Predicate Devices

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The device description and performance studies focus on standard molecular biology techniques (PCR, melt-curve analysis) and statistical analysis of results, with no mention of AI or ML algorithms.

Therapeutic

No
This device is for in vitro diagnostic use, intended to identify microsatellite instability in colorectal cancer tumors, which aids in identifying potential Lynch syndrome and guiding further genetic testing. It is explicitly stated that "The clinical performance of this device to guide treatment decision for MSI high patients has not been established" and "The Idylla™ MSI Test should not be used for diagnosis of CRC," indicating it is not a therapeutic device.

Diagnostic

Yes

The first line of the "Intended Use / Indications for Use" section explicitly states, "For in vitro diagnostic use," and the device is intended for the qualitative identification of microsatellite instability (MSI) in colorectal cancer (CRC) tumors, which indicates its role in disease characterization and aiding in identifying potential Lynch syndrome.

SaMD (Software as a Medical Device)

No

The device description clearly states it includes hardware components (Idylla™ Console, Idylla™ Instruments, Idylla™ Cartridges) and performs physical processes like sample preparation, PCR amplification, and detection. While software (MSI TTP) is involved in directing the process and analyzing data, it is integral to a larger hardware system.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

Here's why:

Explicit Statement: The "Intended Use / Indications for Use" section explicitly states: "For in vitro diagnostic use."
Nature of the Test: The device performs analysis on biological samples (FFPE tissue sections) in vitro (outside of the living body) to provide diagnostic information about the patient's condition (microsatellite instability in CRC tumors).
Intended Purpose: The test is intended to aid healthcare professionals in the qualitative identification of MSI, which is indicative of mismatch repair deficiency and can help identify potential Lynch syndrome. This is a diagnostic purpose.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for 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, 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.

Product codes (comma separated list FDA assigned to the subject device)

PZJ

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.
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.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

colorectal cancer (CRC) tumors, colorectal tissue sections

Indicated Patient Age Range

Not Found

Intended User / Care Setting

healthcare professionals

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

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.

Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)

Analytical Performance Data

Analytical Specificity

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.

Analytical Sensitivity

Limit of Detection (LoD):
Initial LoD estimation using contrived samples: The estimated LoD was 30% allelic frequency. 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.
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%).
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). 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. The 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).

Reproducibility

Inter-lab Reproducibility Study: Conducted at three laboratory sites. Seven individual clinical specimens (MSI-H and MSS FFPE colorectal (CRC) tissue sections) were tested at all three sites with three lots of Idylla™ MSI Test Cartridges, on the Idylla™ Platform. 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. All three sites generated identical and correct MSI status calls across the sites for all seven clinical samples tested (100% concordance). Reproducibility data showed no relevant effects of the different variation sources analyzed (inter-Instrument, inter-lot Cartridge and inter-day).

Interfering Substances

Interference Testing: Performed using seven FFPE colorectal cancer clinical samples (MSI-H and MSS) with commonly encountered interfering substances such as hemoglobin (2 mg/mL), triglycerides (37 mmol/L), and paraffin (one additional 10 um paraffin blank section). Each sample was tested with an interfering substance in five replicates. All seven samples tested with interference substances 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.
Mucin Interference: Evaluated using nineteen individual FFPE clinical samples with various levels of mucinous cell content (0% - 60%). Each mucinous sample was tested in three replicates. 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 indicated that samples with mucinous cell content up to 60% have no impact on the performance.
Necrosis Interference: Evaluated using four FFPE clinical samples with various levels of necrosis content (66-73%). 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.

Clinical Performance Data

Study type: Method comparison study and comparison to germline NGS.
Sample size: 143 samples (123 sequential cohort from two hospital biobanks; 20 enrichment cohort were confirmed Lynch cases from the Colorectal Cancer Family Registry).
Key Results:
Idylla™ MSI Test Vs OncoMate™ MSI Dx Analysis System (All samples combined):
PPA of 96.88% (95% CI, 83.78 - 99.92)
NPA of 99.07% (95% CI, 94.95 - 99.98)
OPA of 98.57% (94.93 – 99.83)

Idylla™ MSI Test Vs OncoMate™ MSI Dx Analysis System (Sequential cohort):
PPA of 100% (95% CI, 78.2 - 100.00)
NPA of 99.07% (95% CI, 94.95 - 99.98)
OPA of 99.19% (95% CI, 95.55 - 99.98)

Idylla™ MSI Test Vs OncoMate™ MSI Dx Analysis System (Enrichment cohort):
PPA of 94.12% (95% CI, 71.31 – 99.85)

