GENESEEQPRIME NGS Tumor Profiling Assay (FFPE) (GS6005)

K250003 · Geneseeq Technology, Inc. · PZM · Aug 29, 2025 · Pathology

Device Facts

• Record ID: K250003
• Device Name: GENESEEQPRIME NGS Tumor Profiling Assay (FFPE) (GS6005)
• Applicant: Geneseeq Technology, Inc.
• Product Code: PZM · Pathology
• Decision Date: Aug 29, 2025
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.6080
• Device Class: Class 2
• Attributes: AI/ML

Intended Use

The GENESEEQPRIME NGS Tumor Profiling Assay (FFPE) is a qualitative in vitro diagnostic test kit that uses next generation sequencing of DNA isolated from formalin-fixed paraffin-embedded tumor tissue from previously diagnosed patients with solid malignant neoplasms to detect tumor gene alterations in a broad multi gene panel. This test is intended to provide tumor mutation profiling information on somatic variants, including single nucleotide variants (SNVs), insertions and deletions (indels), one amplification, four translocations, microsatellite instability (MSI), and tumor mutation burden (TMB). Information provided by GENESEEQPRIME NGS Tumor Profiling Assay (FFPE) is intended to be used by qualified health care professionals in accordance with professional guidelines in oncology. Results from GENESEEQPRIME NGS Tumor Profiling Assay (FFPE) are not intended to be prescriptive or conclusive for labeled use of any specific therapeutic product.

Device Story

The GENESEEQPRIME NGS Tumor Profiling Assay (FFPE) is an in vitro diagnostic kit for tumor mutation profiling. It processes genomic DNA extracted from FFPE tumor tissue samples. The workflow involves DNA fragmentation, end repair, adapter ligation, PCR amplification, and hybrid capture using biotinylated DNA probes targeting 425 cancer-related genes. Enriched libraries are sequenced on the Illumina NextSeq 550Dx platform. The GENESIS software performs data management, demultiplexing, alignment to the hg19/GRCh37 reference genome, and variant calling. The system identifies SNVs, indels, ERBB2 amplifications, specific gene translocations (ALK, RET, ROS1, NTRK1), MSI status, and TMB. The device is used in clinical laboratory settings by qualified professionals. Output reports provide genomic alteration data to assist oncologists in understanding tumor profiles, though results are not prescriptive for specific therapies. The assay benefits patients by providing comprehensive tumor profiling to inform clinical decision-making in accordance with oncology guidelines.

Clinical Evidence

Accuracy was evaluated using 503 FFPE samples across 40 tumor types, comparing results to orthogonal methods (NGS, FISH, IHC, WES). Overall PPA for SNVs/indels was 92.44% (95% CI: 91.45%-93.33%). Specificity for ERBB2 amplification was 100% (NPA). MSI status concordance with IHC was 97.50% PPA and 90.38% NPA. TMB correlation with WES was 0.9298 (Pearson). Reproducibility was validated across three laboratories with high concordance (>97% for variants above LoD).

Technological Characteristics

Hybrid capture NGS assay targeting 425 genes. Materials include library prep reagents, hybridization probes, and magnetic beads. Requires Illumina NextSeq 550Dx sequencer. Software (GENESIS) performs automated analysis. Validated for FFPE DNA input (50-500ng). Sterilization not applicable (reagent kit). Connectivity: Standalone system (no wireless transmission).

Indications for Use

Indicated for previously diagnosed patients with solid malignant neoplasms. Used by qualified healthcare professionals to provide tumor mutation profiling information on somatic variants (SNVs, indels, ERBB2 amplification, ALK/RET/ROS1/NTRK1 translocations, MSI, and TMB) from FFPE tumor tissue.

Regulatory Classification

Identification

A next generation sequencing (NGS) based tumor profiling test is a qualitative in vitro diagnostic test intended for NGS analysis of tissue specimens from malignant solid neoplasms to detect somatic mutations in a broad panel of targeted genes to aid in the management of previously diagnosed cancer patients by qualified health care professionals.

