g., primer design, melt curve analysis parameters) would have been established through a combination of scientific literature, internal studies using characterized cell lines, and reference clinical samples with known MSI status (likely determined by established methods like IHC or other PCR-based MSI tests).
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February 27, 2023
Image /page/0/Picture/1 description: The image shows the logo of the U.S. Food and Drug Administration (FDA). The logo consists of two parts: on the left, there is a seal with an abstract design, and on the right, there is the text "FDA U.S. FOOD & DRUG ADMINISTRATION" in blue. The text is arranged in three lines, with "FDA" in a larger font and enclosed in a blue square.
Biocartis NV Elnaz Jokar Regulatory Affairs Manager Generaal De Wittelaan 11 B3 Mechelen, Antwerpen 2800 Belgium
Re: K211181
Trade/Device Name: Idylla MSI Test Regulation Number: 21 CFR 864.1866 Regulation Name: Lynch syndrome test systems Regulatory Class: Class II Product Code: PZJ Dated: October 21, 2022 Received: October 21, 2022
Dear Elnaz Jokar:
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 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
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- 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 (QS) 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,
Zivana Tezak-fragale -S
Zivana Tezak, PhD Branch Chief Division of Molecular Genetics and Pathology OHT7: Office of In Vitro Diagnostics Office of Product Evaluation and Quality Center for Devices and Radiological Health
Enclosure
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Indications for Use
510(k) Number (if known) K211181
Device Name Idylla™ MSI Test
Indications for Use (Describe) For in vitro diagnostic use. For use on the Biocartis Idylla™ System only.
The Idylla™ MSI Test, for use on the Idylla™ System, uses formalin-fixed, paraffin-embedded (FFPE) tissue sections of human CRC tumor, from which nucleic acids are liberated, then analyzed using PCR amplification of seven monomorphic biomarkers (ACVR2A, BTBD7, DID01, MRE11, RYR3, SEC31A and SULF2) and subsequent melt-curve analysis. The Idylla™MSI Test reports results as either microsatellite stable (MSS), or microsatellite instability high (MSI-H) or invalid.
Idylla™ MSI Test is indicated for use by healthcare professionals for the qualitative identification of microsatellite instability (MSI) in colorectal cancer (CRC) tumors, indicative of mismatch repair deficiency, as an and in the identification of potential Lynch syndrome to help identify patients that would benefit from additional genetic testing to diagnose Lynch syndrome.
The results from the Idylla™ MSI Test should be interpreted by healthcare professionals in conjunction with other clinical findings, family history, and other laboratory data. The Idyla™ MSI Test should not be used for diagnosis of CRC. The clinical performance of this device to guide treatment decision for MSI high patients has not been established.
| Type of Use (Select one or both, as applicable) | |
|---|---|
| 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/1 description: The image shows the logo for Biocartis. The logo features a blue water droplet above the company name. The water droplet has a shadow and is surrounded by a network of lines and dots. The company name, "BIOCARTIS", is written in blue, sans-serif font.
510(k) Summary
| Applicant | Biocartis NVGeneraal De Wittelaan 11 B2800 Mechelen, Belgium |
|---|---|
| Contact Person | Elnaz JokarManager, Regulatory Affairs |
| Email: | ejokar@biocartis.com |
| Preparation Date: | February 27, 2023 |
1. Device
| Device Trade Name: | Idylla™ MSI Test |
|---|---|
| Common Name: | Idylla™ MSI Test |
| Regulatory Section: | 21 CFR §864.1866 - Lynch Syndrome test systems |
| Classification: | II |
| Product Code: | PZJ - Lynch Syndrome test system |
| Review Division: | 88 – Pathology |
| Proposed Intended Use: | For in vitro diagnostic use.For use on the Biocartis Idylla™ System only.The Idylla™ MSI Test, for use on the Idylla™ System, uses formalin-fixed, paraffin-embedded (FFPE) tissue sections of human CRC tumor, from which nucleic acids are liberated, then analyzed using PCR amplification of seven monomorphic biomarkers (ACVR2A, BTBD7, DIDO1, MRE11, RYR3, SEC31A and SULF2) and subsequent melt-curve analysis. The Idylla™ MSI Test reports results as either microsatellite stable (MSS), or microsatellite instability high (MSI-H) or invalid.Idylla™ MSI Test is indicated for use by healthcare professionals for the qualitative identification of microsatellite instability (MSI) in colorectal cancer (CRC) tumors, indicative of mismatch repair deficiency, as an aid in the identification of potential Lynch syndrome to |
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| help identify patients that would benefit fromadditional genetic testing to diagnose Lynchsyndrome. | |
|---|---|
| The results from the Idylla™ MSI Test should beinterpreted by healthcare professionals inconjunction with other clinical findings, familyhistory, and other laboratory data. The Idylla™MSI Test should not be used for diagnosis ofCRC. | |
| The clinical performance of this device to guidetreatment decision for MSI high patients hasnot been established. | |
| Special Instrument Requirements | Idylla™ System manufactured by Biocartis,NV |
| Proposed Predicate Device | OncoMate™ MSI Dx Analysis SystemK200129 |
2. Device Description
The Biocartis Idylla™ System covers the entire process from sample to result with fully integrated sample preparation followed by PCR amplification and high-resolution melting detection of the targeted sequences. The Idylla™ System consists of the Idylla™ Console connected to one or more Idylla™ Instruments (up to eight instruments). Idylla™ Cartridges, designed for specific applications, can be processed by the Idylla System using test specific software (Test Type Package, MSI TTP). The Idylla™ MSI Test procedure and data analysis are validated for FFPE tissue sections.
The Idylla™ MSI Test detects a novel panel of seven monomorphic biomarkers.
The Idylla™ MSI Test Cartridges are ready-for-use and contain the necessary reagents to perform sample preparation, PCR amplification and high-resolution detection, starting from insertion of FFPE tissue sections. The MSI TTP directs the processing of the sample within the cartridge.
The process steps in the Idylla™ MSI Test are:
- FFPE liquefaction and cell lysis: After insertion of the FFPE tissue section into the cartridge, a combination of chemical reagents, enzymes, heat, and High Frequency Ultrasound (HIFU) induces deparaffinization, disruption of the tissue and lysis of the cells. The nucleic acids are liberated for subsequent PCR amplification.
