Predicate Devices

Reference Devices

No Reference Device(s) K/DEN numbers were found in the text. The section for "Reference Device(s)" explicitly states "Not Found".

AI/ML (Artificial Intelligence / Machine Learning)

No
The device description details a standard PCR-based workflow with data analysis based on comparing allele sizes using defined thresholds (≥2.75 bp). There is no mention of AI, ML, or any learning algorithms in the device description, intended use, or performance studies. The "Interpretive Software" appears to apply a rule-based classification based on the measured allele size differences.

Therapeutic

No
The device is a diagnostic test intended to detect microsatellite instability as an aid in identifying probable Lynch syndrome, not to treat a condition.

Diagnostic

Yes

The "Intended Use / Indications for Use" section explicitly states that the system is "intended to detect the deletion of mononucleotides" and "is indicated in patients diagnosed with colorectal cancer (CRC) to detect microsatellite instability (MSI) as an aid in the identification of probable Lynch syndrome." These statements clearly describe a diagnostic purpose.

SaMD (Software as a Medical Device)

No

The device is a system that includes a qualitative multiplex PCR test kit, DNA extraction kits and instruments, a genetic analyzer, and interpretive software. While software is a component, it is not the sole component of the medical device.

IVD (In Vitro Diagnostic)

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

Here's why:

Intended Use: The intended use explicitly states it's a "qualitative multiplex polymerase chain reaction (PCR) test intended to detect the deletion of mononucleotides in 5 microsatellite loci... using matched tumor and normal DNA obtained from formalin fixed, paraffin-embedded (FFPE) colorectal tissue sections." This describes a test performed in vitro (outside the body) on biological samples (DNA from tissue).
Purpose: The test is used "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." This is a diagnostic purpose, providing information about a patient's condition or predisposition.
Device Description: The description details a laboratory workflow involving DNA extraction, PCR amplification, fragment analysis, and software interpretation, all characteristic of an in vitro diagnostic test.
Performance Studies: The document includes various performance studies (Method Comparison, Reproducibility, Limit of Detection, etc.) which are standard for demonstrating the analytical and clinical performance of an IVD.
Predicate Device: The mention of a predicate device (VENTANA MMR IHC panel) is common for IVDs undergoing regulatory review, indicating a comparison to an already cleared IVD.

All these elements strongly indicate that the OncoMate™ MSI Dx Analysis System is an In Vitro Diagnostic device.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

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, 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® 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.

Product codes

PZJ

Device Description

The OncoMate™ MSI Dx Analysis System assay encompasses a complete workflow for MSI determination, from DNA extraction to data analysis. DNA is extracted from FFPE colorectal tissue samples (normal and tumor from the same patient) using the Maxwell® CSC DNA FFPE Kit and Maxwell® CSC Instrument. Double-stranded DNA (dsDNA) is then quantified using a fluorescence-based dsDNA quantification system of the user's choice. Next, amplification products are generated through multiplex PCR amplification of DNA microsatellite markers using the OncoMate™M MSI Dx Analysis System amplification kit. The PCR products are then mixed with Hi-Di™ Formamide and Size Standard 500 and heat-denatured. The resulting single-stranded DNA fragments are separated by size and detected via fluorescence using an Applied Biosystems® 3500Dx Genetic Analyzer. Following capillary electrophoresis, allele sizes from the CRC tumor DNA and the normal DNA are calculated and compared for each of the microsatellite markers using OncoMate™ MSI Dx Interpretive Software. If the length of two or more of the five mononucleotide-repeat marker alleles is changed by >=2.75 base pairs (bp), the tumor is classified as MSI-H; if the allele length is changed for only one marker, or if the difference in allele lengths at the five markers is =3 base pair resolution and demonstrated sufficient measurement precision to detect single-base-pair differences.

Limit of Blank Study:

Study Type: Analytical study to confirm blank samples do not produce positive MSI-H results.
Sample Size: Four known MSS samples tested across 3 amplification kit lots, 2 operators, and 60 replicates each (total 1,440 test results).
Key Results: All 1,440/1,440 tests (100%) resulted in MSS final interpretive results. 99.99% (7,199/7,200) of mononucleotide locus allele calls were "Stable". One instance of one locus (NR-21) being called unstable did not affect the final MSS result. The system provides highly reproducible MSS results, unaffected by lot or operator.

Limit of Detection (LOD) Study:

Study Type: Analytical sensitivity determination.
Sample Size: DNA from six MSI-H tumor and matched normal samples, plus a titration series of 2800M Control DNA. Tested at DNA inputs of 0.2, 0.5, 1.0, 2.0, 2.5 ng (20 replicates each), and tumor contents of 5%, 10%, 15%, and 20% on one sample, and 20% and 30% on three samples (CRC-066, CRC-079, CRC-084).
Key Results: The LOD was established at 30% tumor content based on concordance across all loci using the recommended 1ng DNA input and samples adjusted by blending with matched normal tissue DNA.
- For 1ng DNA input and 20% tumor content:
  - 2800M (MSS): 100% concordant for all 5 loci.
  - CRC-066 (MSI-H), CRC-079 (MSI-H), CRC-213 (MSI-H): 100% concordant for all 5 loci.
  - CRC-076 (MSI-H): 42.5% concordant for NR-24 (17/40), 100% for others.
  - CRC-081 (MSI-H): 90% concordant for NR-21 (36/40), 97.5% for MONO-27 (39/40), 100% for others.
  - CRC-084 (MSI-H): 15% concordant for BAT-25 (6/40), 5% for NR-24 (2/40), 100% for others.
- For 20% tumor content:
  - CRC-066 (0.5ng): NR21 100%, BAT-26 100%, BAT-25 95%, NR-24 90%, MONO-27 85%.
  - CRC-066 (1.0ng, 2.0ng): 100% for all loci.
  - CRC-079 (0.5ng, 1.0ng, 2.0ng): 100% for all loci except MONO-27 at 0.5ng (85%).
  - CRC-084 (0.5ng): BAT-25 10%, NR-24 0%.
  - CRC-084 (1.0ng): BAT-25 5%, NR-24 0%.
  - CRC-084 (2.0ng): BAT-25 0%, NR-24 0%.
- For 30% tumor content:
  - CRC-066 (0.5ng, 1.0ng, 2.0ng): 100% for all loci.
  - CRC-079 (0.5ng, 1.0ng, 2.0ng): 100% for all loci.
  - CRC-084 (0.5ng): NR-24 40%.
  - CRC-084 (1.0ng): BAT-25 95%, NR-24 0%.
  - CRC-084 (2.0ng): NR-24 0%.

Analytical Specificity Study:

Study Type: Evaluation of primer target specificity and thermal cycler compatibility.
Sample Size: 2800M Control DNA replicates amplified in duplicate on three different thermal cycler models (Applied Biosystems Veriti® 96-Well Thermal Cycler, Eppendorf MasterCycler® Nexus SX1 Thermal Cycler, and BioRad C1000 Touch™ Thermal Cycler).
Key Results: 100% agreement (36/36) between expected and observed MSS calls. No interference observed with different thermal cyclers. Primer Blast analysis showed target specificity of primer pairs.

Interfering Substances Study:

Study Type: Assessment of potential influence of common interfering substances.
Sample Size: Lysates from 4 tumor and matched normal samples spiked with hemoglobin, triglycerides, or mucin. 4 tumor and matched normal samples incubated with proteinase K for varying times. 12 tumor samples with 0-75% necrotic tissue. Aliquots of extracted DNA spiked with ethanol or guanidine hydrochloride.
Key Results: No statistically significant impact on assay performance by chaotropic salts (50uM guanidine hydrochloride), ethanol (5%), necrotic tissue (0-75%), hemoglobin (2mg/ml), triglycerides (37mM), mucin (1mg/ml), or proteinase K digestion time (20, 30, or 40 minutes). All samples returned the expected result.

Cross Contamination Study:

Study Type: Evaluation of sample-to-sample cross-contamination.
Sample Size: Extracted DNA from MSI-H and MSS samples (tumor and matched normal), and reagent blanks, arranged in a grid design across 96-well plates over 10 days.
Key Results: 100% concordance between interpretive result and expected results for 470 samples. No interference with data interpretation was observed, indicating the system is not susceptible to sample-to-sample cross-contamination.