Special Controls

*Classification.* Class II (special controls). The special controls for this device are:(1) Premarket notification submissions must include the following information: (i) A detailed description of all somatic mutations that are intended to be detected by the test and that are adequately supported in accordance with paragraph (b)(1)(v) of this section and reported in the test results in accordance with paragraph (b)(2)(iv) of this section, including: (A) A listing of mutations that are cancer mutations with evidence of clinical significance. (B) As appropriate, a listing of mutations that are cancer mutations with potential clinical significance. (ii) The indications for use must specify the following: (A) The test is indicated for previously diagnosed cancer patients. (B) The intended specimen type(s) and matrix ( *e.g.,* formalin-fixed, paraffin-embedded tumor tissue).(C) The mutation types ( *e.g.,* single nucleotide variant, insertion, deletion, copy number variation or gene rearrangement) for which validation data has been provided.(D) The name of the testing facility or facilities, as applicable. (iii) A detailed device description including the following: (A) A description of the test in terms of genomic coverage, as follows: ( *1* ) Tabulated summary of all mutations reported, grouped according to gene and target region within each gene, along with the specific cDNA and amino acid positions for each mutation.( *2* ) A description of any within-gene targeted regions that cannot be reported and the data behind such conclusion.(B) Specifications for specimen requirements including any specimen collection devices and preservatives, specimen volume, minimum tumor content, specimen handling, DNA extraction, and criteria for DNA quality and quantity metrics that are prerequisite to performing the assay. (C) A detailed description of all test components, reagents, instrumentation, and software required. Detailed documentation of the device software including but not limited to, software applications and hardware-based devices that incorporate software. (D) A detailed description of the methodology and protocols for each step of the test, including description of the quality metrics, thresholds, and filters at each step of the test that are implemented for final result reporting and a description of the metrics for run-failures, specimen-failures, invalids, as applicable. (E) A list of links provided by the device to the user or accessed by the device for internal or external information ( *e.g.,* decision rules or databases) supporting clinical significance of test results for the panel or its elements in accordance with paragraphs (b)(1)(v) and (b)(2)(vi) of this section.(F) A description of internal and external controls that are recommended or provided and control procedures. The description must identify those control elements that are incorporated into the testing procedure. (iv) Information demonstrating analytical validity of the device according to analytical performance characteristics, evaluated either specifically for each gene/mutation or, when clinically and practically justified, using a representative approach based on other mutations of the same type, including: (A) Data that adequately supports the intended specimen type ( *e.g.,* formalin-fixed, paraffin-embedded tumor tissue), specimen handling protocol, and nucleic acid purification for specific tumor types or for a pan-tumor claim.(B) A summary of the empirical evidence obtained to demonstrate how the analytical quality metrics and thresholds were optimized. (C) Device precision data using clinical samples to adequately evaluate intra-run, inter-run, and total variability. The samples must cover all mutation types tested (both positive and negative samples) and include samples near the limit of detection of the device. Precision must be assessed by agreement within replicates on the assay final result for each representative mutation, as applicable, and also supported by sequencing quality metrics for targeted regions across the panel. (D) Description of the protocols and/or data adequately demonstrating the interchangeability of reagent lots and multiplexing barcodes. (E) A description of the nucleic acid assay input concentration range and the evidence to adequately support the range. (F) A description of the data adequately supporting the limit of detection of the device. (G) A description of the data to adequately support device accuracy using clinical specimens representing the intended specimen type and range of tumor types, as applicable. ( *1* ) Clinical specimens tested to support device accuracy must adequately represent the list of cancer mutations with evidence of clinical significance to be detected by the device.( *2* ) For mutations that are designated as cancer mutations with evidence of clinical significance and that are based on evidence established in the intended specimen type (*e.g.,* tumor tissues) but for a different analyte type (*e.g.,* protein, RNA) and/or a measurement (*e.g.,* incorporating a score or copy number) and/or with an alternative technology (*e.g.,* IHC, RT-qPCR, FISH), evidence of accuracy must include clinically adequate concordance between results for the mutation and the medically established biomarker test (*e.g.,* evidence generated from an appropriately sized method comparison study using clinical specimens from the target population).( *3* ) For qualitative DNA mutations not described in paragraph (b)(1)(iv)(G)(*2* ) of this section, accuracy studies must include both mutation-positive and wild-type results.(H) Adequate device stability information. (v) Information that adequately supports the clinical significance of the panel must include: (A) Criteria established on what types and levels of evidence will clinically validate a mutation as a cancer mutation with evidence of clinical significance versus a cancer mutation with potential clinical significance. (B) For representative mutations of those designated as cancer mutations with evidence of clinical significance, a description of the clinical evidence associated with such mutations, such as clinical evidence presented in professional guidelines, as appropriate, with method comparison performance data as described in paragraph (b)(1)(iv)(G) of this section. (C) For all other mutations designated as cancer mutations with potential clinical significance, a description of the rationale for reporting. (2) The 21 CFR 809.10 compliant labeling and any product information and test report generated, must include the following, as applicable: (i) The intended use statement must specify the following: (A) The test is indicated for previously diagnosed cancer patients. (B) The intended specimen type(s) and matrix ( *e.g.,* formalin-fixed, paraffin-embedded tumor tissue).(C) The mutation types ( *e.g.,* single nucleotide variant, insertion, deletion, copy number variation or gene rearrangement) for which validation data has been provided.(D) The name of the testing facility or facilities, as applicable. (ii) A description of the device and summary of the results of the performance studies performed in accordance with paragraphs (b)(1)(iii), (b)(1)(iv), and (b)(1)(v) of this section. (iii) A description of applicable test limitations, including, for device specific mutations validated with method comparison data to a medically established test in the same intended specimen type, appropriate description of the level of evidence and/or the differences between next generation sequencing results and results from the medically established test ( *e.g.,* as described in professional guidelines).(iv) A listing of all somatic mutations that are intended to be detected by the device and that are reported in the test results under the following two categories or equivalent designations, as appropriate: “cancer mutations panel with evidence of clinical significance” or “cancer mutations panel with potential clinical significance.” (v) For mutations reported under the category of “cancer mutations panel with potential clinical significance,” a limiting statement that states “For the mutations listed in [cancer mutations panel with potential clinical significance or equivalent designation], the clinical significance has not been demonstrated [with adequate clinical evidence ( *e.g.,* by professional guidelines) in accordance with paragraph (b)(1)(v) of this section] or with this test.”(vi) For mutations under the category of “cancer mutations panel with evidence of clinical significance,” or equivalent designation, link(s) for physicians to access internal or external information concerning decision rules or conclusions about the level of evidence for clinical significance that is associated with the marker in accordance with paragraph (b)(1)(v) of this section.

Predicate Devices

• PGDx elio Tissue Complete (K192063)

Submission Summary (Full Text)

{0}

FDA U.S. FOOD & DRUG ADMINISTRATION

August 29, 2025

Geneseeq Technology Inc.
Xue Wu
Chief Executive Officer
Suite 1802, 393 University Ave
Toronto, ON M5G 1E6
Canada

Re: K250003
Trade/Device Name: GENESEEQPRIME NGS Tumor Profiling Assay (FFPE) (GS6005)
Regulation Number: 21 CFR 866.6080
Regulation Name: Next Generation Sequencing Based Tumor Profiling
Regulatory Class: Class II
Product Code: PZM
Dated: December 30, 2024
Received: January 2, 2025

Dear Xue Wu:

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 (the 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 available 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.

Additional information about changes that may require a new premarket notification are provided in the FDA guidance documents entitled "Deciding When to Submit a 510(k) for a Change to an Existing Device"

U.S. Food & Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993
www.fda.gov

{1}

K250003 - Xue Wu
Page 2

(https://www.fda.gov/media/99812/download) and "Deciding When to Submit a 510(k) for a Software Change to an Existing Device" (https://www.fda.gov/media/99785/download).

Your device is also subject to, among other requirements, the Quality System (QS) regulation (21 CFR Part 820), which includes, but is not limited to, 21 CFR 820.30, Design controls; 21 CFR 820.90, Nonconforming product; and 21 CFR 820.100, Corrective and preventive action. Please note that regardless of whether a change requires premarket review, the QS regulation requires device manufacturers to review and approve changes to device design and production (21 CFR 820.30 and 21 CFR 820.70) and document changes and approvals in the device master record (21 CFR 820.181).

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 (reporting of medical device-related adverse events) (21 CFR Part 803) for devices or postmarketing safety reporting (21 CFR Part 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reporting-combination-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 Part 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR Parts 1000-1050.

All medical devices, including Class I and unclassified devices and combination product device constituent parts are required to be in compliance with the final Unique Device Identification System rule ("UDI Rule"). The UDI Rule requires, among other things, that a device bear a unique device identifier (UDI) on its label and package (21 CFR 801.20(a)) unless an exception or alternative applies (21 CFR 801.20(b)) and that the dates on the device label be formatted in accordance with 21 CFR 801.18. The UDI Rule (21 CFR 830.300(a) and 830.320(b)) also requires that certain information be submitted to the Global Unique Device Identification Database (GUDID) (21 CFR Part 830 Subpart E). For additional information on these requirements, please see the UDI System webpage at https://www.fda.gov/medical-devices/device-advice-comprehensive-regulatory-assistance/unique-device-identification-system-udi-system.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR 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-devices/medical-device-safety/medical-device-reporting-mdr-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/medical-devices/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-devices/device-advice-comprehensive-regulatory-assistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email ([e-mail]) or phone [phone] or 301-796-7100).