- PCR using biomarker-specific primers: All necessary PCR reagents are present in a stable formulation and are used to amplify seven biomarkers indicative for MSI status.
- Detection and analysis: Detection of these specific targets is performed using fluorescently labeled molecular beacons after PCR amplification. These beacons differentially melt from the wild type or mutated amplicons with increasing temperature. The fluorescence differences at melting temperatures are further analyzed by the MSI TTP and translated into genetic calls on biomarker level and MSI status on sample level.
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Image /page/5/Picture/1 description: The image shows the logo for Biocartis. The logo features a blue water droplet above the company name. The droplet has a shadow and is connected to a network of lines and dots. The company name, "BIOCARTIS", is written in a bold, sans-serif font.
- Reporting: At the end of the run, the result, reporting the MSI status, the number of mutated biomarkers, and an MSI score range in the analyzed sample is displayed on the console screen.
The biomarkers detected by the Idylla™ MSI Test are listed in the Table 1 below.
Table 1: Biomarkers
| Biomarkers |
|---|
| ACVR2A |
| BTBD7 |
| DIDO1 |
| MRE11 |
| RYR3 |
| SEC31A |
| SULF2 |
3. Comparison to Predicate Device
Table 2: Comparison to Predicate Device
| Item | Subject Device:Idylla™ MSI TestK211181 | Predicate Device:OncoMate™ MSI Dx AnalysisSystemK200129 |
|---|---|---|
| Similarities | ||
| Regulation | 21 CFR §864.1866 | 21 CFR §864.1866 |
| Product Code | PZJ | PZJ |
| Device Class | Class II | Class II |
| Intended Use | The Idylla™ MSI Test, for use on theIdylla™ System, uses formalin-fixed,paraffin-embedded (FFPE) tissuesections of human CRC tumor, fromwhich nucleic acids are liberated, thenanalyzed using PCR amplification ofseven monomorphic biomarkers(ACVR2A, BTBD7, DIDO1, MRE11, RYR3,SEC31A and SULF2) and subsequentmelt-curve analysis. The Idylla™ MSITest reports results as eithermicrosatellite stable (MSS), ormicrosatellite instability high (MSI-H)or invalid. | The OncoMate™ MSI Dx AnalysisSystem is a qualitative multiplexpolymerase chainreaction (PCR) test intended todetect the deletion ofmononucleotides in 5 microsatelliteloci (BAT-25, BAT-26, NR-21, NR-24and MONO-27) using matchedtumor and normal DNA obtainedfrom formalin fixed, paraffin-embedded (FFPE) colorectal tissuesections.The OncoMate™ MSI Dx AnalysisSystem is for use with the AppliedBiosystems® |
| Item | Subject Device:Idylla™ MSI TestK211181 | Predicate Device:OncoMate™ MSI Dx AnalysisSystemK200129 |
| Idylla™ MSI Test is indicated for use byhealthcare professionals for thequalitative identification ofmicrosatellite instability (MSI) incolorectal cancer (CRC) tumors,indicative of mismatch repairdeficiency, as an aid in theidentification of potential Lynchsyndrome to help identify patients thatwould benefit from additional genetictesting to diagnose Lynch syndrome.The results from the Idylla™ MSI Testshould be interpreted by healthcareprofessionals in conjunction with otherclinical findings, family history, andother laboratory data. The Idylla™ MSITest should not be used for diagnosisof CRC.The clinical performance of this deviceto guide treatment decision for MSIhigh patients has not been established. | 3500Dx Genetic Analyzer andOncoMate™ MSI Dx InterpretiveSoftware.The OncoMate™ MSI Dx AnalysisSystem is indicated in patientsdiagnosed withcolorectal cancer (CRC) to detectmicrosatellite instability (MSI) as anaid in the identification of probableLynch syndrome to help identifypatients that would benefit fromadditional genetic testing todiagnose Lynch syndrome.Results from the OncoMate™ MSI DxAnalysis System should beinterpreted byhealthcare professionals inconjunction with other clinicalfindings, family history, andother laboratory data.The clinical performance of thisdevice to guide treatment decisionfor MSI high patientshas not been established. | |
| Special Conditions for UseStatements | Rx- For prescription use.For In Vitro Diagnostic use. | Same |
| Specimen | FFPE Tumor Tissue | Same |
| Results | MSS or MSI-H | Same |
| Target Population | Patients diagnosed with CRC | Same |
| Differences | ||
| Technology | PCR based microsatellite measurementin tumor DNA | PCR based microsatellitemeasurement in normal and tumorDNA |
| Software | MSI TTP | OncoMate ™ MSI Dx InterpretiveSoftware |
| Targets | ACVR2A, BTBD7, DIDO1, MRE11, RYR3,SEC31A, and SULF2 | 5 Mononucleotide tracts BAT25,BAT26, ΜΟΝΟ27, NR21 and NR24 |
| External Controls | 2 cell line-based FFPE sectionsdesigned to represent MSS and MSI-H | DNA extracted from MSS human cellline |
| Internal Control | The melt analysis result of the sampleprocessing control in each of the PCRreactions is used to check for adequateexecution of the complete processfrom sample to result | None |
| Item | Subject Device:Idylla™ MSI TestK211181 | Predicate Device:OncoMate™ MSI Dx AnalysisSystemK200129 |
| Instrument | Idylla™ System | Applied BioSystems 3500 DxGenetic Analyzer |
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4. Analytical Performance Data
4.1 Analytical Specificity
The Idylla™ MSI Test qualitatively detects a novel panel of seven monomorphic biomarkers. Specifically, the 1-bp deletion of a certain biomarker is considered the mutant allele, while the normal length (no deletion) is considered the wild type (WT) allel. These mutants are frequently present in samples with MSI-H status.
In silico analysis of the human genome sequence did not identify for any of the oligonucleotide primers outside the MSI marker genes that could possibly result in non-specific detection. Therefore, any cross- reactivity of the MSI primers can be excluded.
Moreover, in silico analysis of the human genome sequence did not identify significant interference of known mutations with any of the oligonucleotide primers or beacons of the Idylla™ MSI Test that could possibly result in non-specific detection. Therefore, any cross-reactivity due to mutations can be excluded.