Reproducibility Study:

Study Type: Precision and reproducibility evaluation across multiple sites, operators, runs, days, replicates, and assay kit lots.
Sample Size: Seven CRC samples (4 MSI-H, 3 MSS), a negative control and a positive control. Tested by 2 operators at each of 3 sites (1 internal, 2 external) on 3 instruments, using 3 reagent lots over 2 runs/day on 3 nonconsecutive days.
Key Results: Positive Percent Agreements (PPAs) for MSI-H ranged from 89.6% to 97.9%, and Negative Percent Agreements (NPAs) for MSS ranged from 97.2% to 100%. Overall reproducibility PPA (95% CI) was 95.5% (92.4-97.6%) and NPA (95% CI) was 99.5% (97.4-100%). Study demonstrated reproducibility across sites, operators, days, lots, and runs.

Shelf Life Stability Studies:

Study Type: Determination of stability for the OncoMate™ MSI Dx Analysis System and OncoMate™ 5C Matrix Standard.
Key Results:
- Open Vial Reagent Stability (OncoMate™ MSI Analysis System amplification kit): 4 months stability at +2 to 10°C, supporting a 3-month claim.
- Open Vial Reagent Stability (OncoMate™ 5C Matrix Standard): 4 months stability at +2 to 10°C, supporting a 3-month claim. Diluted Matrix stability was 7 days, supporting a 6-day claim.
- Real Time Stability at -30°C to -10°C (OncoMate™ MSI Analysis System amplification kit): 24 months stability in frozen storage.
- Real Time Stability at -30°C to -10°C followed by 4 months at +2 to 10°C (OncoMate™ MSI Analysis System amplification kit): 20 months frozen storage followed by 4 months refrigerated storage, for a total of 24 months.
- Real Time Stability at -30°C to -10°C (OncoMate™ 5C Matrix Standard): 24 months stability for frozen storage.
- Real Time Stability at -30°C to -10°C followed by 4 months at +2°C to 10°C (OncoMate™ 5C Matrix Standard): 20 months frozen storage followed by 4 months refrigerated storage, for a total of 24 months.

Method Comparison Study (OncoMate™ MSI Dx Analysis System vs. VENTANA MMR IHC Panel):

Study Type: Evaluation of accuracy and usability in identifying microsatellite instability.
Sample Size: 154 samples.
Key Results:
- All Samples:
  - PPA: 97.8% (44/45), 95% CI: 88.4-99.6
  - NPA: 97.2% (106/109), 95% CI: 92.2-99.1
  - OPA: 97.4% (150/154), 95% CI: 93.5-99.0
- Sequential Cohort (130 samples):
  - PPA: 95.8% (23/24), 95% CI: 79.8-99.3
  - NPA: 100.0% (106/106), 95% CI: 96.5-100.0
  - OPA: 99.2% (129/130), 95% CI: 95.8-99.9
- Enrichment Cohort (24 samples):
  - PPA: 100.0% (21/21), 95% CI: 84.5-100.0
  - NPA: 0.0% (0/3), 95% CI: 0.0-56.1
  - OPA: 87.5% (21/24), 95% CI: 69.0-95.7
The OncoMate™ MSI Dx Analysis System effectively identified tumors with MMR deficiency and showed strong agreement with the VENTANA MMR IHC panel.

Method Comparison Study (OncoMate™ MSI Dx Analysis System vs. NGS Mismatch Repair Gene Mutations Results):

Study Type: Confirmation of identification of Lynch syndrome patients.
Sample Size: 154 samples (18 confirmed Lynch syndrome cases).
Key Results:
- All Samples:
  - PPA: 94.4% (17/18), 95% CI: 74.2-99.0
  - NPA: 77.9% (106/136), 95% CI: 70.3-84.1
  - OPA: 79.9% (123/154), 95% CI: 72.8-85.4
- Enrichment Cohort (24 samples):
  - PPA: 100.0% (16/16), 95% CI: 80.6-100
  - NPA: 0.0% (0/8), 95% CI: 0.0-32.4
  - OPA: 66.7% (16/24), 95% CI: 46.7-82.0
- Sequential Cohort (130 samples):
  - PPA: 50.0% (1/2), 95% CI: 9.5-90.5
  - NPA: 82.8% (106/128), 95% CI: 75.3-88.4
  - OPA: 82.3% (107/130), 95% CI: 74.8-87.9
The OncoMate™ MSI Dx Analysis System effectively identified tumors with confirmed germline MMR mutations indicative of Lynch syndrome.

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

Method Comparison: OncoMate™ MSI Dx Analysis System vs. IHC Results

PPA: 97.8% (All Samples), 95.8% (Sequential Cohort), 100.0% (Enrichment Cohort)
NPA: 97.2% (All Samples), 100.0% (Sequential Cohort), 0.0% (Enrichment Cohort)
OPA: 97.4% (All Samples), 99.2% (Sequential Cohort), 87.5% (Enrichment Cohort)

Method Comparison: OncoMate™ MSI Dx Analysis System vs. NGS Mismatch Repair Gene Mutations Results

PPA: 94.4% (All Samples), 100.0% (Enrichment Cohort), 50.0% (Sequential Cohort)
NPA: 77.9% (All Samples), 0.0% (Enrichment Cohort), 82.8% (Sequential Cohort)
OPA: 79.9% (All Samples), 66.7% (Enrichment Cohort), 82.3% (Sequential Cohort)

Predicate Device(s):

DEN170030

Reference Device(s):

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information

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

Image /page/0/Picture/0 description: The image shows the logo of the U.S. Food and Drug Administration (FDA). The logo consists of two parts: the Department of Health & Human Services logo on the left and the FDA logo on the right. The FDA logo is a blue square with the letters "FDA" in white, followed by the words "U.S. FOOD & DRUG ADMINISTRATION" in blue.

Promega Corporation Pamela Swatkowski Regulatory Consultant 2800 Woods Hollow Rd Madison, Wisconsin 53711

July 26, 2021

Re: K200129

Trade/Device Name: OncoMate MSI Dx Analysis System Regulation Number: 21 CFR 864.1866 Regulation Name: Lynch Syndrome Test Systems Regulatory Class: Class II Product Code: PZJ Dated: November 30, 2020 Received: December 1, 2020

Dear Pamela Swatkowski:

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 medical devices and radiation-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, PhD Chief Molecular Genetics Branch Division of Molecular Genetics and Pathology OHT7: Office of In Vitro Diagnostics and Radiological Health Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

2

Indications for Use

510(k) Number (if known) K200129

Device Name OncoMate™ MSI Dx Analysis System

Indications for Use (Describe)

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, 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® 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.

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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3

510(k) SUMMARY

1 COMPANY AND CONTACT INFORMATION

Company Name:	Promega Corporation
Address:	2800 Woods Hollow Rd
Madison, WI 53711
Telephone:	608.274.4330
Contact Person:	Ron Wheeler
Sr. Director, Quality and Regulatory Affairs
Date of Summary:	20 July 2021
DEVICE IDENTIFICATION
Trade (Proprietary) Name:	OncoMateTM MSI Dx Analysis System
Common (Usual) Name:	OncoMateTM MSI Dx Analysis System
Classification Name:	Lynch Syndrome Test System
Product Code:	PZJ
Regulation Number:	21 CFR 864.1866
Regulatory Classification:	Class II
Panel:	88 - Pathology

3 PREDICATE DEVICE

2

VENTANA MMR IHC Panel

4

4 DEVICE DESCRIPTION

OncoMate™ MSI Dx Analysis System Overview 4.1

The OncoMate™ MSI Dx Analysis System assay encompasses a complete workflow for MSI determination, from DNA extraction to data analysis (Figure 1). DNA is extracted from FFPE colorectal tissue samples (normal and tumor from the same patient) using the Maxwell® CSC DNA FFPE Kit and Maxwell® CSC Instrument. Double-stranded DNA (dsDNA) is then quantified using a fluorescence-based dsDNA quantification system of the user's choice. Next, amplification products are generated through multiplex PCR amplification of DNA microsatellite markers using the OncoMate™M MSI Dx Analysis System amplification kit. The PCR products are then mixed with Hi-Di™ Formamide and Size Standard 500 and heat-denatured. The resulting single-stranded DNA fragments are separated by size and detected via fluorescence using an Applied Biosystems® 3500Dx Genetic Analyzer. Following capillary electrophoresis, allele sizes from the CRC tumor DNA and the normal DNA are calculated and compared for each of the microsatellite markers using OncoMate™ MSI Dx Interpretive Software. If the length of two or more of the five mononucleotide-repeat marker alleles is changed by ≥2.75 base pairs (bp), the tumor is classified as MSI-H; if the allele length is changed for only one marker, or if the difference in allele lengths at the five markers is 3 base pairs. Two sets of seven synthetic DNA fragments ("resolution markers") were analyzed during this study. These fragments consist of dye-labeled amplicons of known size that are separated by 1bp within each set, with the two sets designed to bracket the upper (Large) and lower (Small) ends of the amplicon size range of the MSI markers (83-168bp).