{2}

K250003 - Xue Wu
Page 3

Sincerely,

Zivana Tezak-fragale -S

Zivana Tezak, Ph.D.
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

{3}

FORM FDA 3881 (8/23)
Page 1 of 1
PSC Publishing Services [phone]
EF

| DEPARTMENT OF HEALTH AND HUMAN SERVICES Food and Drug Administration Indications for Use | Form Approved: OMB No. 0910-0120 Expiration Date: 07/31/2026 See PRA Statement below. |
| --- | --- |
| 510(k) Number (if known) K250003 | |
| Device Name GENESEEQPRIME NGS Tumor Profiling Assay (FFPE) | |
| Indications for Use (Describe) The GENESEEQPRIME NGS Tumor Profiling Assay (FFPE) is a qualitative in vitro diagnostic test kit that uses next generation sequencing of DNA isolated from formalin-fixed paraffin-embedded tumor tissue from previously diagnosed patients with solid malignant neoplasms to detect tumor gene alterations in a broad multi gene panel. This test is intended to provide tumor mutation profiling information on somatic variants, including single nucleotide variants (SNVs), insertions and deletions (indels), one amplification, four translocations, microsatellite instability (MSI), and tumor mutation burden (TMB). | |
| Information provided by GENESEEQPRIME NGS Tumor Profiling Assay (FFPE) is intended to be used by qualified health care professionals in accordance with professional guidelines in oncology. Results from GENESEEQPRIME NGS Tumor Profiling Assay (FFPE) are not intended to be prescriptive or conclusive for labeled use of any specific therapeutic product. | |
| 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) | |
| CONTINUE ON A SEPARATE PAGE IF NEEDED. | |
| This section applies only to requirements of the Paperwork Reduction Act of 1995. *DO NOT SEND YOUR COMPLETED FORM TO THE PRA STAFF EMAIL ADDRESS BELOW.* | |
| The burden time for this collection of information is estimated to average 79 hours per response, including the time to review instructions, search existing data sources, gather and maintain the data needed and complete and review the collection of information. Send comments regarding this burden estimate or any other aspect of this information collection, including suggestions for reducing this burden, to: Department of Health and Human Services Food and Drug Administration Office of Chief Information Officer Paperwork Reduction Act (PRA) Staff [e-mail] | |
| "An agency may not conduct or sponsor, and a person is not required to respond to, a collection of information unless it displays a currently valid OMB number." | |

{4}

1

# 510(k) Summary

## I. Introduction:

A. Applicant:
Geneseeq Technology Inc.

B. Proprietary and Established Names:
GENESEEQPRIME NGS Tumor Profiling Assay (FFPE)

C. 510(k) Number
K250003

D. Regulatory Information:

| Product Code | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| PZM | Class II | 21 CFR 866.6080 – Next Generation Sequencing Based Tumor Profiling Assay | Pathology |

## II. Submission/Device Overview:

A. Purpose for Submission:
New Device

B. Measurand (quantity intended to be measured):
Somatic single nucleotide variants (SNVs), insertions and deletions (Indels), select amplifications and translocations, microsatellite instability (MSI), and tumor mutation burden (TMB) in human genomic DNA obtained from formalin-fixed paraffin embedded tumor tissue. For a complete list of genes, please refer to the device user manual for details.

C. Type of Test:
Next-generation sequencing tumor profiling test

## III. Intended Use/Indications for Use:

A. Intended Use:
The GENESEEQPRIME NGS Tumor Profiling Assay (FFPE) is a qualitative *in vitro* diagnostic test kit that uses next generation sequencing of DNA isolated from formalin-fixed paraffin-embedded tumor tissue from previously diagnosed patients with solid malignant neoplasms to detect tumor gene alterations in a broad multi gene panel. This test is intended to provide tumor mutation profiling information on somatic variants, including single nucleotide variants (SNVs), insertions and deletions (indels), one amplification, four translocations, microsatellite instability (MSI), and tumor mutation burden (TMB).

{5}

Information provided by GENESEEQPRIME NGS Tumor Profiling Assay (FFPE) is intended to be used by qualified health care professionals in accordance with professional guidelines in oncology. Results from GENESEEQPRIME NGS Tumor Profiling Assay (FFPE) are not intended to be prescriptive or conclusive for labeled use of any specific therapeutic product.

B. Indications for Use:
Same as above

C. Special Conditions for Use Statement(s):
For Prescription Use

For in vitro diagnostic use.

D. Special Instrument Requirements:
Illumina NextSeq® 550Dx Sequencer

IV. Device/System Characteristics:

A. Device Description:

1. Targeted Genes of Interest List
The complete list of 425 targeted genes for the GENESEEQPRIME NGS Tumor Profiling Assay (FFPE) is provided in the device user manual.

2. Reagents
The GENESEEQPRIME NGS Tumor Profiling Assay (FFPE) (Ref. No. GS6005, hereafter referred to as "GENESEEQPRIME") includes the following components as listed in Table 1.

Table 1. GENESEEQPRIME kit: Reagent Components and Storage Condition.

| Library Preparation and Target Enrichment Kit, Box 1 of 3 (Store at -25°C to -15°C) | | | |
| --- | --- | --- | --- |
| Cap Label | Component Name | Cap Color | Volume (μL) |
| LB1 | ER/AT Buffer | Yellow | 130 |
| LE1 | ER/AT Enzyme Mix | Yellow | 55 |
| LE2 | DNA Ligase | Green | 180 |
| LB2 | Ligation Buffer | Green | 540 |
| PM | PCR Master Mix | Pink | 1200 |
| PR | PCR Primers | Pink | 240 |
| HP | Hybridization Probes | Red | 15 |
| BL1 | DNA Blockers | Red | 120 |
| BL2 | Adaptor Blocker | Red | 12 |
| HB1 | Hybridization Buffer 1 | Red | 45 |
| HB2 | Hybridization Buffer 2 | Red | 18 |

{6}

3

| WB1 | Wash Buffer 1 | White | 180 |
| --- | --- | --- | --- |
| WB2 | Wash Buffer 2 | White | 120 |
| WB3 | Wash Buffer 3 | White | 120 |
| WB4 | Wash Buffer 4 | White | 240 |
| BW | Beads Wash Buffer | White | 1500 |
| NC | Negative Control | Blue | 55 |
| PC | Positive Control | Blue | 55 |
| UDI Adaptor for Illumina, Box 2 of 3 (Store at -25°C to -15°C) | | | |
| Cap Label | Component Name | Volume | Concentration |
| DA01-DA30 | UDI Adaptor 1-30 | 6.5 μL/tube | 15 μM |
| Purification and Capture Beads, Box 3 of 3 (Store at 2 to 8°C) | | | |
| Cap Label | Component Name | | Volume |
| CB | Capture Beads | | 300 μL |
| PB | Purification Beads | | 9.0 mL |

3. **Material Required but Not Provided:**
A list of materials required for upstream preparation of samples for sequencing is but not included as part of the GENESEEQPRIME assay is shown in Table 2.