Limit of Blank (LoB): The LoB study was conducted to evaluate the non-specific amplification and crossreactivity between the mutant and wild type targets. The LoB study results demonstrate that the Idylla™ MSI Test can correctly generate an 'Invalid' MSI status call when there is no sample in the Cartridge. This study also demonstrates that there is no cross-reactivity between the MSI mutant primers and beacons with MSI wild type DNA. The Test can correctly generate the MSI status 'MSS' when a wild type sample is tested with the Idylla™ MSI Test.
4.2 Analytical Sensitivity
Analytically the Limit of Detection (LoD) is defined as the lowest average mutant/total allele ratio (of weighted combined MSI biomarkers based on prevalence) generating a positive MSI status in 95% of cases (at a 95% confidence level). The initial LoD estimation was determined using contrived samples from cell lines designed to be representative of an average heterozygous clinical sample.
For clinical samples, LoD has been defined as the proportion of cells minimally required to obtain a correct MSI call. Estimation was done using four clinical MSI-H samples, diluted to various neoplastic cell contents with MSS samples. Final LoD confirmation studies were performed using seven clinical samples (MSI-H and MSS) at approximately 33% neoplastic cell content level.
Estimation of LoD with reference samples: The LoD estimation study was performed using reference cell lines (0% and 100% allelic frequency (AF) samples), by a titration experiment using varying levels of biomarker allelic frequency (5% - 50% mimicking heterozygous clinical samples with 10- 100% neoplastic
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cell content) at two different sample input levels (high and low input) representative for clinical samples. This study result showed the estimated LoD was 30% allelic frequency. Additionally, the study results showed that the Idylla™ MSI Test generated correct MSI status calls with estimated allelic frequency LoD using contrived specimens, ranging from 15 to 30% for low input background in the individual biomarkers as demonstrated in Table 4 below.
| Allelic Frequency(% MSI-H) | Number ofReplicatesTested | LOD Estimation Results | |
|---|---|---|---|
| Low Sample InputNumber of Correct Calls (%) | High Sample InputNumber of Correct Calls (%) | ||
| 50% | 24 | 24/24 (100%) | 24/24 (100%) |
| 30% | 24 | 24/24 (100%) | 24/24 (100%) |
| 20% | 24 | 24/24 (100%) | 24/24 (100%) |
| 15% | 24 | 24/24 (100%) | 24/24 (100%) |
| 10% | 24 | 22/24 (91.7%) | 24/24 (100%) |
| 5% | 24 | 4/24 (16.7%) | 0/24 (0%) |
Table 3: Estimation of LoD Study Results
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| Sample dilution series | Individual biomarker results (Point estimate or Hit rate), 95% CI | |||||||
|---|---|---|---|---|---|---|---|---|
| (AF%) | BTBD7 | RYR3 | SEC31A | ACVR2A | DIDO1 | MRE11 | SULF2 | |
| High Input | 5% MSI-H | 4% | 0% | 0% | 0% | 0% | 0% | 0% |
| Background | (1/24) | (0/24) | (0/24) | (0/24) | (0/24) | (0/24) | (0/24) | |
| 0.74- | 0.00- | 0.00- | 0.00- | 0.00- | 0.00- | 0.00- | ||
| 20.24% | 13.80% | 13.80% | 13.80% | 13.80% | 13.80% | 13.80% | ||
| 10% MSI-H | 100% | 88% | 100% | 100% | 75% | 12% | 83% | |
| (24/24) | (21/24) | (24/24) | (24/24) | (18/24) | (3/24) | (20/24) | ||
| 86.20- | 69.00- | 86.20- | 86.20- | 55.10- | 4.34- | 64.15- | ||
| 100% | 95.66% | 100% | 100% | 88.00% | 31.00% | 93.32% | ||
| 15% MSI-H | 100% | 100% | 100% | 100% | 92% | 42% | 100% | |
| (24/24) | (24/24) | (24/24) | (24/24) | (22/24) | (10/24) | (24/24) | ||
| 86.20- | 86.20- | 86.20- | 86.20- | 74.15- | 24.47- | 86.20- | ||
| 100% | 100% | 100% | 100% | 97.68% | 61.17% | 100% | ||
| 20% MSI-H | 100% | 100% | 100% | 100% | 100% | 92% | 100% | |
| (24/24) | (24/24) | (24/24) | (24/24) | (24/24) | (22/24) | (24/24) | ||
| 86.20- | 86.20- | 86.20- | 86.20- | 86.20- | 74.15- | 86.20- | ||
| 100% | 100% | 100% | 100% | 100% | 97.68% | 100% | ||
| 30% MSI-H | 100% | 100% | 100% | 100% | 100% | 100% | 100% | |
| (24/24) | (24/24) | (24/24) | (24/24) | (24/24) | (24/24) | (24/24) | ||
| 86.20- | 86.20- | 86.20- | 86.20- | 86.20- | 86.20- | 86.20- | ||
| 100% | 100% | 100% | 100% | 100% | 100% | 100% | ||
| 50% MSI-H | 100% | 100% | 100% | 100% | 100% | 100% | 100% | |
| (24/24) | (24/24) | (24/24) | (24/24) | (24/24) | (24/24) | (24/24) | ||
| 86.20- | 86.20- | 86.20- | 86.20- | 86.20- | 86.20- | 86.20- | ||
| 100% | 100% | 100% | 100% | 100% | 100% | 100% | ||
| Low Input | 5% MSI-H | 17% | 8% | 4% | 17% | 4% | 0% | 8% |
| Background | (4/24) | (2/24) | (1/24) | (4/24) | (1/24) | (0/24) | (2/24) | |
| 6.68- | 2.32- | 0.74- | 6.68- | 0.74- | 0.00- | 2.32- | ||
| 35.85% | 25.85% | 20.24% | 35.85% | 20.24% | 13.80% | 25.85% | ||
| 10% MSI-H | 83% | 50% | 88% | 75% | 88% | 17% | 75% | |
| (20/24) | (12/24) | (21/24) | (18/24) | (21/24) | (4/24) | (18/24) | ||
| 64.15- | 31.43- | 69.00- | 55.10- | 69.00- | 6.68- | 55.10- | ||
| 93.32% | 68.57% | 95.66% | 88.00% | 95.66% | 35.85% | 88.00% | ||
| 15% MSI-H | 88% | 88% | 100% | 96% | 92% | 33% | 83% | |
| (21/24) | (21/24) | (24/24) | (23/24) | (22/24) | (8/24) | (20/24) | ||