13

L

The resolution markers were subjected to capillary electrophoresis using an Applied Biosystems® 3500 Dx Genetic Analyzer and analyzed with OncoMate™ MSI Dx Interpretive Software. Resolution markers were analyzed either mixed only with the Size Standard 500 or mixed separately with two MSS tumor samples and the Size Standard 500. While the OncoMate™ MSI Dx Interpretive Software was not designed to identify the resolution markers, it was critical to demonstrate the System software's ability to determine fragment size and precision. The sizing precision of individual resolution fragments was characterized (Table 4), and size differences between all fragments separated by 3bp were calculated and averaged. Observed mean differences were compared with predicted values.

There were no invalid results or samples requiring reinjection or reamplification testing for this study.

Resolution fragments were sized precisely, with standard deviations ranging from 0.07-0.13bp. Mean absolute differences calculated for fragments separated by 3bp were also precise, ranging from 3.06-3.40bp and 3.05-3.35bp for observed and predicted values, respectively. Accordingly, the study met the objective of ≥3 base pair resolution and, in fact, demonstrated measurement precision of individual resolution markers (standard deviations ≤0.13 base pairs) that was sufficient to detect single-base-pair differences in size.

| Resolution
Marker | N | Minimum | Maximum | Median | Mean | Standard
deviation | 95%-CI | |
|----------------------|----|---------|---------|--------|--------|-----------------------|----------------|--------|
| | | | | | | Lower
Limit | Upper
Limit | |
| Large | 20 | 180.60 | 180.95 | 180.77 | 180.75 | 0.10 | 180.71 | 180.80 |
| Large_2 | 20 | 181.67 | 182.02 | 181.89 | 181.83 | 0.10 | 181.78 | 181.88 |
| Large_3 | 20 | 182.75 | 182.97 | 182.85 | 182.86 | 0.08 | 182.83 | 182.90 |
| Large_4 | 20 | 183.70 | 184.04 | 183.88 | 183.85 | 0.09 | 183.80 | 183.89 |
| Large 5 | 20 | 184.66 | 184.99 | 184.83 | 184.80 | 0.09 | 184.76 | 184.85 |
| Large_6 | 20 | 185.73 | 186.06 | 185.90 | 185.88 | 0.10 | 185.83 | 185.93 |
| Large_7 | 20 | 186.80 | 187.01 | 186.91 | 186.91 | 0.07 | 186.88 | 186.94 |
| Small | 20 | 84.29 | 84.71 | 84.62 | 84.57 | 0.13 | 84.51 | 84.63 |
| Small 2 | 20 | 85.52 | 85.95 | 85.83 | 85.79 | 0.13 | 85.72 | 85.85 |
| Small 3 | 20 | 86.66 | 87.14 | 86.95 | 86.92 | 0.13 | 86.86 | 86.98 |
| Small 4 | 20 | 87.71 | 88.15 | 87.99 | 87.97 | 0.13 | 87.91 | 88.03 |
| Small 5 | 20 | 88.73 | 89.18 | 89.04 | 89.02 | 0.12 | 88.96 | 89.07 |
| Small 6 | 20 | 89.96 | 90.40 | 90.25 | 90.21 | 0.13 | 90.15 | 90.27 |
| Small 7 | 20 | 91.06 | 91.55 | 91.39 | 91.34 | 0.13 | 91.28 | 91.41 |

Table 4: Descriptive Statistics for Resolution Marker Base Pair Size

7.3 Limit of Blank

A Limit of Blank study was conducted to confirm a blank (an MSS sample in this study) did not produce positive MSI-H results. The study tested four known MSS samples across three amplification kit lots, two operators and 60 replicates of each MSS samplea total of 1,440 test results.

005 510k Summary Original 510(k) Application

14

There were 2 samples for which the initial test result was invalid. These samples were reinjected per protocol, and after reinjection were resolved. No reamplification testing was required for this study.

All tests (1,440/1,440; 100%) resulted in MSS final interpretive results. The 1,440 test results represent 7,200 mononucleotide locus allele calls. For the mononucleotide loci, 99.99% (7.199/7.200) of the marker stability calls were "Stable". There was a single instance of one locus, NR-21, being called unstable. A single unstable locus results in an MSS final interpretive result, and the one unstable locus did not affect the final test result. In conclusion, the OncoMate-114 MSI Dx Analysis System provides MSS results that are highly reproducible and were not affected by lot or operator.

7.4 Limit of Detection

The analytical sensitivity of the OncoMate™ MSI Dx Analysis System was determined using extracted DNA isolated from six MSI-H tumor and matched normal samples, as well as a titration series of the 2800M Control DNA. The 2800M Control DNA samples were treated as MSS samples for analysis using the OncoMate™ MSI Dx Interpretive Software. To create 20% tumor content (the minimum tumor content required), the tumor samples were mixed with the matched normal samples as needed. A subsequent study investigated higher tumor content. The MSI-H samples and the MSS samples were tested at 0.2, 0.5, 1.0, 2.0 and 2.5ng DNA per amplification reaction with 20 replicates for each sample and DNA amount (five MSI-H cases × 20 replicates). To evaluate the influence of turnor content on limit of detection (LOD), one tumor sample was combined with the matched normal sample to simulate a tumor content of 5%, 10%, 15% and 20%.

Each sample in the study was tested on at least 1 instrument with two OncoMate MSI Dx Analysis system lots at 6 different concentrations by two operators (2 replicates/operator/ sample) and over 5 days for a total of 20 replicates per sample to identify the LOD. Because there were no failures based upon the amount of DNA tested, additional testing was necessary to identify the LOD. New sample dilutions were prepared to test a 0.1ng per reaction DNA input using samples with a 20% tumor content, as well as a 1ng per reaction DNA input using a sample containing 2.5% tumor.

A summary of the interpretive results for the MSI-H and MSS cases are summarized in Table 5 for the DNA input study, and for MSI-H cases in the tumor content study in Table 6. Table 7 displays a summary of reference result vs. interpretive result and locus status by sample ID. The results for all samples tested at 1ng DNA Input and 20% Tumor Content are shown. Table 8 displays a summary of reference result vs. interpretive result and locus status by sample ID. The results for all samples tested at 0.5, 1.0, and 2.0ng DNA input and 20 and 30% tumor content are shown in Table 8.

The LOD for the OncoMate™ MSI Dx Analysis System was determined across two dimensions: 1) the total amount of input DNA used for the assay and 2) the fraction of tumor DNA present in the sample.

The LOD for the OncoMate™ MSI Dx Analysis System was established at 30% tumor content based on concordance across all loci when using the recommended 1ng DNA 005 510k Summary Original 510(k) Application Confidential

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input and samples where tumor content was adjusted by blending with DNA extracted from matched normal tissue.

| Reagent Lot | Lot by Final
DNA Input Result (MSI-H – Diluted
Sample Set) | Result (MSS – Diluted Sample
Set) | | |
|-------------|---- | | | Lot 1 | 0.1 | | 0.2 | | 0.50 | | 1 | | 2 | | 2.50 | Lot 2 | 0.1 | | 0.2 | | 0.50 | | 1 | | 2 | | 2.50 | All | 0.1 | | 0.2 | | 0.50 | | 1 | | 2 | | 2.50 (ng) | OncoMate™ Interpretive
| | OncoMate™ Interpretive
----------------------------|------------------------------------------------------------------|---------|-------|----------------------------------------------------------------|---------|-------|
| MSI-H | No Call | Total | MSS | No Call | Total |
| 20 | 80 | 100 | 0 | 20 | 20 |
| 99 | 11 | 100 | 20 | 0 | 20 |
| 100 | 0 | 100 | 20 | 0 | 20 |
| 100 | 0 | 100 | 20 | 0 | 20 |
| 100 | 0 | 100 | 20 | 0 | 20 |
| 100 | 0 | 100 | 20 | 0 | 20 |
| 24 | 76 | 100 | 0 | 20 | 20 |
| 100 | 0 | 100 | 20 | 0 | 20 |
| 100 | 0 | 100 | 20 | 0 | 20 |
| 100 | 0 | 100 | 20 | 0 | 20 |
| 100 | 0 | 100 | 20 | 0 | 20 |
| 100 | 0 | 100 | 20 | 0 | 20 |
| 44 | 156 | 200 | 0 | 40 | 40 |
| 199 | 1 | 200 | 40 | 0 | 40 |
| 200 | 0 | 200 | 40 | 0 | 40 |
| 200 | 0 | 200 | 40 | 0 | 40 |
| 200 | 0 | 200 | 40 | 0 | 40 |
| 200 | 0 | 200 | 40 | 0 | 40 |