Table 2. Materials required, but not provided

| Name | Recommendations |
| --- | --- |
| FFPE DNA extraction kit | User's choice (column-based or beads-based) |
| dsDNA quantification kit | User's choice (Fluorometric method) |
| Library quantification kit | User's choice (qPCR method) |
| Molecular biology grade nuclease-free water | User's choice |
| TE Buffer (10 mM Tris, 1 mM EDTA, pH 8.0) | User's choice |
| Low EDTA TE Buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0) | User's choice |
| 1N NaOH | User's choice |
| 100% ethanol, molecular biology grade | User's choice |
| 200 mM Tris-HCl pH 8.0 | User's choice |
| NextSeq 550Dx High Output Reagent Kits v2.5 (300 Cycles) | Illumina, 20028871 |
| Disposable pipet basin | User's choice |
| Areosol barrier, nuclease-free, low retention sterile pipette tips (1000ul, 200ul, 20ul, 10ul) | User's choice |

{7}

# 4. GENESIS Software:

The GENESIS by GENESEEQ™ (hereafter referred to as "GENESIS") software necessary for the GENESEEQPRIME assay (software version is displayed on the user interface and on reports) is provided by Geneseeq Technology Inc. (Geneseeq) to perform sample information management, sequencing data analysis and test report generation. The software is only compatible with Illumina's NextSeq550 Dx sequencers. The raw data is maintained on the system during data analysis and report generation using redundant disk storage, and the system does not automatically delete or modify the raw data in any way. However, the minimal-requirement server necessary to run the software stores only sample information and reports; it does not provide long-term storage or backup of raw sequencing data. The software saves sample information and reports only and does not provide backup of raw sequencing data. If users need to reduce the amount of storage that raw data occupies, they need super admin privilege to perform raw data deletion via the system interface, and can only do so after successful report generation, with a confirmation prompt. Improper use will result in no report being generated.

# 5. Instrument:

Sequencing libraries prepared from the GENESEEQPRIME assay must be sequenced on Illumina NextSeq 550Dx sequencing platform. Other instruments that are required for use but not included in the GENESEEQPRIME assay are listed in Table 3.

Table 3. Instruments required, not provided

| Equipment | Notes |
| --- | --- |
| Centrifugal vacuum concentrator | Uses vacuum centrifugal force to evaporate liquid and concentrate DNA. |
| Sonicator | Mechanically shears DNA to the appropriate size. |
| Fluorometer | Uses detection of target-specific fluorescence to provide quantification of samples prior to library preparation and sequencing. Separate fluorometers are required in pre-PCR and post-PCR areas. |
| DNA fragment analyzer | Automated sample processing determines size, quantity, and purity for quick library QC. |
| Magnetic stand | Designed for paramagnetic bead precipitation from standard and deep 96-well microplates. Separate magnetic stands are required in pre-PCR and post-PCR areas. |
| qPCR machine | For library quantification. |

{8}

| Thermal cyclers | One 96-well dual-block thermal cycler (or two 96-well single block thermal cyclers) is required in the post-PCR areas. |
| --- | --- |
| Vortex mixer | Separate vortex mixers are required in pre-PCR and post- PCR areas. |
| Thermomixer | Thermomixer capable of temperatures ranging from 20 °C to 70 °C and shaking at 1700 rpm. Two thermomixers or two thermal cyclers (or one thermal cycler with multiple thermal blocks) are required in the pre-PCR area and one thermomixer is required in the post-PCR area. |
| Microcentrifuge | Tabletop micro-centrifuge or mini-centrifuge capable of holding 0.5 mL to 2.0 mL tubes. Separate micro- or mini- centrifuges are required in pre-PCR and post-PCR areas. |
| Single-channel pipettors (P-2, P-10, P-20, P-200, P-1000) | Separate sets of pipettors are required in pre-PCR and post-PCR areas. Pipettors should be calibrated regularly and verified accurate within 5% of stated volume. |
| Multi-channel pipettor (P-20, P-200) | Separate sets of pipettors are required in pre-PCR and post-PCR areas. Pipettors should be calibrated regularly and verified accurate within 5% of stated volume. |

# 6. Sample Preparation:

The GENESEEQPRIME assay requires genomic DNA isolated from formalin-fixed, paraffin-embedded (FFPE) tissue sample using a validated commercially available DNA extraction method (column-based or beads-based). The concentration of the extracted genomic DNA can be measured by using a fluorescence quantification method. The total DNA yield of FFPE sample should be no less than $50\mathrm{ng}$ . The GENESEEQPRIME assay has been validated with FFPE sample stored at room temperature $(15 - 25^{\circ}\mathrm{C})$ for up to 5 years and extracted genomic DNA samples stored at $-25$ to $-15^{\circ}\mathrm{C}$ for up to one year. Longer storage time may compromise the testing results. FFPE tissue specimen should be processed and stored using standardized anatomical pathology protocol (Table 4).

At least $20\%$ of the nucleated cell within the specimen should be represented in tumor tissue for GENESEQPRIME assay. Any sample containing less than $20\%$ tumor content can be macro-dissected before use. Using samples with less than $20\%$ tumor content may compromise the testing results. The tumor volume and minimum tumor content needed to obtain sufficient DNA for testing to achieve the necessary quality performance are shown in the Table 4 below. The recommended number of FFPE sections of each sample for DNA extraction varies depending on the tissue sample size, please see Table 5 for suggestions.

Genomic DNA is extracted from tissue specimens according to the extraction kit protocol. DNA is quantified and concentrated if necessary. The amount of DNA required to perform the test is $50 - 500\mathrm{ng}$ . DNA shearing is conducted per protocol and a quality control check is performed. Average fragment size should be $\sim 300\mathrm{bp}$ .

{9}

Sheared DNA is stored at $-20^{\circ}\mathrm{C}$ if not proceeding directly to library preparation. The DNA can be stored at $37^{\circ}\mathrm{C}$ for 10-20 minutes, stored at $2 - 8^{\circ}\mathrm{C}$ for 24 hours, or at $-20^{\circ}\mathrm{C}$ for longer periods.

Table 4. Specimen Handling and Processing for Validated Specimen Types

| Tissue Type | Volume | Minimum Tumor Proportion | Macrodissection requirements | Limitations | Storage |
| --- | --- | --- | --- | --- | --- |
| FFPE sections | 2-15 unstained sections, 10 microns thick | ≥ 20% tumor proportion based on proportion of tumor nuclei in total viable nuclei in the selected tumor area | Any sample containing less than 20% tumor content can be macro-dissected before use. | Archival FFPE material >5 years post-resection is not suitable for analysis | Room temperature |

Table 5. Tissue sample size and FFPE section specification for GENESEEQPRIME assay

| Surface area of tissue sample (A) | Unstained FFPE slides or curls |
| --- | --- |
| A ≥ 1.0 cm * 1.0 cm | 2-5 slides/curls at 5-10 μm |
| 0.5 cm * 0.5 cm ≤ A < 1.0 cm * 1.0 cm | 5-10 slides/curls at 5-10 μm |
| A < 0.5 cm * 0.5 cm | 10-15 slides/curls at 5-10 μm |

# 7. DNA Extraction:

The kit does not provide reagents for genomic DNA extraction and fragmentation. However, both magnetic beads-based and spin column-based DNA extraction protocols have been validated for the kit. Genomic DNA is isolated from FFPE sections prepared from FFPE tumor tissue samples. The amount and the purity of DNA samples are measured to meet the minimum required standards of the test. The obtained genomic DNA is then mechanically sheared into short DNA fragments with desired length using ultrasonic devices to be read on Illumina sequencing platforms.

# 8. Library Preparation:

The GENESEEQPRIME assay workflow begins with fragmentation and purification of the genomic DNA. Genomic DNA is first quantified using a fluorometric method. DNA is then fragmented to a desired size of $\sim 300$ bp base pairs using sonication and purified using magnetic beads. DNA fragments then undergo end repair, A tailing, and adapter ligation, followed by another round of purification.