| 69.00- | 69.00- | 86.20- | 79.76- | 74.15- | 17.97- | 64.15- | ||
| 95.66% | 95.66% | 100% | 99.26% | 97.68% | 53.29% | 93.32% | ||
| 20% MSI-H | 100% | 96% | 100% | 100% | 96% | 58% | 92% | |
| (24/24) | (23/24) | (24/24) | (24/24) | (23/24) | (14/24) | (22/24) | ||
| 86.20- | 79.76- | 86.20- | 86.20- | 79.76- | 38.83- | 74.15- | ||
| 100% | 99.26% | 100% | 100% | 99.26% | 75.53% | 97.68% | ||
| 30% MSI-H | 100% | 100% | 100% | 100% | 100% | 96% | 100% | |
| (24/24) | (24/24) | (24/24) | (24/24) | (24/24) | (23/24) | (24/24) | ||
| 86.20- | 86.20- | 86.20- | 86.20- | 86.20- | 79.76- | 86.20- | ||
| 100% | 100% | 100% | 100% | 100% | 99.26% | 100% | ||
| 50% MSI-H | 100% | 100% | 100% | 100% | 100% | 100% | 100% | |
| (24/24) | (24/24) | (24/24) | (24/24) | (24/24) | (24/24) | (24/24) | ||
| 86.20- | 86.20- | 86.20- | 86.20- | 86.20- | 86.20- | 86.20- | ||
| 100% | 100% | 100% | 100% | 100% | 100% | 100% |
Table 4: LoD Estimation Hit Rates and 95% Cl per Biomarker and per Sample in High and Low Input
Clinical LoD estimation: The clinical LoD was estimated using four FFPE samples by a titration experiment using varying levels of MSI-H neoplastic cell content (5-50%). The call rates for sample 4 were variable:
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this sample had a ddPCR measurement which was well below the validated linear range of the ddPCR method before dilutions were made. This sample was also originally characterized as a challenging sample with only 2 positive markers. Therefore, the call rate for this sample could also be indicative for the performance of challenging samples.
| % Neoplastic cell content | Correct MSI-H Call (Hit rate) | Results(correct calls) | |||
|---|---|---|---|---|---|
| Sample 1 | Sample 2 | Sample 3 | Sample 4 | ||
| 50% | 6/6 | 6/6 | 6/6 | 6/6 | 24/24 |
| 40% | 6/6 | 6/6 | 6/6 | 6/6 | 24/24 |
| 30% | 6/6 | 6/6 | 6/6 | 4/6 | 22/24 |
| 20% | 6/6 | 6/6 | 6/6 | 5/6 | 23/24 |
| 10% | 6/6 | 6/6 | 6/6 | 5/6 | 23/24 |
| 5% | 0/6 | 3/6 | 6/6 | 1/6 | 10/24 |
| Table 5: Estimation of Clinical LoD Results | |||
|---|---|---|---|
| -- | -- | -- | --------------------------------------------- |
Clinical LoD confirmation: The clinical LoD was confirmed using seven FFPE clinical samples (MSI-H, MSS and borderline samples) at approximately 33% neoplastic cell content, and the minimum required tissue level (25 mm² per 10 um section). The samples were tested across two cartridge lots in ten replicates per lot (n=20) over five days on multiple Instruments (total n=140 from all seven samples). As presented in Table 6 below, all seven clinical samples (MSI-H and MSS) including those with neoplastic cell content of approximately 33%, and the lowest sample input size of 25 mm² per 10 µm section, generated reports with 100% correct calls. This study confirms the clinical analytical sensitivity (LoD) of the Idylla™ MSI Test at around 33% neoplastic cell content in clinical samples at the lowest sample input size (25 mm² per 10 µm section). A secondary analysis of the data was performed on biomarker level as shown in Table 7 for the MSI-H samples and in Table 8 for the MSS samples.
| Table 6: Clinical Lod Confirmation Study Results (Clinical Samples Tested At Approximately 33 % Neoplastic Cell | |
|---|---|
| Content And The Lowest Sample Input) |
| Sample ID | Cartridge Lot | Total Runs | MSI Status Results) | (Idylla Concordant (Correct) Call % |
|---|---|---|---|---|
| Sample 1 (MSI-H) | Lot 1 | 40 | MSI-H | 100% (80/80) |
| Sample 2 (MSI-H) | ||||
| Sample 3 (MSI-H) | ||||
| Sample 4 (MSI-H) | Lot 2 | 40 | ||
| Sample 5 (MSS) | Lot 1 | 30 | MSS | 100% (60/60) |
| Sample 6 (MSS) | ||||
| Sample 7 (MSS) | Lot 2 | 30 |
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| SAMPLE | INDIVIDUAL BIOMARKER RESULTS (POINT ESTIMATE OR HIT RATE*) | ||||||
|---|---|---|---|---|---|---|---|
| 95% CI AT 33% NEOPLASTIC CELL CONTENT | |||||||
| BTBD7 | RYR3 | SEC31A | ACVR2A | DIDO1 | MRE11 | SULF2 | |
| Sample 1 | 100%(20/20 mut)83.89-100% | 95.00%(19/20 wt)76.39-99.11% | 100%(20/20 mut)83.89-100% | 100%(20/20 mut)83.89-100% | 70%(14/20 mut)48.1-85.45% | 95.00%(19/20 wt)76.39-99.11% | 100%(20/20 mut)83.89-100% |
| Sample 2 | 100%(20/20 mut)83.89-100% | 100%(20/20 mut)83.89-100% | 100%(20/20 mut)83.89-100% | 100%(20/20 mut)83.89-100% | 100%(20/20 mut)83.89-100% | 95.00%(19/20 mut)76.39-99.11% | 65%(13/20 mut)43.29-81.88% |
| Sample 3 | 100%(20/20 mut)83.89-100% | 100%(20/20 mut)83.89-100% | 85%(17/20 wt)63.96-94.76% | 100%(20/20 wt)83.89-100% | 100%(20/20 mut)83.89-100% | 100%(20/20 wt)83.89-100% | 100%(20/20 wt)83.89-100% |
| Sample 4 | 100%(20/20 mut)83.89-100% | 100%(20/20 wt)83.89-100% | 35%(7/20 wt)18.12-57.00% | 100%(20/20 mut)83.89-100% | 100%(20/20 mut)83.89-100% | 100%(20/20 mut)83.89-100% | 100%(20/20 mut)83.89-100% |
Table 7: LoD Confirmation Study: Point Estimate And 95% Cl Presented Per Biomarker And Per Sample For MSI-H Samples (Clinical Specimens)
*Hit rates of biomarkers n/N: mutant detected/total observations
Table 8: LoD Confirmation Study: Mutant Hit Rates And 95% Cl Presented Per Biomarker And Per Sample For MSS Samples