Table 5: Interpretive Result Frequency for the DNA Concentration Study, by
Reagent Lot

1 One No Call due to low allele peak height detected.

16

Lot by Tumor content (%)		OncoMate™ Interpretive Result
		MSI-H	MSS	Total
Lot 1	2.5	0	20	20
	5	20	0	20
	10	20	0	20
	15	20	0	20
	20	20	0	20
Lot 2	2.5	0	20	20
	5	20	0	20
	10	20	0	20
	15	20	0	20
	20	20	0	20
All	2.5	0	40	40
	5	40	0	40
	10	40	0	40
	15	40	0	40
	20	40	0	40

Table 6: Interpretive Result Frequency for the Tumor Concentration Study, by Reagent Lot

Table 7: Summary of Reference Result vs. Interpretive Result and Locus Status with 95% Wilson-Score CI by Sample (1ng DNA Input and 20% Tumor Content)

| Sample
(MSI-Status) | ng
DNA
input | 20% Tumor content | | | | | |
|------------------------|-----------------------|-----------------------------------------------------------------|--------------------------------------------------------------------|--------------------------------------------------------------------|--------------------------------------------------------------------|-------------------------------------------------------------------|------------------------------------------------------------------|
| | | NR21(n/N)
% Concordant
to reference
result (95%
CI) | BAT-26 (n/N)
% Concordant
to reference
result (95%
CI) | BAT-25 (n/N)
% Concordant
to reference
result (95%
CI) | NR-24 (n/N)
% Concordant
to reference
result (95%
CI) | Mono-27 (n/N)
% Concordant to
reference result
(95% CI) | |
| 2800M
(MSS) | 1.0 | 40/40; 100%
(91.2-100) | 40/40; 100%
(91.2-100) | 40/40; 100%
(91.2-100) | 40/40; 100%
(91.2-100) | 40/40; 100%
(91.2-100) | |
| CRC-066
(MSI-H) | 1.0 | 40/40; 100%
(91.2-100) | 40/40; 100%
(91.2-100) | 40/40; 100%
(91.2-100) | 40/40; 100%
(91.2-100) | 40/40; 100%
(91.2-100) | |
| CRC-076
(MSI-H) | 1.0 | 40/40; 100%
(91.2-100) | 40/40; 100%
(91.2-100) | 40/40; 100%
(91.2-100) | 17/40; 42.5%
(28.5-57.8) | 40/40; 100%
(91.2-100) | |
| CRC-079
(MSI-H) | 1.0 | 40/40; 100%
(91.2-100) | 40/40; 100%
(91.2-100) | 40/40; 100%
(91.2-100) | 40/40; 100%
(91.2-100) | 40/40; 100%
(91.2-100) | |
| CRC-081
(MSI-H) | 1.0 | 36/40; 90%
(77.0-96.0) | 40/40; 100%
(91.2-100) | 40/40; 100%
(91.2-100) | 40/40; 100%
(91.2-100) | 39/40; 97.5
(87.1-99.6) | |
| CRC-084
(MSI-H) | 1.0 | 40/40; 100%
(91.2-100) | 40/40; 100%
(91.2-100) | 6/40; 15%
(7.1--29.1) | 2/40; 5%
(1.4-16.5) | 40/40; 100%
(91.2-100) | |
| CRC-213
(MSI-H) | 1.0 | 40/40; 100%
(91.2-100) | 40/40; 100%
(91.2-100) | 40/40; 100%
(91.2-100) | 40/40; 100%
(91.2-100) | 40/40; 100%
(91.2-100) | |
| Sample
(All MSI-H) | %
Tumor
content | ng
DNA
input | NR21 (n/N) %
Concordant to
reference
result (95%
CI) | BAT-26 (n/N)
% Concordant
to reference
result (95%
CI) | BAT-25 (n/N)
% Concordant
to reference
result (95%
CI) | NR-24 (n/N)
% Concordant
to reference
result (95%
CI) | MONO-27 (n/N)
% Concordant to
reference result
(95% CI) |
| CRC-066 | 20 | 0.5 | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 19/20; 95%
(76.4-99.1) | 18/20; 90%
(69.9-99.2) | 17/20; 85%
(64.0-94.8) |
| | 20 | 1.0 | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) |
| | 20 | 2.0 | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) |
| CRC-066 | 30 | 0.5 | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) |
| | 30 | 1.0 | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) |
| | 30 | 2.0 | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) |
| CRC-079 | 20 | 0.5 | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 17/20; 85%
(64.0-94.8) |
| | 20 | 1.0 | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) |
| | 20 | 2.0 | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) |
| CRC-079 | 30 | 0.5 | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) |
| | 30 | 1.0 | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) |
| | 30 | 2.0 | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) |
| CRC-084 | 20 | 0.5 | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 2/20; 10%
(2.8-30.1) | 0/20; 0%
(0-16.1) | 20/20; 100%
(83.9-100) |
| | 20 | 1.0 | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 1/20; 5%
(0.9--23.6) | 0/20; 0%
(0-16.1) | 20/20; 100%
(83.9-100) |
| | 20 | 2.0 | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 0/20; 0%
(0-16.1) | 0/20; 0%
(0-16.1) | 20/20; 100%
(83.9-100) |
| CRC-084 | 30 | 0.5 | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 8/20; 40%
(21.9-61.3) | 20/20; 100%
(83.9-100) |
| | 30 | 1.0 | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 19/20; 95%
(76.4-99.1) | 0/20; 0%
(0-16.1) | 20/20; 100%
(83.9-100) |
| | 30 | 2.0 | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 20/20; 100%
(83.9-100) | 0/20; 0%
(0-16.1) | 20/20; 100%
(83.9-100) |

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Table 8: Summary of Reference Result vs. Interpretive Result and Locus Status with 95% Wilson-Score CI by Sample (Supplemental Study, All DNA Inputs, 20 and 30% Tumor Content)

7.5 Analytical Specificity

Primer pairs for the seven OncoMate™ MSI Dx Analysis System markers were checked for target specificity using the publicly available BLASTn and Primer BLAST search tools on the US National Center for Biotechnology Information website

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(https://ncbi.nlm.nih.gov/tools/primer-blast/. accessed 01/14/2020: https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE TYPE=BlastSearch &LINK LOC=blasthome, accessed 07/24/2021; Ye et al., 2012). The primers share 100% identity with their intended targets, and BLAST results predicted specific target amplification.

The OncoMate™ MSI Dx Analysis System primers were assessed for off-target homology with ≥80% identity. Seven of the 14 primers exhibited off-target matches with >80% identity. However, no off-target homology was identified that would interfere with assay interpretation.

In summary, the labeled primers are specific for the template of interest because of 3' terminal mismatches and/or depressed annealing temperatures with off-target template. Also, by design, less-specific primers are unlabeled. Accordingly, non-specific product generated with the unlabeled primers would not be detected by the analytical system and would not interfere with the assay. In addition, the assay only considers fluorescent products within the range of 60 through 300bp. Therefore, any non-specific product observed outside of this range would not affect the assay results, and possible effects of non-specific amplification with labeled primers would be limited to the dye channel of the specific label. Finally, there were no reproducible, unexpected amplification artifacts observed during product development or during the analytical or clinical studies. These theoretical and empirical data support the Primer BLAST analysis showing target specificity of the primer pairs.

An analytical specificity study evaluated primer specificity of the OncoMate™ MSI Dx Analysis System and demonstrated compatibility of the system with different thermal cycler models.

The 2800M Control DNA was used, and replicates were treated as MSS samples for analysis using the OncoMate™ MSI Dx Interpretive Software. Samples were amplified in duplicate with the OncoMate™ MSI Dx Analysis System amplification kit using 1ng, 2ng or 4ng of DNA on each of three different thermal cycler models. The thermal cyclers (Applied Biosystems Veriti® 96-Well Thermal Cycler, Eppendorf MasterCycler® Nexus SX1 Thermal Cycler and BioRad C1000 Touch™ Thermal Cycler) all fall within the following required performance specifications:

Maximum Block Ramp Rate: 3.9℃/second to 5℃/second Temperature Accuracy: ±0.25°C (at ≥90°C) Temperature Uniformity: 90°C) Heated lid capable of reaching 103-105℃

There were no invalid results or samples requiring reinjection or reamplification testing for this study.

There was 100% agreement (36/36) between the expected and observed MSS call for all samples. There were no artifacts observed that interfered with the system's ability to provide the expected interpretive result when using different thermal cvclers.