Idylla™ MSI Test Vs Germline NGS for MMR Genes (All samples combined):
PPA of 92% (95% CI, 73.97 – 99.02)
NPA of 89.81% (95% CI, 82.50 – 94.80)
OPA of 90.22% (95% CI, 83.99 – 94.20)

Idylla™ MSI Test Vs Germline NGS for MMR Genes (Sequential cohort):
PPA of 80% (95% CI, 28.36 - 99.49)
NPA of 89.81% (95% CI, 82.51 - 94.80)
OPA of 89.38% (95% CI, 82.18 - 94.39)

Idylla™ MSI Test Vs Germline NGS for MMR Genes (Enrichment cohort):
PPA of 95% (95% CI, 75.13 – 99.87)

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

See "Summary of Performance Studies" above for PPA, NPA, and OPA.

Predicate Device(s): If the device was cleared using the 510(k) pathway, identify the Predicate Device(s) K/DEN number used to claim substantial equivalence and list them here in a comma separated list exactly as they appear in the text. List the primary predicate first in the list.

OncoMate™ MSI Dx Analysis System Gender K200129

Reference Device(s): Identify the Reference Device(s) K/DEN number and list them here in a comma separated list exactly as they appear in the text.

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information for the subject device only (e.g. presence / absence, what scope was granted / cleared under the PCCP, any restrictions, etc).

Not Found

Regulation Number and Section

§ 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.

Summary

0

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

1

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

2

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 NV
Generaal De Wittelaan 11 B
2800 Mechelen, Belgium |
|-------------------|----------------------------------------------------------------------|
| Contact Person | Elnaz Jokar
Manager, 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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Image /page/4/Picture/1 description: The image shows the logo for BIOCARTIS. The logo features a blue water droplet above the word "BIOCARTIS" in blue, bold letters. Behind the water droplet is a network of gray lines connecting small blue circles.

| | 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 Idylla™
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. |
| Special Instrument Requirements | Idylla™ System manufactured by Biocartis,
NV |
| Proposed Predicate Device | OncoMate™ MSI Dx Analysis System
K200129 |

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.

5

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 Test
K211181 | Predicate Device:
OncoMate™ MSI Dx Analysis
System
K200129 |
|------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| 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 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. | The OncoMate™ MSI Dx Analysis
System is a qualitative multiplex
polymerase chain
reaction (PCR) test intended to
detect the deletion of
mononucleotides in 5 microsatellite
loci (BAT-25, BAT-26, NR-21, NR-24
and MONO-27) using matched
tumor and normal DNA obtained
from formalin fixed, paraffin-
embedded (FFPE) colorectal tissue
sections.
The OncoMate™ MSI Dx Analysis
System is for use with the Applied
Biosystems® |
| Item | Subject Device:
Idylla™ MSI Test
K211181 | Predicate Device:
OncoMate™ MSI Dx Analysis
System
K200129 |
| | 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 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 Idylla™ 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. | 3500Dx Genetic Analyzer and
OncoMate™ MSI Dx Interpretive
Software.
The OncoMate™ MSI Dx Analysis
System is indicated in patients
diagnosed with
colorectal cancer (CRC) to detect
microsatellite instability (MSI) as an
aid in the identification of probable
Lynch syndrome to help identify
patients that would benefit from
additional genetic testing to
diagnose Lynch syndrome.
Results from the OncoMate™ MSI Dx
Analysis System should be
interpreted by
healthcare professionals in
conjunction with other clinical
findings, family history, and
other laboratory data.
The clinical performance of this
device to guide treatment decision
for MSI high patients
has not been established. |
| Special Conditions for Use
Statements | 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 measurement
in tumor DNA | PCR based microsatellite
measurement in normal and tumor
DNA |
| Software | MSI TTP | OncoMate ™ MSI Dx Interpretive
Software |
| 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 sections
designed to represent MSS and MSI-H | DNA extracted from MSS human cell
line |
| Internal Control | The melt analysis result of the sample
processing control in each of the PCR
reactions is used to check for adequate
execution of the complete process
from sample to result | None |
| Item | Subject Device:
Idylla™ MSI Test
K211181 | Predicate Device:
OncoMate™ MSI Dx Analysis
System
K200129 |
| Instrument | Idylla™ System | Applied BioSystems 3500 Dx
Genetic 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 of
Replicates
Tested | LOD Estimation Results | |
|--------------------------------|-----------------------------------|-------------------------------------------------|--------------------------------------------------|
| | | Low Sample Input
Number of Correct Calls (%) | High Sample Input
Number 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