| SAMPLE | INDIVIDUAL BIOMARKER RESULTS (POINT ESTIMATE OR HIT RATE*)95% CI AT 33% NEOPLASTIC CELL CONTENT | ||||||
|---|---|---|---|---|---|---|---|
| BTBD7 | RYR3 | SEC31A | ACVR2A | DIDO1 | MRE11 | SULF2 | |
| Sample 5 | 100%(20/20 wt)83.89-100% | 100%(20/20 wt)83.89-100% | 100%(20/20 wt)83.89-100% | 100%(20/20 wt)83.89-100% | 100%(20/20 wt)83.89-100% | 100%(20/20 wt)83.89-100% | 100%(20/20 wt)83.89-100% |
| Sample 6 | 100%(20/20 wt)83.89-100% | 100%(20/20 wt)83.89-100% | 100%(20/20 wt)83.89-100% | 100%(20/20 wt)83.89-100% | 100%(20/20 wt)83.89-100% | 100%(20/20 wt)83.89-100% | 100%(20/20 wt)83.89-100% |
| Sample 7 | 100%(20/20 wt)83.89-100% | 100%(20/20 wt)83.89-100% | 100%(20/20 wt)83.89-100% | 100%(20/20 wt)83.89-100% | 100%(20/20 wt)83.89-100% | 100%(20/20 wt)83.89-100% | 100%(20/20 wt)83.89-100% |
*Hit rates of biomarkers n/N: mutant detected/total observations
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4.3 Reproducibility
The inter-lab reproducibility study was conducted at three laboratory sites. Seven individual clinical specimens (MSI-H and MSS FFPE colorectal (CRC) tissue sections) that satisfied the sample requirement conditions were tested at all three sites with three lots of Idylla™ MSI Test Cartridges, on the Idylla™ Platform. This study was designed to evaluate the impact of lot-to-lot, instrument/operator, inter-day and inter-lab variability on the Idylla™ MSI Test. At each site, each sample was tested by one operator on four Instruments with one replicate per Instrument per day, for three non-consecutive days using three lots of Idylla™ MSI Test Cartridges, resulting in a total of twelve replicates per sample per site. Overall, this study produced 36 results (tests) in total per sample from all three sites. All 252 valid runs from the three sites were used for data analysis. As presented in Table 9 below, all three sites generated identical and correct MSI status calls across the sites for all seven clinical samples tested. Reproducibility data showed no relevant effects of the different variation sources analyzed (inter-Instrument, inter-lot Cartridge and inter-day) in this study. A secondary analysis of the data from the multi-site reproducibility study using FFPE clinical samples was performed for every biomarker and for each of the specimens. Table 10 provides a global overview of the analysis for every sample, including the agreement for both MSI status and individual biomarkers with 95% CI.
| Clinical Sample | Number ofReplicatesTested | Reproducibility Results | |
|---|---|---|---|
| Number of Correct MSIStatus Calls | Concordance Rate (%) | ||
| Sample 1 (MSI-H) | 36 | 36/36 | 100% |
| Sample 2 (MSI-H) | 36 | 36/36 | 100% |
| Sample 3 (MSI-H) | 36 | 36/36 | 100% |
| Sample 4 (MSI-H) | 36 | 36/36 | 100% |
| Sample 5 (MSS) | 36 | 36/36 | 100% |
| Sample 6 (MSS) | 36 | 36/36 | 100% |
| Sample 7 (MSS) | 36 | 36/36 | 100% |
| Within Site | |||
| Site 1 | 84 | 84/84 | 100% |
| Site 2 | 84 | 84/84 | 100% |
| Site 3 | 84 | 84/84 | 100% |
| All Sites | 252 | 252 / 252 | 100% |
Table 9: Reproducibility Study Results
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| Sample | ReferenceMSIstatus | TissueSize(mm²) | %NeoplasticCells intissue | Agreementtoreferencestatus% PPA(MUT) andNPA (WT)(Hit rate),95% CI | BTBD7 | RYR3 | SEC31A | ACVR2A | DIDO1 | MRE11 | SULF2 | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Individual biomarker results (Point estimate (Hit rate), 95% CI) | ||||||||||||
| Sample1 | MSI-H | 75 | 30-40 | 100%(36/36) | 100%(36/36MUT) | 100%**(36/36MUT) | 100%(36/36WT) | 100%(36/36MUT) | 100%(36/36MUT) | 100%(36/36MUT) | 100%(36/36MUT) | |
| 90.36-100% | 90.36-100% | 90.36-100% | 90.36-100% | 90.36-100% | 90.36-100% | 90.36-100% | 90.36-100% | |||||
| Sample2* | MSI-H | 30 | 50-60 | 100%(36/36) | 77.78%(28/36WT) | 97.22%**(35/36MUT) | 100%(36/36WT) | 100%(36/36MUT) | 100%(36/36MUT) | 100%(36/36MUT) | 86.11%(31/36WT) | |
| 90.36-100% | 61.92-88.28% | 85.83-99.51% | 90.36-100% | 90.36-100% | 90.36-100% | 90.36-100% | 71.34-93.92% | |||||
| Sample3 | MSI-H | 160 | 30-40 | 100%(36/36) | 80.56%(29/36MUT) | 97.22%(35/36MUT) | 97.22%(35/36MUT) | 100%(36/36MUT) | 100%(36/36MUT) | 94.44%(34/36MUT) | 97.22%(35/36MUT) | |
| 90.36-100% | 64.97-90.25% | 85.83-99.51% | 85.83-99.51% | 90.36-100% | 90.36-100% | 81.86-98.46% | 85.83-99.51% | |||||
| Sample4 | MSI-H | 125 | 40 | 100%(36/36) | 66.67%(24/36MUT) | 100%(36/36MUT) | 100%(36/36MUT) | 100%(36/36MUT) | 100%(36/36MUT) | 86.11%(31/36MUT) | 97.22%(35/36WT) | |
| 90.36-100% | 50.33-79.79% | 90.36-100% | 90.36-100% | 90.36-100% | 90.36-100% | 71.34-93.92% | 85.83-99.51% | |||||
| Sample5 | MSS | 37 | 35 | 100%(36/36) | 100%(36/36WT) | 100%(36/36WT) | 100%(36/36WT) | 100%(36/36WT) | 100%(36/36WT) | 100%(36/36WT) | 100%(36/36WT) | |
| 90.36-100% | 90.36-100% | 90.36-100% | 90.36-100% | 90.36-100% | 90.36-100% | 90.36-100% | 90.36-100% | |||||
| Sample6 | MSS | 35 | 50 | 100%(36/36) | 100%(36/36WT) | 100%(36/36WT) | 100%(36/36WT) | 100%(36/36WT) | 100%(36/36WT) | 100%(36/36WT) | 100%(36/36WT) | |