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7.6 Interfering Substances

A study was performed to establish the potential influence of interfering substances on the performance of the OncoMate™ MSI Dx Analysis System, specifically chaotropic salts, alcohol, proteinase K treatment time, necrotic tissue, hemoglobin, triglycerides and mucin. DNA was extracted from sample curls (0.1-2mm3 tissue). DNA extraction was performed for each sample at each condition tested using the Maxwell® CSC Instrument and Maxwell® CSC DNA FFPE Kit.

In the first series of experiments, lysates from four tumor and matched normal samples were spiked with hemoglobin (2mg/ml final concentration), triglycerides (37mM final concentration) or mucin (1mg/ml final concentration) prior to DNA extraction. Following analysis with the OncoMate™ MSI Dx Analysis System, all samples yielded the expected result.

Four tumor and matched normal samples (two replicates per sample for a total of 16 extractions) were incubated at 56°C in the presence of proteinase K for 20 minutes, 30 minutes (recommended condition) or 40 minutes prior to purification. All samples yielded the expected result.

Twelve tumor samples with necrotic tissue ranging from 0-75% and matched normal samples were tested. All samples yielded the expected result.

To evaluate the impact of potential carry-over alcohol or guanidine salts from the DNA purification process, aliquots of the extracted DNA from tumor and matched normal samples with varying amounts of tissue necrosis (0-75%) were spiked with ethanol (5% final concentration), guanidine hydrochloride (50μM final concentration) or water prior to amplification and analysis. All samples yielded the expected result.

There were 13 Invalid results initially obtained across the Interfering Substances testing. All 13 samples were reinjected resulting in 5 samples being resolved. The remaining 8 samples were resolved after reamplification.

In conclusion, the OncoMate™ MSI Dx Analysis System showed no statistically significant impact on assay performance by the interfering substances, specifically chaotropic salts (50uM guanidine hydrochloride), ethanol (5%), necrotic tissue (0-75%), hemoglobin (2mg/ml), triglycerides (37mM), and mucin (1mg/ml) or proteinase K digestion time [20, 30 (standard) or 40 minutes] tested in this study (Table 9).

20

	Interpretive Result				Total
	MSI-H	MSS
	N	%	N	%	N	%
Sample Treatment
20min at 56°C	4	50.0	4	50.0	8	100
30min at 56°C	4	50.0	4	50.0	8	100
40min at 56°C	4	50.0	4	50.0	8	100
Ethanol spike	8	50.0	8	50.0	16	100
Guanidine spike	8	50.0	8	50.0	16	100
Hemoglobin	4	50.0	4	50.0	8	100
Mucin	4	50.0	4	50.0	8	100
Necrosis	14	58.3	10	41.7	24	100
Triglycerides	4	50.0	4	50.0	8	100
Water spike	8	50.0	8	50.0	16	100
Total	62	51.7	58	48.3	120	100

Table 9: OncoMate™ MSI Dx Analysis System Sample Treatment by Interpretative Result

1 All samples returned the expected result (MSS or MSI-H) in interfering substances experiments.

7.7 Cross Contamination

The sample-to-sample cross-contamination in the OncoMate™ MSI Dx Analysis System was evaluated using extracted DNA from an MSI-H sample and an MSS sample, including both the tumor and its matched normal samples and reagent blanks were interspersed in a grid design across 96-well plates. In this plate layout, amplification, capillary electrophoresis and analysis were performed each day for a total of 10 days. Expected results included MSI-H, MSS and No Call test results. The No Call test result was expected for the reagent blanks. The results were not averaged, and any observed carryover was reported.

There was a total of 3 Invalid results initially obtained in the study. After reinjection all 3 sample results were resolved. No reamplification was performed for this study.

There was 100% concordance between the interpretative result and the expected results for the 470 samples analyzed, and no interference with data interpretation was observed. The study concluded the OncoMate™ MSI Dx Analysis System was not susceptible to sample-to-sample cross-contamination.

7.8 Reproducibility

Precision and reproducibility for the OncoMate™ MSI Dx Analysis System was evaluated across multiple sites, operators, runs, days, replicates and assay kit lots. Data were assessed for between-site, between-operator, between- run, between-day, within-run and between-lot repeatability and precision.

21

The test panel consisted of seven CRC samples (four MSI-H and three MSS cases), a negative amplification control (Water, Amplification Grade) and a positive amplification control (2800M Control DNA). Two of the MSI-H samples were adjusted to a 20% tumor burden by mixing DNA extracted from the tumor sample with matched normal DNA. The samples were blinded and distributed to the operators at each of the test sites. The panel of samples was tested by two operators located at each of three sites on three instruments (one at each site). Two external sites and one internal site served as the three test sites. Three reagent lots were used for two runs per day, on three nonconsecutive days.

The Positive Percent Agreements (PPAs) for MSI-H and Negative Percent Agreements (NPAs) for MSS interpretative results versus expected results (Table 10) were reproducible for site. operator. day, lot and run. The PPAs for site, operator, day, lot and run ranged from 89.6 – 97.9% and the NPAs ranged from 97.2 - 100%, demonstrating reproducibility for each factor tested. The overall reproducibility PPA (95% Cl) and NPA (95% CI) were 95.5% (92.4 - 97.6%) and 99.5%% (97.4-100%), respectively.

Table 11 provides the absolute and relative frequency of interpretive results by sample ID.

A summary of PPA with 95% Wilson-Score CI for interpretive results and locus status vs reference result is shown in Table 12.

A total of 13 results were initially Invalid during testing. All 13 samples were reinjected resulting in 8 samples that resolved and 5 samples that did not resolve. Upon reamplification 3 of the samples were resolved. A total of 2 samples remained Invalid after reamplification and are included in the agreement analysis below.

In conclusion, this study demonstrated that the OncoMate™ MSI Dx Analysis System and reagents are reproducible between and across sites, operators, days, lots, and runs.

| Factor | Item | PPA
% (#/n) | 95% CI | NPA
% (#/n) | 95% CI |
|------------|------|----------------|-------------|----------------|-------------|
| Site 1 | 1 | 96.9 (93/96) | 91.1 - 99.4 | 98.6 (71/72) | 92.5 - 100 |
| Site 2 | 2 | 92.7 (89/96) | 85.6 - 97.0 | 100 (72/72) | 95 - 100 |
| Site 3 | 3 | 96.9 (93/96) | 91.1 - 99.4 | 100 (72/72) | 95 - 100 |
| Operator 1 | 1 | 97.9 (47/48) | 88.9 - 100 | 100 (36/36) | 90.3 - 100 |
| Operator 2 | 2 | 95.8 (46/48) | 85.8 - 99.5 | 97.2 (35/36) | 85.5 - 99.9 |
| Operator 3 | 3 | 89.6 (43/48) | 77.3 - 96.5 | 100 (36/36) | 90.3 - 100 |
| Operator 4 | 4 | 95.8 (46/48) | 85.8 - 99.5 | 100 (36/36) | 90.3 - 100 |
| Operator 5 | 5 | 97.9 (47/48) | 88.9 - 100 | 100 (36/36) | 90.3 - 100 |
| Operator 6 | 6 | 95.8 (46/48) | 85.8 – 99.5 | 100 (36/36) | 90.3 - 100 |
| Day 1 | 1 | 93.8 (90/96) | 86.9 - 97.7 | 98.6 (71/72) | 92.5 - 100 |
| Day 2 | 2 | 95.8 (92/96) | 89.7 - 98.8 | 100 (72/72) | 95 - 100 |
| Day 3 | 3 | 96.9 (93/96) | 91.1 - 99.4 | 100 (72/72) | 95 - 100 |

Table 10: Summarv of PPA and NPA for Interpretative Result versus Reference Result

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| Factor | Item | PPA
% (#/n) | 95% CI | NPA
% (#/n) | 95% CI |
|---------|------|----------------|-------------|----------------|------------|
| | | | | | |
| Lot 1 * | 1 | 95.8 (92/96) | 89.7 – 98.8 | 100 (72/72) | 95 - 100 |
| Lot 2 * | 2 | 94.8 (91/96) | 88.3 - 98.3 | 98.6 (71/72) | 92.5 - 100 |
| Lot 3 * | 3 | 95.8 (92/96) | 89.7 - 98.8 | 100 (72/72) | 95 - 100 |
| Run A * | A | 94.4 (136/144) | 89.4 - 97.6 | 100 (108/108) | 96.6 - 100 |
| Run B * | B | 96.5 (139/144) | 92.1 - 98.9 | 99.1 (107/108) | 95.0 - 100 |
| Total | | 95.5 (275/288) | 92.4 - 97.6 | 99.5 (215/216) | 97.4 - 100 |