9

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 of
Replicates
Tested | Reproducibility Results | |
|------------------|-----------------------------------|---------------------------------------|----------------------|
| | | Number of Correct MSI
Status 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 | Reference
MSI
status | Tissue
Size
(mm²) | %
Neoplastic
Cells in
tissue | Agreement
to
reference
status
% PPA
(MUT) and
NPA (WT)
(Hit rate),
95% CI | BTBD7 | RYR3 | SEC31A | ACVR2A | DIDO1 | MRE11 | SULF2 | |
|--------------|----------------------------|-------------------------|---------------------------------------|---------------------------------------------------------------------------------------------------|--------------------------|--------------------------------|------------------------------------------------------------------|-------------------------------|------------------------|--------------------------|--------------------------|--|
| | | | | | | | Individual biomarker results (Point estimate (Hit rate), 95% CI) | | | | | |
| Sample
1 | MSI-H | 75 | 30-40 | 100%
(36/36) | 100%
(36/36
MUT) | 100%
**
(36/36
MUT) | 100%
(36/36
WT) | 100%
(36/36
MUT) | 100%
(36/36
MUT) | 100%
(36/36
MUT) | 100%
(36/36
MUT) | |
| | | | | 90.36-
100% | 90.36-
100% | 90.36-
100% | 90.36-
100% | 90.36-
100% | 90.36-
100% | 90.36-
100% | 90.36-
100% | |
| Sample
2* | MSI-H | 30 | 50-60 | 100%
(36/36) | 77.78%
(28/36
WT) | 97.22%
**
(35/36
MUT) | 100%
(36/36
WT) | 100%
(36/36
MUT) | 100%
(36/36
MUT) | 100%
(36/36
MUT) | 86.11%
(31/36
WT) | |
| | | | | 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% | |
| Sample
3 | MSI-H | 160 | 30-40 | 100%
(36/36) | 80.56%
(29/36
MUT) | 97.22%
(35/36
MUT) | 97.22%
(35/36
MUT) | 100%
(36/36
MUT) | 100%
(36/36
MUT) | 94.44%
(34/36
MUT) | 97.22%
(35/36
MUT) | |
| | | | | 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% | |
| Sample
4 | MSI-H | 125 | 40 | 100%
(36/36) | 66.67%
(24/36
MUT) | 100%
(36/36
MUT) | 100%
(36/36
MUT) | 100%
(36/36
MUT) | 100%
(36/36
MUT) | 86.11%
(31/36
MUT) | 97.22%
(35/36
WT) | |
| | | | | 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% | |
| Sample
5 | MSS | 37 | 35 | 100%
(36/36) | 100%
(36/36
WT) | 100%
(36/36
WT) | 100%
(36/36
WT) | 100%
(36/36
WT) | 100%
(36/36
WT) | 100%
(36/36
WT) | 100%
(36/36
WT) | |
| | | | | 90.36-
100% | 90.36-
100% | 90.36-
100% | 90.36-
100% | 90.36-
100% | 90.36-
100% | 90.36-
100% | 90.36-
100% | |
| Sample
6 | MSS | 35 | 50 | 100%
(36/36) | 100%
(36/36
WT) | 100%
(36/36
WT) | 100%
(36/36
WT) | 100%
(36/36
WT) | 100%
(36/36
WT) | 100%
(36/36
WT) | 100%
(36/36
WT) | |
| | | | | 90.36-
100% | 90.36-
100% | 90.36-
100% | 90.36-
100% | 90.36-
100% | 90.36-
100% | 90.36-
100% | 90.36-
100% | |
| Sample
7* | MSS | 110 | 40 | 100%
(36/36) | 100%
(36/36
WT) | 100%
(36/36
WT) | 100%
(36/36
WT) | 88.89%
**
(32/36
WT) | 100%
(36/36
WT) | 100%
(36/36
WT) | 100%
(36/36
WT) | |
| | | | | 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*
Total
With Each
Interference | 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 µm
Section) | 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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Image /page/15/Picture/1 description: The image shows the logo for BIOCARTIS. The logo features a blue water droplet above the word "BIOCARTIS." The water droplet has three circles inside of it. There are also several lines connecting circles above the word "BIOCARTIS."

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™ MSI
Test | ONCOMATE™ MSI DX | MSI-H | MSS | Invalid | No
Call | 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 | Point
Estimate
(%) | 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™ MSI

Test	ONCOMATE™ MSI
	MSI-
H	MSS	Invalid	No
Call	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 NGS
Results | | | |
|------------------|-------------------------|-------------------|---------|-------|
| | Lynch
Positive | Lynch
Negative | 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 | Point
Estimate | 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™ MSI

Test	Germline NGS results
	Lynch
positive	Lynch
negative	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
	Lynch
negative	Lynch
negative*	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.