| 90.36-100% | 90.36-100% | 90.36-100% | 90.36-100% | 90.36-100% | 90.36-100% | 90.36-100% | 90.36-100% | |||||
| Sample7* | MSS | 110 | 40 | 100%(36/36) | 100%(36/36WT) | 100%(36/36WT) | 100%(36/36WT) | 88.89%**(32/36WT) | 100%(36/36WT) | 100%(36/36WT) | 100%(36/36WT) | |
| 90.36-100% | 90.36-100% | 90.36-100% | 90.36-100% | 74.69 -95.59 | 90.36-100% | 90.36-100% | 90.36-100% |
Table 10: Summary of PPA (MUT) and 95% Cl for MSI Status and Individual Biomarkers in FFPE Clinical Samples
- Borderline samples per Idylla™ Test v2.0 initial screening results
** While reproducibility for this marker across 36 replicates is > 85%, the biomarker call observed is discordant to the biomarker status of the clinical sample that was obtained during sample characterization.
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4.4 Interfering Substances
Interference testing was performed using seven FFPE colorectal cancer clinical samples (MSI-H and MSS) that met the sample requirement criteria, including commonly encountered interfering substances such as hemoglobin, triglycerides, and paraffin. For hemoglobin and triglycerides, interference was tested at the highest possible concentration levels (2 mg/mL and 37 mmol/L, respectively) per CLSI guidelines Interference Testing in Clinical Chemistry, EP07-A2. To assess the paraffin interference, one additional 10 um paraffin blank section was added to the cartridge along with the sample. Each sample was tested with an interfering substance in five replicates using a single lot of Idylla™ MSI Cartridges. Additionally, the samples were tested without an interfering substance as a control. The results obtained for each interfering substance were checked for a correct MSI status call, and compared with the control results (runs without interfering substance) for the proportion (%) of concordance with control results. As presented in Table 11 below, all seven samples tested with interference substances i.e., hemoglobin, triglycerides and excess paraffin generated correct MSI status calls, showed 100% concordance with control results (with no interference substance). Therefore, hemoglobin, triglycerides, and paraffin have no impact on the Idylla™ MSI Test.
| Interference Substance | Runs*TotalWith EachInterference | Interference Test Results | |||
|---|---|---|---|---|---|
| Substance | Correct MSI Status Calls(%) | Concordance With Control Results(%) | |||
| Control (No Interferent) | રે રેત | 35 / 35 (100%) | |||
| Hemoglobin (2 Mg / ml) | રે રે | 35 / 35 (100%) | 100% | ||
| Triglycerides (37 Mm) | રે રે | 35 / 35 (100%) | 100% | ||
| Paraffin (1 X 10 µmSection) | રે રે | 35 / 35 (100%) | 100% |
Mucin (i.e., % mucinous cells in FFPE sample) impact on the Idylla™ MSI Test was evaluated using nineteen individual FFPE clinical samples with various levels of mucinous cell content (0% - 60%). These samples included both MSI-H and MSS samples that met sample requirement criteria for the test. Each mucinous sample was tested in three replicates using one lot of Idylla™ MSI Test Cartridges. The results obtained were summarized for the mucinous cell content for each sample tested and their valid MSI status call. The highest mucinous cell content (%) tested, at which level the sample still generated a valid and correct MSI status call is considered as the tested interference limit for mucinous content impact on the Idylla™ MSI Test on Idylla™ Systems. All nineteen mucinous samples results showed correct MSI calls for approximately 96.5% runs (55 of 57 runs) performed, except one sample which generated 'Invalid' results for 2 out of 3 runs performed. This study also indicated that samples with mucinous cell content up to 60% have no impact on the performance of the Idylla™ MSI Test, and can still generate correct MSI status calls.
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The impact of necrosis content >50% on the Idylla™ MSI Test was evaluated using four FFPE clinical samples with various levels of necrosis content (66-73%). The samples included both MSI-H and MSS samples. Each sample was tested in triplicate with and without necrosis content. All four samples generated a correct MSI status call for testing with and without necrosis. Therefore, necrosis content up to 73% has no impact on the Idylla™ MSI Test.
5. Clinical Performance Data
A method comparison study design was utilized to demonstrate the diagnostic accuracy of the Idylla™ MSI Test against the OncoMate™ MSI Dx Analysis System for the detection of MSI status. A comparison of the Idylla™ MSI Test results with results from a germline Next Generation Sequencing (NGS) for DNA mismatch repair (MMR) genes was performed to confirm identification of Lynch cases. The study analysis included a total of 143 samples; of which 123 were sequentially selected (sequential cohort) from two hospital biobanks and 20 (enrichment cohort) were confirmed Lynch cases obtained from the Colorectal Cancer Family Registry. Statistical analysis was performed to calculate PPA, NPA and associated Clopper-Pearson 95% confidence intervals (CI) with valid results from testing 143 samples on the Idylla™ MSI Test, OncoMate™ MSI and germline NGS.