*Not all lots tested in a run

	Table 11: Absolute and Relative Frequencies for Interpretative Results by Sample

		Reference Result MSI-H			MSS			Total
Interpretative Result	Sample ID	N	%	N	%	Invalid	N		%	N	%	Invalid	N
	AS REP 01	59	81.9	12	16.7	1	1.4	0	0	0	0	0	0	72
	AS REP 02	72	100	0	0	0	0	0	0	0	0	0	0	72
	AS REP 03	72	100	0	0	0	0	0	0	0	0	0	0	72
	AS REP 04	72	100	0	0	0	0	0	0	0	0	0	0	72
	AS REP 05	0	0	0	0	0	0	0	0	71	98.6	1	1.4	72
	AS REP 06	0	0	0	0	0	0	0	0	72	100	0	0	72
	AS REP 07	0	0	0	0	0	0	0	0	72	100	0	0	72
	Total	275	95.5	12	4.2	1	0.3	0	0	215	99.5	1	0.5	504

Table 12: Summary of PPA and 95% Wilson-Score CI for Interpretative Result and
Locus Status

| Sample | Reference
MSI
status | Agreement to
reference
status (n/N)
% PPA (95%
CI) | NR21
(n/N)
% Agreement
(95% CI) | BAT-26
% Agreement
(95% CI) | BAT-25
% Agreement
(95% CI) | NR-24
% Agreement
(95% CI) | Mono-27
% Agreement
(95% CI) |
|-----------|----------------------------|----------------------------------------------------------------|------------------------------------------|-----------------------------------|-----------------------------------|----------------------------------|------------------------------------|
| AS_REP_01 | MSI-H | 59/71; 83.1%
(72.7-90.1) | 71/71; 100%
(94.9-100) | 71/71; 100%
(94.9-100) | 27/71; 38.0%
(27.6-49.7) | 71/71; 100%
(94.9-100) | 53/71; 74.6%
(63.4-83.3) |
| AS_REP_02 | MSI-H | 72/72; 100%
(94.9-100) | 72/72; 100%
(94.9-100) | 72/72; 100%
(94.9-100) | 72/72; 100%
(94.9-100) | 72/72; 100%
(94.9-100) | 72/72; 100%
(94.9-100) |
| AS_REP_03 | MSI-H | 72/72; 100%
(94.9-100) | 72/72; 100%
(94.9-100) | 72/72; 100%
(94.9-100) | 72/72; 100%
(94.9-100) | 72/72; 100%
(94.9-100) | 72/72; 100%
(94.9-100) |
| AS_REP_04 | MSI-H | 72/72; 100%
(94.9-100) | 72/72; 100%
(94.9-100) | 72/72; 100%
(94.9-100) | 72/72; 100%
(94.9-100) | 72/72; 100%
(94.9-100) | 72/72; 100%
(94.9-100) |
| AS_REP_05 | MSS | 71/71; 100%
(94.9-100) | 71/71; 100%
(94.9-100) | 71/71; 100%
(94.9-100) | 71/71; 100%
(94.9-100) | 71/71; 100%
(94.9-100) | 71/71; 100%
(94.9-100) |
| AS_REP_06 | MSS | 72/72; 100%
(94.9-100) | 72/72; 100%
(94.9-100) | 72/72; 100%
(94.9-100) | 72/72; 100%
(94.9-100) | 71/71; 100%
(94.9-100) | 72/72; 100%
(94.9-100) |
| AS_REP_07 | MSS | 72/72; 100%
(94.9-100) | 72/72; 100%
(94.9-100) | 72/72; 100%
(94.9-100) | 72/72; 100%
(94.9-100) | 72/72; 100%
(94.9-100) | 72/72; 100%
(94.9-100) |

005_510k Summary
Original 510(k) Application

23

7.9 Shelf Life

Studies were conducted to determine the stability of the Promega OncoMate™ MSI Dx Analysis System and Promega OncoMate™ 5C Matrix Standard.

Prior to execution of the real time or open vial studies, lots of the reagent kits were subjected to freeze-thaw cycles by cycling between the recommended storage temperature of -10° to -30°C and ambient temperature until thawed. The OncoMate™ MSI Dx Analysis System amplification kit reagents were subjected to five freeze-thaw cycles and OncoMate™ 5C Matrix Standard was subjected to one freeze-thaw cycle.

Following freeze-thaw, the reagent lots were then transferred into shipping containers at -10° to -30°C on dry ice and shipped overnight by aircraft to a remote site. The samples were returned to Promega and stored in the shipping containers for a total of 5 days with sufficient dry ice to maintain -10° to -30°C. The reagents were then transferred to a -10° to -30℃ freezer until used for stability testing.

7.9.1 Open Vial Studies

Open Vial Reagent Stability (OncoMate™ MSI Analysis System amplification kit)

The purpose of this study was to determine the open vial shelf life stability of the OncoMate™ MSI Dx Analysis System amplification kit reagents at +2 to 10° C in real time.

DNA was extracted from four MSI-High and four MSS CRC tumor samples with matched normal samples, quantitated, adjusted to 0.5ng/ul, and 10ul aliquots were provided to the study operator. The study operator did not know the MSI status of the samples. Samples were tested by one operator and instrument using three reagent lots, two of the lots were subjected to freeze-thaw and shipment. Tests were conducted at time zero, followed by each lot being tested at four additional time points of 1, 2, 3, and 4 months. Two repeats were performed for each time point.

Across all time points a total of 17 results were initially called Invalid during testing. All 17 samples were reinjected, and 16 samples were resolved. The single remaining Invalid result was resolved upon reamplification.

The open vial shelf life stability of the OncoMate™ MSI Dx Analysis System amplification kit reagents was determined to be four (4) months, supporting the intended claim of 3 months stability at +2 to 10°C.

Open Vial Reagent Stability (OncoMate™ 5C Matrix Standard)

The purpose of this study was to determine the open vial shelf life stability of the OncoMate™ 5C Matrix Standard at +2 to 10° C in real time.

24

One OncoMate™ 5C Matrix Standard reagent lot, subjected to freeze-thaw and shipping, was tested at time zero, followed by testing at four additional time points of 1, 2, 3, and 4 months. Two repeats were performed for each time point.

The open vial short-term stability of diluted OncoMate™ 5C Matrix Standard was performed at time zero and 7 days using three lots of reagents. Two of the lots were subjected to freeze/thaw and shipping. The open vial short-term stability study was performed 7 days after the Matrix Standard was diluted to support a 6 day stability claim for the diluted Matrix Standard.

The OncoMate™ 5C Matrix Standard was used to perform spectral calibration on the Applied Biosystems 3500 Dx Genetic Analyzer as described in the Technical Manual (TM542). The result of the spectral calibration is a multicomponent matrix, which is applied during sample detection to compensate for spectral overlap among the dyes and separate the raw fluorescent signals into individual dye signals. The Quality Value (Q value) for each capillary was measured by the instrument software. A Q score of ≥0.95 is indicative of a successful spectral calibration.

The open vial shelf life stability of the OncoMate™ 5C Matrix Standard was determined to be four (4) months, supporting the intended claim of 3-month stability at +2 to 10°C.

The open vial stability of diluted Matrix was determined to be seven (7) days, supporting the intended claim of six (6) days at +2 to 10°C. No difference was observed based on lot, regardless of whether the lot was subjected to freeze-thaw and shipping.

7.9.2 Real Time Studies

Real Time Stability at -30°C to -10°C (OncoMate™ MSI Analysis System amplification kit)

The purpose of this study was to determine the real time stability of the OncoMate™ MSI Dx Analysis System amplification kit reagents stored at -30°C to -10°C in real time. The purpose of these experiments was to confirm the reagents are stable after storage at -30°C to -10°C.

DNA was extracted from four MSI-High and four MSS CRC tumor samples with matched normal samples, quantitated, adjusted to 0.5ng/ul and provided to the study operator. The study operator did not know the MSI status of the samples were tested by one operator and instrument using three reagent lots in corrugate packaging and three reagents lots in paperboard packaging. Two lots in each package type were subjected to freeze-thaw and shipping, of the OncoMate™ MSI Dx Analysis System at time zero, followed by each lot being thawed and tested at 6, 13, 19, and 25 months. Two repeats were performed for each time point.

Across all time points a total of 7 results were called Invalid during testing. All 7 samples were reinjected, and 6 samples were resolved. The remaining sample was resolved upon reamplification.

005 510k Summary Original 510(k) Application

25

The Real-Time Long-Term Stability of the OncoMate™ MSI Dx System amplification kit reagents was determined to be 24 months at frozen storage in both types of packaging based on the data collected to date. No difference was observed based on OncoMate™ MSI Dx Analysis System amplification kit lot.