For the Illumina sequencer to capture DNA fragments for clonal amplification and sequencing reactions, Illumina compatible DNA adaptors need to be added onto the end of fragmented DNA. Each adaptor contains unique index composed of 8 nucleotides to distinguish one sample from another, which enables multiplexing and sequencing multiple libraries in one sequencing run. Excessive adaptors that are not

{10}

ligated to DNA will be cleaned by size selection using the magnetic beads. The number of PCR cycles is commensurate with the DNA input amount. Each sample library must be $\geq 10\ \mathrm{ng}/\mu\mathrm{l}$ ($\geq 200\ \mathrm{ng}$ total amount) in order to proceed to the following Target Enrichment steps.

9. **Hybrid Capture NGS:**
DNA libraries with different indexes are pooled together, are denatured by heating and subjected to hybridization with single-stranded DNA oligonucleotides (also called ‘probes’) in a length of $\sim 120$ bp. Probes are biotinylated to allow their binding to streptavidin-coated magnetic beads and washing out of unbound DNA. Capture libraries are enriched using PCR amplification. Target enrichment libraries must be quantified using the KAPA Library Quantification Kit. Successful target enrichment is indicated by a total output volume &gt; 9 ng at a concentration of $\geq 0.5\ \mathrm{ng}/\mu\mathrm{l}$.

10. **Sequencing**
Sequencing libraries prepared from this kit are sequenced on Illumina NextSeq 550Dx sequencing platforms. Sample libraries are quantified and normalized into library enrichment pools of up to 8 samples each. A maximum of 28 samples with one positive control and one negative control can be run in one sequencing batch. Pooled sample libraries should be quantified using a qPCR method prior to sequencing.

11. **Data Analysis**
a) **Data Management System (DMS):** Sequencing data is automatically processed using the GENESIS software that tracks sample names, sample metadata and processing status from sequencing through to analysis and reporting. Reports of identified alterations are available in a web-based user interface for download. Sequencing and sample metrics, including sample and sequencing quality, are available in the final report output.
b) **Demultiplexing &amp; FASTQ Generation:** Demultiplexing of BCL files is done by adapter sequences indicated on the samples sheet. Paired FASTQ files are generated which contains sequence and base quality score information. The FASTQ formatted data files are used for subsequent processing of samples.
c) **Run QC check:** Quality control for each sequencing run is determined by cluster density and Q30 read proportion. Cluster density measures the number of clusters on a flow cell, with a sequencer cluster density $\geq 135$ being the passing threshold. For each run, the proportion of total reads with Q30 must be greater than or equal to $80\%$.
d) **Read Alignment &amp; BAM Generation:** To map sequence reads for each sample to the human reference genome (hg19/GRCh37), genome alignment is carried out. The resulting alignments are stored as Binary Alignment Map (BAM) files, presenting information about read placement in relation to the reference genome along with quality scores. Subsequently, the aligned BAM files undergo additional processing

7

{11}

within a pipeline to pinpoint genomic alterations.

e) Sample QC checks: A bioinformatic analysis of genome haplotypes is conducted on samples to screen for potential contamination. This involves examining predefined SNP sites representative of populations and individuals. Samples displaying multiple haplotypes are deemed potentially contaminated and samples with higher than 4% contamination is flagged as failed. Additionally, sequence coverage is evaluated across the panel, necessitating that at least 90% of targeted regions have a minimum coverage exceeding 100x.

f) Mutation calling: A fully automated pipeline for bioinformatic analysis is used to identify genomic alterations, including SNVs, indels, ERBB2 amplifications, ALK translocations, RET translocations, ROS1 translocations, NTRK1 translocations, MSI status, and TMB score.

i. SNVs and Indels: Identification of variants, insertions and deletions are filtered according to variant allele frequency, allele depth, and variant coverage. For non-hotspot SNVs and indels, at least 2% allele frequency and 5 mutant reads is required. For a hotspot variant, at least a 1% VAF and 4 supporting mutant reads is required. Clinically significant variants must have an allele frequency of greater than 0.4% and at least 4 supporting mutant reads.

ii. Amplification: GENESEEQPRIME assay is only validated for reporting ERBB2 amplifications. Only amplifications that have equal to or more than a 1.8-fold change will be reported.

iii. Translocation: This assay only reports the presence or absence of translocations involving four hotspot genes (ALK, RET, ROS1, and NTRK1). For each translocation with a hotspot gene and its canonical partner, a minimum of 6 fusion supporting reads is required. Alternatively, if the partner gene is a non-canonical partner, a minimum of 12 fusion supporting reads is required.

iv. Microsatellite Status: Microsatellite status for each sample is evaluated based on the mutation status of 61 microsatellite sites from within the regions of interest. A sample is deemed to have microsatellite instability (MSI-H) when at least 16% of the detected sites are unstable or contain specific mutations signatures.

v. Tumor Mutation Burden (TMB): TMB is calculated based on detected sequence mutations and indels. Filtering of sequence mutations is performed to exclude low mutant allele fraction mutations (&lt;2% VAF), common somatic driver mutations, and common germline mutations. Both synonymous and nonsynonymous alterations are considered for the mutation load. TMB is reported as the number of mutations per megabase (Muts/Mb).

8

{12}

9

12. Controls:

The GENESEEQPRIME assay kit includes a negative control (NC) and a positive control (PC) to monitor the performance of the instruments and the reagents. All external control samples should be processed the same as the testing samples and should pass all quality control criteria, and all expected mutations should be detected in the PC sample, but not the NC sample.

a) Negative Control:

A non-cancerous cell line (NA18535) is included in the reagent kit and must be included on the sequencing run. The purpose of the negative control is to validate the quality of the sequencing run. No variants of interest should be detected in the negative control sample for sequencing run to pass quality control thresholds.

b) Positive Control:

The positive control sample is included in each kit. The positive control contains 7 alterations of interest (BRAF V600E, EGFR L858R, EGFR Exon 19 Deletion, KRAS G13D, TPM3~NTRK1 translocation, CD74~ROS1 translocation, ERBB2 amplification) and must be included on every sequencing run. All 7 alterations must be detected in the positive control sample for a sequencing run to pass quality control.

13. Result reporting:

a) Somatic single nucleotide variants and short insertion/deletion mutations are reported under one of two categories: "Variants with evidence of clinical significance" or "Variants with potential of clinical significance". The two categories are based on the supporting level of clinical evidence listed on the document "CDRH's Approach to Tumor Profiling Next Generation Sequencing Tests". Common germline mutations and polymorphisms are masked from the final report if they appear in 1000g (version 201508), ExAC (version 0.3nontcga), or gnomAD (version r2.0.1). Variants determined to be germline based on the Tumor-Only Sequencing module are also excluded from reporting.

b) Reporting of microsatellite status for samples is either high microsatellite instability (MSI-H), microsatellite stable (MSS), or uncertain status (Indeterminate).

c) Tumor mutational burden (TMB) is reported in terms of mutations per megabase (mut/mb). Driver mutations are not included in the count of mutations. Both synonymous and non-synonymous mutations are included in the count of mutations.

d) Presence of copy number variations on ERBB2 and gene translocations on four genes (ALK, RET, ROS1, NTRK1) are reported. These alterations are reported under one of two categories: "Variants with evidence of clinical significance" or "Variants with potential of clinical significance". The two categories are based on the supporting level of clinical evidence listed on the document "CDRH's Approach to Tumor Profiling Next Generation Sequencing Tests".