Idylla™ MSI Test Vs OncoMate™ MSI Dx Analysis System
For MSI status, MSS and MSI-H, point estimates of agreements calculated were PPA of 96.88% (95% CJ, 83.78 - 99.92), and NPA of 99.07% (95% Cl, 94.95 - 99.98), between the Idylla™ MSI Test and the OncoMate™ MSI for all samples, i.e., sequential and enrichment cohorts combined. The concordance and percent agreement results are presented in Tables 12 and 13 below.
| Idylla™ MSITest | ONCOMATE™ MSI DX | MSI-H | MSS | Invalid | NoCall | Total |
|---|---|---|---|---|---|---|
| MSI-H | 31 | 1** | 0 | 3 | 35 | |
| MSS | 1* | 107 | 0 | 0 | 108 | |
| Invalid | 0 | 0 | 0 | 0 | 0 | |
| Total | 32 | 108 | 0 | 3 | 143 |
| Table 12: Concordance for MSI status between Idylla™ MSI Test and the OncoMate™ MSI Dx | |||
|---|---|---|---|
| Analysis System for all samples. |
*: One (1) sample that tested MSS by Idylla™ MSI Test and MSI-H by the OncoMate™ MSI Dx Analysis System is a confirmed Lynch case by NGS.
**: One (1) sample that tested MSI-H by ldylla™ MSI by the OncoMate™ MSI Dx Analysis System is a confirmed Lynch case by NGS.
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Table 13: Percent Agreements between Idylia™ MSI Test and the OncoMate™ MSI Dx System for all samples tested.
| Measure | Rate | PointEstimate(%) | 95% CI |
|---|---|---|---|
| PPA | 31/32 | 96.88 | 83.78 - 99.92 |
| NPA | 107/108 | 99.07 | 94.95 - 99.98 |
| OPA | 138/140 | 98.57 | 94.93 – 99.83 |
Concordance and agreement analysis categorized by sequential and enrichment cohorts are provided in Tables 14 to 17 below.
Table 14: Concordance for MSI status between Idylla™ MSI Test and the OncoMate™ MSI Dx Analysis System for sequential cohort.
| Idylla™ MSI Test | ONCOMATE™ MSI | ||||
|---|---|---|---|---|---|
| MSI-H | MSS | Invalid | No Call | Total | |
| MSI-H | 15 | 1 | 0 | 0 | 16 |
| MSS | 0 | 107 | 0 | 0 | 107 |
| Invalid | 0 | 0 | 0 | 0 | 0 |
| Total | 15 | 108 | 0 | 0 | 123 |
Table 15: Percent agreement for MSI status between Idylla™ MSI Test and the OncoMate™ MSI Dx Analysis System for sequential cohort.
| Measure | Rate | Point Estimate(%) | 95%CI |
|---|---|---|---|
| PPA | 15/15 | 100 | 78.2 - 100.00 |
| NPA | 107/108 | 99.07 | 94.95 - 99.98 |
| OPA | 122/123 | 99.19 | 95.55 - 99.98 |
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Image /page/17/Picture/1 description: The image shows the logo for Biocartis. The logo features a blue water droplet above a network of interconnected lines and dots. Below the graphic is the word "BIOCARTIS" in a bold, sans-serif font, also in blue.
| Idylla™ MSITest | ONCOMATE™ MSI | ||||
|---|---|---|---|---|---|
| MSI-H | MSS | Invalid | NoCall | Total | |
| MSI-H | 16 | 0 | 0 | 3 | 19 |
| MSS | 1 | 0 | 0 | 0 | 1 |
| Invalid | 0 | 0 | 0 | 0 | 0 |
| Total | 17 | 0 | 0 | 3 | 20 |
Table 16: Concordance for MSI status between Idylla™ MSI Test and the OncoMate™ MSI Dx Analysis System for enrichment cohort.
| Table 17: Percent agreement for MSI status between Idylla™ MSI Test and the OncoMate™ |
|---|
| MSI Dx Analysis System for enrichment cohort. |
| Measure | Rate | Point Estimate | 95%CI |
|---|---|---|---|
| PPA | 16/17 | 94.12 | 71.31 – 99.85 |
| NPA* | 0/0 | N/A | N/A |
| OPA | 16/17 | 94.12 | 71.31 – 99.85 |
*: All samples in the enrichment cohort are confirmed Lynch cases expected to be MSI-H.
Idylla™ MSI Test Vs Germline NGS for MMR Genes
The PPA between the Idylla™ MSI Test against germline NGS of MMR genes for all samples, i.e., sequential and enrichment cohorts combined, was 92%, and NPA was 89.81%. The NPA is less informative since Lynch syndrome negative samples by germline NGS can still exhibit microsatellite instability (MSI-H) due to sporadic somatic mutations in one or more of the MMR genes (sporadic dMMR) (Chen, W. et al. 2017 Diagn. Pathol. 12, 24). Concordance and agreement result between the Idylla™ MSI Test and germline NGS for MMR genes is presented in Tables 18 and 19 below.
Table 18: Concordance for MSI status between Idylla™ MSI Test and germline NGS for MMR genes for all samples.
| Idylla™ Msi Test | Germline NGSResults | |||
|---|---|---|---|---|
| LynchPositive | LynchNegative | Invalid | Total | |
| MSI-H | 23 | 11 | 1 | 35 |
| MSS | 2 | 97 | 9 | 108 |
| Invalid | 0 | 0 | 0 | 0 |
| Total | 25 | 108 | 10 | 143 |
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Image /page/18/Picture/1 description: The image shows the logo for BIOCARTIS. The logo features a blue water droplet above the word "BIOCARTIS" in blue font. The water droplet has a gradient of light to dark blue, and there are three circles inside the droplet. Behind the water droplet is a network of gray lines that connect to four circles.
| Measure | Rate | PointEstimate | 95% CI |
|---|---|---|---|
| PPA | 23/25 | 92.00% | 73.97 – 99.02 |
| NPA | 97/108 | 89.81% | 82.50 – 94.80 |
| OPA | 120/133 | 90.22% | 83.99 – 94.20 |
Table 19: Percent Agreements Between Idylla™ MSI Test and Germline NGS For All Samples
The concordance and agreement result between the Idylla™ MSI Test and germline NGS of MMR genes categorized by sequential cohort and enrichment cohort is presented in Tables 20 - Table 23 below. The PPA for identification of Lynch syndrome cases was 80% and 95% with sequential and enrichment cohorts, respectively.