Real Time Stabilitv at -30°C to -10°C followed bv 4 months at +2 to 10°C (OncoMate™ MSI Analysis System amplification kit)

DNA was extracted from four MSI-High and four MSS CRC tumor samples with matched normal samples, quantitated, adjusted to 0.5ng/ul and provided to the study operator. The study operator did not know the MSI status of the samples were tested by one operator and instrument using three reagent lots in corrugate packaging and three reagents lots in paperboard packaging. Two lots in each package type were subjected to freeze-thaw and shipping of the OncoMate™ MSI Dx Analysis System amplification kit at time zero and after 4 months storage at -30°C to -10°C. Another set of samples were stored at -30°C to -10°C for 0, 2, 9, 15, and 21 months. Each storage period at -30°C to -10°C was followed by storage for 4 months at the customer in-use temperature of +2 to 10°C. Two repeats were performed for each time point.

Across all time points a total of 10 results were called Invalid during testing. All 10 samples were reinjected, and 6 samples were resolved. The remaining 4 samples were resolved upon reamplification.

The Real Time Shelf Life Stability of the OncoMate™ MSI Dx Analysis System amplification kit reagents was determined to be frozen storage for 20 months followed by refrigerated storage for four (4) months, for a total of 24 months based on the data collected to date. No difference was observed based on OncoMate™M MSI Dx Analysis System amplification kit lot.

Real Time Stability at -30°C to -10°C (OncoMate™ 5C Matrix Standard)

The purpose of this study was to determine the real time stability of the OncoMate™ 5C Matrix Standard stored at -30°C to -10°C in real time.

Samples were tested by one operator and instrument using three reagent lots, two lots subjected to freeze-thaw and shipping, at time zero and after 4 months storage at -30°C to -10°C. Samples were tested after storage at -30°C to -10°C for 0, 6, 13, 19, and 25 months. Two repeats were performed for each time point.

The OncoMate™ 5C Matrix Standard was used to perform spectral calibration on the Applied Biosystems 3500 Dx Genetic Analyzer as described in the Technical Manual (TM542). The result of the spectral calibration is a multicomponent matrix, which is applied during sample detection to compensate for spectral overlap among the dyes and separate the raw fluorescent signals into individual dye signals. The Quality Value (Q value) for each capillary was measured by the instrument software. A O score of ≥0.95 is indicative of a successful spectral calibration.

26

The Shelf Life Stability of the OncoMate™ 5C Matrix Standard was determined to 24 months for frozen storage based on the data collected to date. No difference was observed based on OncoMate 5C Matrix Standard lot.

Real Time Stability at -30°C to -10°C followed by 4 months at +2°C to 10°C (OncoMate™ 5C Matrix Standard)

Another set of samples were stored at -30°C to -10°C for 2, 9, 15, and 21 months. Each storage period at -30°C to -10°C was followed by storage for 4 months at the customer in-use temperature of +2 to 10℃. Two repeats were performed for each time point.

The OncoMate™ 5C Matrix Standard was used to perform spectral calibration on the Applied Biosystems 3500 Dx Genetic Analyzer as described in the Technical Manual (TM542). The result of the spectral calibration is a multicomponent matrix, which is applied during sample detection to compensate for spectral overlap among the dyes and separate the raw fluorescent signals into individual dye signals. The Quality Value (O value) for each capillary was measured by the instrument software. A Q score of ≥0.95 is indicative of a successful spectral calibration.

The Real-Time Long-Term Stability of the OncoMate™ 5C Matrix Standard was determined to be frozen storage for 20 months followed by storage at +2 to 10°C for four (4) months, for a total of 24 months based on the data collected to date. No difference was observed based on OncoMate™ 5C Matrix Standard lot.

7.10 Method Comparison Study

The primary objective of the method comparison study was to evaluate the accuracy and usability of the OncoMate™ MSI Dx Analysis System in identifying microsatellite instability in the clinical setting. Tumors from Lynch syndrome patients have a high likelihood of having an MSI-H status (4, 5, 19), and therefore MSI-H status can identify patients where further genetic testing for Lynch syndrome is recommended. A method comparison was performed between the OncoMate™ MSI Dx Analysis System and the VENTANA MMR IHC Panel, the predicate device to identify Lynch syndrome candidates, which stains for the presence of DNA mismatch repair proteins. A comparison of the OncoMate™ MSI Dx Analysis System result to germline Next Generation Sequencing for DNA mismatch repair genes (NGS MMR genes) was performed to confirm identification of Lynch syndrome patients.

The study was performed by testing a sequential series of colorectal cancer patient samples that were enriched with a second set of suspected Lynch syndrome samples, for a total of 154 cases. Sample curls, generated from FFPE tissue blocks, were provided to an external laboratory to perform immunohistochemistry. Extracted DNA aliquots of 154 matched CRC DNA samples of unknown MSI status were randomized then analyzed by the OncoMate™ MSI Dx Analysis System and NGS.

Immunohistochemistry was performed on all 154 tumor samples to determine protein expression of the MHL1, MSH2, MSH6, and PMS2 using the VENTANA MMR IHC Panel on the provided sample curls, per the VENTANA instructions for use and the

005 510k Summary Original 510(k) Application

27

laboratory's Standard Operating Procedures. BRAF testing was only performed if the sample exhibited a loss of one of the mismatch repair genes. BRAF testing may provide evidence of the mechanism for the mismatch repair deficiency and therefore differentiate Lynch syndrome candidates from sporadic dMMR cases (4, 16, 20).

DNA from the 154 samples were provided to a reference laboratory for Next Generation Sequencing. These samples underwent DNA sequencing to determine the presence or absence of pathogenic mutations of the mismatch repair genes (MLH1, MSH2, MSH6 and PMS2) and BRAF exon 15. Upon germline testing, 18 Lynch syndrome cases were confirmed. Only pathogenic or likely pathogenic mutations listed in the ClinVar database (21) were accepted as confirmed Lynch syndrome cases.

A total of 2 samples tested using the OncoMate™ MSI Dx Analysis System yielded Invalid results during initial testing. Both samples were resolved upon reinjection.

7.10.1 Method Comparison: OncoMate™ MSI Dx Analysis System vs. IHC Results

The comparison results and agreement analysis between the OncoMate™ MSI Dx Analysis System and the VENTANA IHC MMR Panel for the 154 samples are listed in Table 13. A total of 106 samples were scored as MSS using the OncoMate™ MSI Dx Analysis System and MMR Intact for all four MMR proteins using the VENTANA IHC MMR Panel. Forty-one samples exhibited a loss of IHC staining for at least one of the four MMR proteins (dMMR). Of these 45 samples, 44 samples were scored as MSI-H by the OncoMate™ MSI Dx Analysis System.

Three samples were scored as MSI-H by the OncoMate™ MSI Dx Analysis System but were scored as MMR Intact by IHC staining for all four MMR proteins.

The Positive Percent Agreement (PPA) was 97.8% and the Negative Percent Agreement (NPA) was 97.2%, with an Overall Percent Agreement (OPA) of 97.4% between the two methods. Additional comparison and agreement analysis data stratified by sequential and enrichment cohort can be found in Table 14 and Table 15.

The OncoMate™ MSI Dx Analysis System effectively identified tumors with MMR deficiency and shows strong agreement with the VENTANA MMR IHC panel.

28

OncoMate MSI Dx	MMR Loss	MMR Intact	Invalid	Total
MSI-H	44	3	0	47
MSS	1	106	0	107
Invalid	0	0	0	0
Total	45	109	0	154
Agreement
Type	n/N	%	95% CI
PPA	44/45	97.8	88.4 – 99.6
NPA	106/109	97.2	92.2 99.1
OPA	150/154	97.4	93.5-99.0

Table 13: Comparison and Agreement Analysis of OncoMate™ MSI Dx Analysis System Interpretative Results vs. Ventana MMR IHC (All Samples)

Table 14: Comparison and Agreement Analysis of OncoMate™ MSI Dx Analysis
System Interpretative Results vs. Ventana MMR IHC (Sequential Cohort)

	Ventana MMR IHC results
OncoMate MSI Dx	MMR Loss	MMR Intact	Invalid	Total
MSI-H	23	0	0	23
MSS	1	106	0	107
Invalid	0	0	0	0
Total	24	106	0	130
Agreement
Type	n/N	%	95% CI
PPA	23/24	95.8	79.8 – 99.3
NPA	106/106	100.0	96.5 - 100.0
OPA	129/130	99.2	95.8 - 99.9

Table 15: Comparison and Agreement Analysis of OncoMate™ MSI Dx Analysis
System Interpretative Results vs. Ventana MMR IHC (Enrichment Cohort)

	Ventana MMR IHC results
OncoMate MSI Dx	MMR Loss	MMR Intact	Invalid	Total
MSI-H	21	3	0	24
MSS	0	0	0	0
Invalid	0	0	0	0
Total	21	3	0	24

29

Agreement
Type	n/N	%	95% CI
PPA	21/21	100.0	84.5 - 100.0
NPA	0/3	0.0	0.0 - 56.1
OPA	21/24	87.5	69.0 - 95.7

7.10.2 Method Comparison to NGS Mismatch Repair Gene Mutations Results

A total of 18 samples tested positive for Lynch syndrome, based on detection of a pathogenic or likely pathogenic mutation in one of the mismatch repair genes (MLH1, MSH2, MSH6 or PMS2) and no mutations in BRAF exon 15. The data for all samples are summarized in Table 16. Table 17 and Table 18 summarize the data for the enrichment cohort and sequential cohort, respectively.