{13}

e) The turnaround time for the GENESEEQPRIME assay from DNA to final clinical report is typically 5 business days. This includes all steps in the workflow: library preparation, sequencing, bioinformatics analysis, and report generation.

# 14. Quality metrics:

Reporting of variants considers the quality metrics outlined in Table 6. Quality metrics are assessed across the following categories.

- Batch-level: Quality metrics that are quantified per sequencing run; failing batch-level metrics will prevent all reports for samples in the run from generating. If the positive or negative control fails these criteria, all samples in the sequencing run will not generate IVD reports.
- Sample-level: Metrics that are quantified per sample; generates no IVD report for any sample failing these QC metrics.
- Analyte-level: Metrics that are quantified for individual alteration types and loci. Only variants that pass analyte-level QC are reported.

Table 6. Summary of GENESEEQPRIME Post-Sequencing Quality Control Metrics

| Quality Metric | Level of Qualification | Passing Criteria |
| --- | --- | --- |
| Cluster Density | Batch-level | Sequencer Cluster Density ≥ 130 |
| Q30 Reads | Batch-level | %Q30 (Total) ≥ 80% |
| External Control | Batch-level | 7 known mutations in positive control are detected; No level 2 or hotspot mutations detected in negative control |
| Percent Regions Covered | Sample-level | ≥ 90% exon region with > 100X Dedup Depth |
| Contamination QC | Sample-level | Estimated contamination levels < 4% |
| Select SNVs and Indels with Evidence of Clinical Significance | Analyte-level | Mutant reads ≥ 4; VAF ≥ 0.4% |
| Hotspot SNVs and Indels | Analyte-level | Mutant reads ≥ 4; VAF ≥ 1% |
| Non-hotspot SNVs and Indels | Analyte-level | Mutant reads ≥ 5; VAF ≥ 2% |
| MSI Detection | Analyte-level | MSI Score ≥ 16 |
| ERBB2 Amplification | Analyte-level | Fold change ≥ 1.8 |
| Translocations (ALK, NTRK1, RET, and ROS1) | Analyte-level | Fusion reads ≥ 6 for canonical partner genes; Fusion reads ≥ 12 for non-canonical partner genes |

{14}

# B. Principle of Operation:

The GENESEEQPRIME assay kit is comprised of reagents for sequencing library preparation and target enrichment for 425 cancer-related genes. The starting material for this kit is extracted genomic DNA from FFPE specimen, which will be subjected to fragmentation, end repair, adaptor ligation, polymerase chain reaction (PCR) amplification and DNA probe hybridization capture of human genomic regions of interest. The regions of interest include the exonic regions, select intronic regions, and specific microsatellite regions of 425 genes within the human genome. The targeted DNA fragments are then enriched using magnetic beads followed by PCR amplification. Library quantification and quality control will be performed on the enriched library before library sequencing.

# C. Determination of assay thresholds:

# 1. Exon coverage

To determine the minimum exon coverage necessary for downstream review, a power analysis was conducted on 11 FFPE samples. We found that mutations with a true frequency of $2\%$ (95% CI: 1.02%-3.90%) is detected with $95\%$ statistical power at $&gt;400\mathrm{X}$ coverage. Mutations with a true frequency of $&gt;5\%$ (95% CI: 3.05%-8.10%) can be reliably detected at $&gt;300\mathrm{X}$ (Figure 1).

Figure 1. Power analysis of mutation detection based on exon coverage. Mutations were separated into 4 types based on variant allele frequency (True VAF = 20%, 10%, 5%, and 2%). Lower dotted line indicates a 90% sensitivity in detecting variants and the upper dotted line indicates the 95% sensitivity threshold.

Summary metrics were computed for individual exons within a sample cohort of 3000 normal tissue samples to pinpoint regions prone to sequencing artifacts. These specific regions were subsequently eliminated from the GENESEEQPRIME assay, rendering

{15}

them ineligible for variant analysis or inclusion in reports. No variants with explicit evidence of clinical significance or somatic hotspot mutations are withheld from the report.

Sequence coverage requirement was evaluated in the remaining regions across a cohort of 200 FFPE samples, and $90\%$ of targeted regions ( $\sim 4,756$ of 5,286 regions) were sequenced to a depth of $100\mathrm{X}$ or greater if the deduplicated sample level coverage is $200\mathrm{X}$ . If the deduplicated sample level coverage is $400\mathrm{X}$ , $90\%$ of regions of interest achieved $200\mathrm{X}$ coverage or greater (Figure 2).

A

B

{16}

Figure 2. Percent of regions of interest achieving A) 100X or B) 200X coverage in correlation to sample level depth. Horizontal dotted line indicates 90 percent of regions. The leftmost vertical dotted line in each figure indicates the sample level average deduplicated depth of 200X. The rightmost vertical dotted line in each figure indicates the sample level average deduplicated depth of 400X.

## 2. Sample coverage

Sample level summary metrics of 200 FFPE samples from across 10 tumor types were evaluated to determine the required sample coverage for report generation and analysis. Overall exonic coverage was high in the regions of interest with a high percentage of on target reads. The mean coverage across all targeted regions for the 200 FFPE samples is 978X (± 245X standard deviation).

The assessment of sequence coverage was extended to determine the minimum requirements for the analysis and reporting of variants. A power analysis indicated that a minimum sequence coverage of 100x is essential for accurately detecting mutations with an underlying mutation frequency of 2% or higher. To establish a per-sample threshold, the analysis considered the number of exons in individual samples meeting this coverage criterion. The samples under evaluation encompassed a spectrum of DNA quality estimates. Of the 200 samples evaluated, &gt;96.5% of samples (193 of 200 samples) demonstrated ≥ 100x coverage across at least 90% of targeted regions of interest (Figure 3 and Figure 4). The consistently robust coverage affirms the capacity to tolerate occasional dips in coverage that might arise due to fluctuations in sample quality. Based on this analysis, the threshold sample coverage for analysis and report generation is set at 100X in at least 90% of evaluated regions. This criterion is employed to assess whether a sample has been sequenced deeply enough to warrant analysis and inclusion in the reporting process.

13

{17}


Figure 3. Distribution of mean distinct coverage per sample in GENESEEQPRIME across 200 FFPE samples.
Figure 4. Distribution of mean deduplicated depth per sample and percentage of regions with more than $100\mathrm{X}$ coverage. Dotted line indicates the cut-off of $90\%$ regions with more than $100\mathrm{X}$ coverage.