Table 20: Concordance for MSI status between Idylla™ MSI Test and germline NGS for MMR genes for sequential cohort.
| Idylla™ MSITest | Germline NGS results | |||
|---|---|---|---|---|
| Lynchpositive | Lynchnegative | Invalid | Total | |
| MSI-H | 4 | 11 | 1 | 16 |
| MSS | 1 | 97 | 9 | 107 |
Table 21: Percent agreement for MSI status between Idylla™ MSI Test and germline NGS for MMR genes for sequential cohort.
| Measure | Rate | Point Estimate | 95% CI |
|---|---|---|---|
| PPA | 4/5 | 80% | 28.36 - 99.49 |
| NPA | 97/108 | 89.81% | 82.51 - 94.80 |
| OPA | 101/113 | 89.38% | 82.18 - 94.39 |
Table 22: Concordance for MSI status between Idylla™ MSI Test and germline NGS for MMR genes for enrichment cohort.
| Idylla™ MSI Test | Germline NGS results | |||
|---|---|---|---|---|
| Lynchnegative | Lynchnegative* | Invalid | Total | |
| MSI-H | 19 | N/A | 0 | 19 |
| MSS | 1 | N/A | 0 | 1 |
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Image /page/19/Picture/1 description: The image shows the Biocartis logo. The logo features a blue water droplet above the word "BIOCARTIS". There is a network of lines and dots behind the water droplet.
| Invalid | 0 | N/A | 0 | 0 |
|---|---|---|---|---|
| Total | 20 | N/A | 0 | 20 |
*: All samples from enrichment cohort are confirmed Lynch cases.
Table 23: Percent agreement for MSI status between Idylla™ MSI Test and germline NGS for MMR genes for enrichment cohort.
| Measure | Rate | Point Estimate | 95% CI |
|---|---|---|---|
| PPA | 19/20 | 95% | 75.13 – 99.87 |
| NPA | N/A | N/A | N/A |
| OPA | N/A | N/A | N/A |
6. Conclusion
The Idylla™ MSI Test demonstrates substantially equivalent performance to the predicate OncoMate™ MSI Dx Analysis System.
§ 864.1866 Lynch syndrome test systems.
(a)
Identification. Lynch syndrome test systems are in vitro diagnostic tests for use with tumor tissue to identify previously diagnosed cancer patients at risk for having Lynch syndrome.(b)
Classification. Class II (special controls). The special controls for this device are:(1) Premarket notification submissions must include the following information, as appropriate:
(i) A detailed description of all test components, including all provided reagents, and required but not provided, ancillary reagents.
(ii) A detailed description of instrumentation and equipment, including illustrations or photographs of non-standard equipment or manuals.
(iii) Detailed documentation of the device software, including, but not limited to, standalone software applications and hardware-based devices that incorporate software.
(iv) A detailed description of quality controls including appropriate positive and negative controls that are recommended or provided.
(v) Detailed specifications for sample collection, processing, and storage.
(vi) A detailed description of methodology and assay procedure.
(vii) A description of the assay cut-off (
i.e., the medical decision point between positive and negative results) or other relevant criteria that distinguishes positive and negative results, or ordinal classes of marker expression, including the rationale for the chosen cut-off or other relevant criteria and results supporting validation of the cut-off.(viii) Detailed specification of the criteria for test result interpretation and reporting.
(ix) Detailed information demonstrating the performance characteristics of the device, including:
(A) Data from an appropriate study demonstrating clinical accuracy using well-characterized clinical specimens representative of the intended use population (
i.e., concordance to Deoxyribonucleic Acid (DNA) sequencing results of the Lynch syndrome associated genes or method comparison to the predicate device using samples with known alterations in genes representative of Lynch syndrome). Pre-specified acceptance criteria must be provided and followed.(B) Appropriate device reproducibility data investigating all sources of variance (
e.g., for distributed tests, data generated using a minimum of three sites, of which at least two sites must be external sites). Each site must perform testing over a minimum of 5 nonconsecutive days evaluating a sample panel that spans the claimed measuring range, and includes the clinical threshold. Pre-specified acceptance criteria must be provided and followed.(C) Data demonstrating reader reproducibility, both within-reader and between-reader, assessed by three readers over 3 nonconsecutive days at each site, including a 2 week washout period between reads, as appropriate.
(D) Device precision data using clinical samples spanning the measuring range and controls to evaluate the within-lot, between-lot, within-run, between run, and total variation.
(E) Analytical specificity studies including as appropriate, western blots, peptide inhibition, testing in normal tissues and neoplastic tissues, interference by endogenous and exogenous substances, and cross-reactivity and cross contamination testing.
(F) Device analytical sensitivity data generated by testing an adequate number of samples from individuals with the target condition such that prevalence of the biomarker in the target population is established.
(G) Device stability data, including real-time stability and in-use stability, and stability evaluating various storage times, temperatures, and freeze-thaw conditions, as appropriate.
(H) The staining performance criteria assessed must include overall staining acceptability, background staining acceptability, and morphology acceptability, as appropriate.
(I) Appropriate training requirements for users, including interpretation manual, as applicable.
(J) Identification of risk mitigation elements used by the device, including a description of all additional procedures, methods, and practices incorporated into the instructions for use that mitigate risks associated with testing.
(2) The device's § 809.10(b) of this chapter compliant labeling must include a detailed description of the protocol, including the information described in paragraphs (b)(1)(i) through (viii) of this section, as appropriate, and a detailed description of the performance studies performed and the summary of the results, including those that relate to paragraph (b)(1)(ix) of this section, as appropriate.