Seventeen of the samples (17/18) tested MSI-H with the OncoMate™ MSI Dx Analysis System. One of the samples (1/18) tested MSS with the OncoMate™ MSI Dx Analysis System and exhibited no loss of MMR protein by IHC (Table 16). This sample is referenced as having a likely pathogenic mutation in the PMS2 gene on the ClinVar database (21). This single nucleotide polymorphism (rs267608153) results in a c.903G>T variant that likely results in a splicing defect (25). All 16 of the enrichment cohort samples tested MSI-H with the OncoMate™ MSI Dx Analysis System (Table 17).

The Positive Percent Agreement (PPA) was 94.4% and the Negative Percent Agreement (NPA) was 77.9% between the two methods for all samples (Table 16). The PPA was 100% between the two methods for the enrichment cohort (Table 17). The NPA is less informative than the PPA in a comparison of somatic microsatellite instability to germline mutations in MMR genes, since cases negative for germline, pathogenic Lynch syndrome mutations include MSI-H cases with sporadic, somatic causes for dMMR as well as MSS cases. Somatic mechanisms such as epigenetic silencing and biallelic somatic mutation can lead to dMMR and an MSI-H phenotype (26) without a germline MMR gene mutation. Table 18 provides the comparison and agreement analysis between the two methods for the sequential cohort.

Table 19 provides a comparison of all three testing methods and displays the results for each sample confirmed as Lynch. This table also provides the pathogenic germline mutation, ClinVar accession number, and clinical significance for each sample confirmed as Lynch.

In conclusion, the OncoMate™ MSI Dx Analysis System effectively identified tumors with confirmed germline MMR mutations indicative of Lynch syndrome.

30

OncoMate MSI Dx	DNA Sequencing Results
	Pathogenic Mutation	No Pathogenic Mutation	Invalid	Total
MSI-H	17	30	0	47
MSS	1	106	0	107
Invalid	0	0	0	0
Total	18	136	0	154
Agreement
Type	n/N	%		95% CI
PPA	17/18	94.4		74.2 - 99.0
NPA	106/136	77.9		70.3 - 84.1
OPA	123/154	79.9		72.8 - 85.4

Table 16: Comparison and Agreement Analysis of OncoMate™ MSI Dx Analysis
System Interpretative Results vs. NGS MMR Sequencing (All Samples)

Table 17: Comparison and Agreement Analysis of OncoMate™ MSI Dx Analysis System Interpretative Results vs. NGS MMR Sequencing (Enrichment Cohort)

OncoMate MSI Dx	DNA Sequencing Results
	Pathogenic Mutation	No Pathogenic Mutation	Invalid	Total
MSI-H	16	8	0	24
MSS	0	0	0	0
Invalid	0	0	0	0
Total	16	8	0	24
Agreement
Type	n/N	%	95% CI
PPA	16/16	100.0	80.6 - 100
NPA	0/8	0.0	0.0 - 32.4
OPA	16/24	66.7	46.7 - 82.0

Table 18: Comparison and Agreement Analysis of OncoMate™ MSI Dx Analysis System Interpretative Results vs. NGS MMR Sequencing (Sequential Cohort)

	DNA Sequencing Results
OncoMate MSI Dx	Pathogenic Mutation	No Pathogenic Mutation	Invalid	Total
MSI-H	1	12	0	13
MSS	1	106	0	107
Invalid	0	0	0	0
Total	2	128	0	130

31

Agreement
Type	n/N	%	95% CI
PPA	1/2	50.0	9.5 – 90.5
NPA	106/128	82.8	75.3 – 88.4
OPA	107/130	82.3	74.8 – 87.9

	Table 19: Lynch Syndrome Cases Confirmed During the Method Comparison
Study

| UID
Number | Cohort | OncoMate™
Result | IHC
Status | Pathogenic
germline
mutation¹ | ClinVar
Accession
Number² | Clinical
significance² |
|---------------|----------------------------|----------------------|-------------------------|-------------------------------------|---------------------------------|-------------------------------------------------|
| P00010 | Enrichment | MSI-H | Loss
(MLH1,
PMS2) | MLH1
rs63751642 | VCV000090067.4 | Pathogenic |
| P00060 | Sequential | MSI-H | Loss
(MSH6) | MSH6
rs63750955 | VCV000008932.6 | Pathogenic |
| P00092 | Enrichment | MSI-H | Loss
(PMS2) | PMS2
rs121434629 | VCV000009245.12 | Pathogenic/Likely
pathogenic |
| P00096 | Sequential | MSS | Intact | PMS2
rs267608153 | VCV000091377.9 | Pathogenic/Likely
pathogenic |
| P00109 | Enrichment | MSI-H | Loss
(MSH6) | MSH2
rs63749932 | VCV000036572.7 | Pathogenic |
| P00120 | Enrichment | MSI-H | Loss
(MLH1,
PMS2) | MLH1
rs587779029 | VCV000090295.1 | Pathogenic |
| P00125 | Enrichment | MSI-H | Loss
(MLH1,
PMS2) | MLH1
rs267607767 | VCV000090291.7 | Pathogenic |
| P00126 | Enrichment | MSI-H | Loss
(MLH1,
PMS2) | MLH1
rs876658821 | VCV000230876.3 | Pathogenic |
| P00131 | Enrichment | MSI-H | Intact | MSH6
rs730881830 | VCV000182683.3 | Pathogenic/Likely
pathogenic |
| P00146 | Enrichment | MSI-H | Loss
(MSH6) | PMS2
rs573125799 | VCV000183716.9 | Pathogenic |
| P00153 | Supplemental
Enrichment | MSI-H | Loss
(MLH1,
PMS2) | MLH1
rs267607888 | VCV000090046.3 | Pathogenic/Likely
pathogenic |
| P00158 | Supplemental
Enrichment | MSI-H | Loss
(MLH1,
PMS2) | PMS2
rs876661113 | VCV000234604.9 | Pathogenic |
| P00162 | Supplemental
Enrichment | MSI-H | Loss
(MSH6) | PMS2
rs587781317 | VCV000140847.7 | Likely pathogenic;
Uncertain
significance |
| P00164 | Supplemental
Enrichment | MSI-H | Loss
(MSH6) | MSH2
rs63751044 | VCV000091262.2 | Pathogenic |
| P00171 | Supplemental
Enrichment | MSI-H | Loss
(MSH6) | MSH2
rs63750302 | VCV000090558.1 | Pathogenic |
| P00172 | Supplemental
Enrichment | MSI-H | Loss
(MLH1,
PMS2) | MLH1
rs267607888 | VCV000090046.3 | Pathogenic/Likely
pathogenic |
| UID
Number | Cohort | OncoMateTM
Result | IHC
Status | Pathogenic
germline
mutation1 | ClinVar
Accession
Number2 | Clinical
significance2 |
| P00173 | Supplemental
Enrichment | MSI-H | Loss
(MLH1,
PMS2) | MLH1
rs267607888 | VCV000090046.3 | Pathogenic/Likely
pathogenic |
| P00174 | Supplemental
Enrichment | MSI-H | Loss
(MLH1,
PMS2) | MLH1
rs267607723 | VCV000090061.4 | Pathogenic |

005_510k Summary
Original 510(k) Application

32

1 Lynch syndrome status was confirmed by the detection of a "Pathogenic" or "Likely pathogenic"

germline mutation. Germline mutations are those detected in the normal tissue sample.

2 National Center for Biotechnology Information. ClinVar;

https://www.ncbi.nlm.nih.gov/clinvar/variation/VCV... (accessed Sept. 23, 2020).

7.11 Conclusion

The results of the nonclinical analytical and clinical performance studies summarized above demonstrate that the OncoMate™ MSI Dx Analysis System is substantially equivalent to the predicate device.

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