{18}

15

3. **Determination of assay cutoff**

To ensure high specificity in mutation detection, 30 normal FFPE samples were analyzed under varying stringency levels, initially yielding 273 false positives (209 non-hotspot, 58 hotspot, 6 clinically significant variants). Applying mutation-specific filters—VAF and supporting read thresholds tailored to each category—eliminated all false positives, achieving a 100% rejection rate. Clinically significant variants are reported by GENESEEQPRIME when VAF is ≥0.4% with ≥4 supporting reads, hotspot variants (from COSMIC v96 and internal databases) at ≥1% VAF with ≥4 reads, and non-hotspot variants at ≥2% VAF with ≥5 reads. For ERBB2 copy number variation, the assay reports amplification only when fold change exceeds 1.8x relative to diploid state, based on a cut-off set at 4 standard deviations above the mean from 46 ERBB2-negative samples. Microsatellite instability (MSI) status is determined by the proportion of somatic sites among total tracked sites, with a validated cut-off set at 16% based on ROC analysis of 100 samples. For gene translocations (ALK, RET, ROS1, and NTRK1), baseline fusion read counts from 39 FFPE-negative samples were used to establish detection thresholds: ≥6 reads for canonical partners and ≥12 reads for non-canonical partners.

4. **Somatic Mutation Detection by Tumor only Sequencing (ToSeq) Module**

The GENESIS software includes a machine learning module, ToSeq, which distinguishes somatic from germline SNVs and indels without matched normal samples by evaluating features related to variant location, frequency, pathogenicity, and database presence. Trained on 3,189 tumor-normal pairs and tested on 4,781 independent pairs (7,970 total), ToSeq uses a Distributed Random Forest model that achieved high performance (SNV AUC = 0.9867; indel AUC = 0.9818). In total, 132,734 SNVs and 11,086 indels were assessed, with the ToSeq module demonstrating 95.59% (95% CI: 95.40% - 95.78%) sensitivity and 95.63% (95% CI: 95.43% - 95.82%) positive predictive value against reference classifications.

D. **Substantial Equivalence Information**

1. **Predicate Device Name(s):**

PGDx elio Tissue Complete

2. **Predicate Device 510(k) Number**

K192063

3. **Comparison of technological characteristics with the predicate device**

| Characteristics | Predicate device: PGDx elio tissue complete | Subject Device: GENESEEQPRIME NGS tumor profiling assay (FFPE) |
| --- | --- | --- |
| Similarities | | |

{19}

| Indications for Use | The PGDx elio™ tissue complete assay is a qualitative in vitro diagnostic device that uses targeted next generation sequencing of DNA isolated from formalin-fixed, paraffin-embedded tumor tissue from patients with solid malignant neoplasms to detect tumor gene alterations in a broad multi-gene panel.PGDx elio tissue complete is intended to provide tumor mutation profiling information on somatic alterations (SNVs, small insertions and deletions, one amplification and four translocations), microsatellite instability (MSI) and tumor mutation burden (TMB) for use by qualified healthcare professionals in accordance with professional guidelines in oncology for previously diagnosed cancer patients and is not conclusive or prescriptive for labeled use of any specific therapeutic product. | The GENESEEQPRIME NGS Tumor Profiling Assay (FFPE) is a qualitative in vitro diagnostic test kit that uses next generation sequencing of DNA isolated from formalin-fixed paraffin-embedded tumor tissue from previously diagnosed patients with solid malignant neoplasms to detect tumor gene alterations in a broad multi-gene panel. This test is intended to provide tumor mutation profiling information on somatic variants, including single nucleotide variants (SNVs), insertions and deletions (indels), one amplification, four translocations, microsatellite instability (MSI), and tumor mutation burden (TMB).Information provided by GENESEEQPRIME NGS Tumor Profiling Assay (FFPE) is intended to be used by qualified health care professionals in accordance with professional guidelines in oncology. Results from GENESEEQPRIME NGS Tumor Profiling Assay (FFPE) are not intended to be prescriptive or conclusive for labeled use of any specific therapeutic product. |
| --- | --- | --- |
| Technology | Hybrid Capture | Same |
| Specimen Type | Formalin-fixed, paraffin-embedded (FFPE) tumor tissue from patients with solid malignant neoplasms | Same |
| Target Population | Patients with malignant solid neoplasms | Same |
| Instrument | Illumina NextSeq 550Dx (qualified by PGDx) | Illumina NextSeq 550Dx |
| Test Environment | Kit | Same |
| Differences | | |
| Genes on Panel | 505 genes | 425 genes |
| Variant Types | Somatic Variants including point mutations, small insertions, and small deletions, ERBB2 amplification, 4 | Somatic Variants including point mutations, small insertions, and small deletions, ERBB2 amplification, 4 gene |
| | intrusion, 4 and 5 | intrusion, 4 and 5 |
| Variants | 122 | 122 |

{20}

| | gene translocations (ALK, RET, NTRK2, and NTRK3), MSI and TMB information. | translocations (ALK, RET, NTRK1, and ROS1), MSI and TMB information. |
| --- | --- | --- |
| Black List | 58 genes/exons excluded from reporting due to consistently low coverage and low complexity, and repeat genomic regions in 254 genes | 7 regions from 5 genes and 268 variants from 135 genes are excluded from reporting as recurrent artifacts based on next generation sequencing results of normal samples (blood or FFPE) |
| Determination of Pipeline Threshold | Sequence coverage of >400x provides 95% statistical power for detection of true mutations at 2% MAF (95% CI, 0.8% - 3.5% MAF). For mutations with 5% underlying MAF, sequence coverage of >150x provides 95% statistical power for detection (95% CI, 2.0% - 8.6% MAF). | Sequencing coverage of >400x provides 95% statistical power for detection of true mutations at 2% VAF (95% CI, 1.9% - 2.5% VAF). For mutations with 5% underlying VAF, sequencing coverage of >300x provides 95% statistical power for detection (95% CI, 12.0%, 22.0% VAF, Mean VAF = 17.0%). |
| Assay cut-off | A minimum of 4 or 6 mutant observations and 0.4%, 2%, or 5% mutant allele fraction (MAF) are required depending on sequence coverage and status of the variant as a Variant with Evidence of Clinical Significance, somatic hotspot, or a Variant with Potential Clinical Significance. SNVs with lower bound 95% Confidence Interval <5% MAF based on sequence coverage are excluded from reporting. Common germline mutations present in dbSNP, ExAC, and gnomAD are identified and excluded from reporting. Additional germline mutations with ≥ 3 matches in ExAC and MAF ≥ 20% are also excluded from reporting. | A minimum of 4 or 5 mutant observations and 0.4%, 1%, or 2% variant allele fraction (VAF) are required depending on sequence coverage and status of the variant as a Variant with Evidence of Clinical Significance, somatic hotspot, or a Variant with Potential Clinical Significance. SNVs and Indels <2% VAF based on sequence coverage are excluded from reporting. Common germline mutations present in 1000g, ExAC, and gnomAD are identified and excluded from reporting. GENESIS software includes a self-developed machine learning model used to distinguish germline SNVs and Indels from somatic SNVs and Indels without the need for a matched normal sample. |
| Controls | • Positive Control • No template control (NTC) • Normalized to database of common germline SNPs | • Positive Control • Negative Control • Normalized to database of common germline SNPs |
| Samples per Run (controls excluded) | 15 | 28 |

{21}

18

| Clinical Evidence Curation | Variant calls are organized into Variants with Evidence of Clinical Significance or Variants with Potential Clinical Significance; with Variants with Evidence of Clinical Significance aligning with Tier 1A of the AMP/ASCO/CAP guidelines, based on the selected tumor type for use in tumor profiling. Tumor type selection should align with the clinical diagnosis and all available information. In the case of metastasis of unknown origin, unknown primary site, or uncertainty of the tumor type, ‘Other’ should be selected. | GENESEEQPRIME assay reports variants as either a Variant with Evidence of Clinical Significance or a Variant with Potential Clinical Significance depending on the designated cancer type. Classification of variants adhere to the three-tiered approach for reporting biomarkers as outlined by the CDRH. Variants with evidence of clinical significance fall within tier 1A of the AMP/ASCO/CAP guidelines. |
| --- | --- | --- |

## E. Guidance Documents Referenced

1. CDRH'S APPROACH TO TUMOR PROFILING NEXT GENERATION SEQUENCING TESTS (2017)
2. General Principles of Software Validation; Final Guidance for Industry and FDA Staff (January 11, 2002)
3. Guidance for Industry Cybersecurity for Networked Medical Devices Containing Off-the-Shelf (OTS) Software (January 14, 2005)
4. Guidance for Industry and FDA Staff Statistical Guidance on Reporting Results from Studies Evaluating Diagnostic Tests (March 13, 2007)
5. Content of Premarket Submissions for Management of Cybersecurity in Medical Devices Guidance for Industry and Food and Drug Administration Staff (October 2, 2014)
6. Postmarket Management of Cybersecurity in Medical Devices Guidance for Industry and Food and Drug Administration Staff (December 28, 2016)
7. Content of Premarket Submissions for Device Software Functions Guidance for Industry and Food and Drug Administration Staff (June 14, 2023)
8. Off-The-Shelf Software Use in Medical Devices Guidance for Industry and Food and Drug Administration Staff (August 11, 2023)

## F. Performance Characteristics

### 1. Introduction

This document provides a comprehensive summary of the accuracy, reproducibility, and analytical performance of the GENESEEQPPRIME assay. The outlined performance

{22}

studies evaluate the assay's ability to deliver consistent and accurate results across diverse clinical and laboratory settings.

# 2. Accuracy Study: Method Comparison

Concordance of variant calls, translocation and ERBB2 detection, microsatellite status, and TMB were compared between appropriate comparator methods and GENESEEQPRIME. SNVs, insertions, and deletions are compared to an externally validated orthogonal NGS method. Gene translocations, CNV, and MSI are compared to the corresponding FISH or IHC test as well as an orthogonal NGS method. TMB is compared to whole exome sequencing (WES) and an orthogonal NGS method. In total, there were 503 FFPE samples in accuracy studies. For rare variants not commonly represented in consecutive sampling, samples were identified based on the variant information provided by the Biobank's pre-existing test results. Ethnic origin of sample included in the method comparison study is summarized in Table 7. Samples originated from multiple sites. Samples cover 40 distinct tumor types. Detailed sample invalid rate for each cancer type post-sequencing is presented in Table 8. Five samples failed pre-sequencing quality control for GENESEEQPRIME. No samples were pre-screened using GENESEEQPRIME results.

Table 7. Distribution of ethnic background of all samples used in the method comparison study between GENESEEQPRIME and the orthogonal NGS device.

| Ethnicity | Accuracy | Proportion |
| --- | --- | --- |
| White or Caucasian | 345 | 68.59% |
| Asian | 108 | 21.47% |
| Black or African American | 29 | 5.77% |
| Hispanic or Latino | 10 | 1.99% |
| Native American or Alaskan Native | 1 | 0.20% |
| Other | 8 | 1.59% |
| Unknown | 2 | 0.40% |
| Sum | 503 | 100.00% |

Table 8. Overall post-sequencing sample pass rate for each assay used in the accuracy study sorted by cancer type.

| Cancer Type | GENESEEQPRIME | | |
| --- | --- | --- | --- |
| | Samples passed QC | Total samples tested | Invalid rate |
| Bladder Cancer | 16 | 17 | 5.88% |
| Breast Cancer | 83 | 91 | 8.79% |
| Cervical Cancer | 10 | 11 | 9.09% |
| Cholangiocarcinoma | 3 | 3 | 0.00% |
| Colorectal Cancer | 71 | 72 | 1.39% |

{23}

| Endometrial Cancer | 28 | 30 | 6.67% |
| --- | --- | --- | --- |
| Esophageal Cancer | 5 | 6 | 16.67% |
| Gastric Cancer | 14 | 15 | 6.67% |
| Gastrointestinal Stromal Tumor | 3 | 4 | 25.00% |
| Head and Neck Cancer | 11 | 13 | 15.38% |
| Liver Cancer | 11 | 11 | 0.00% |
| Lung Adenocarcinoma | 8 | 8 | 0.00% |
| Melanoma | 13 | 15 | 13.33% |
| Non-small cell lung cancer | 64 | 66 | 3.03% |
| Others* | 28 | 30 | 6.67% |
| Lung adenosquamous carcinoma | 3 | 3 | 0.00% |
| Ovarian Cancer | 10 | 11 | 9.09% |
| Pancreatic Cancer | 6 | 6 | 0.00% |
| Prostate Cancer | 32 | 32 | 0.00% |
| Renal Cancer | 17 | 18 | 5.56% |
| Skin Cancer | 12 | 14 | 14.29% |
| Soft Tissue Sarcoma | 7 | 8 | 12.50% |
| Thyroid Cancer | 13 | 14 | 7.14% |
| Total | 468 | 498 | 6.02% |

*Other cancer types include central nervous system cancers, fallopian tube, mediastinum, medulloblastoma, penis, testis, vulva, breast cancer (solid tumor), thymus tumor, biliary tract cancer, glioma, lung squamous cell cancer, neuroendocrine tumor, carcinosarcoma, lung-NOS**, lung-SCLC, small intestine cancer, and head and neck cancer.
**NOS: Not otherwise specified

# a) Accuracy for SNVs, Indels, CNV, gene translocations, TMB, MSI

Data was aggregated at the variant level for SNVs and indels, gene level for amplification and translocations, and case level for MSI and TMB. The accuracy is summarized for the entire cohort of 503 samples for each of the assessed types (SNVs, indels, structural variants, MSI, and TMB). Orthogonal methods used consisted of validated Next Generation Sequencing (NGS), Fluorescence In Situ Hybridization (FISH), immunohistochemistry (IHC), and whole exome sequencing (WES). For all analyses, the PPA and NPA were calculated by comparing the concordance between the GENESEEQPRIME Assay and the appropriate comparator to evaluate the degree of concordance between the assays (Table 9).

{24}

Table 9. Accuracy of GENESEEQPRIME

| Variant Category | Orthogonal Method | Analysis Category | PPA (2-sided 95% CI) | NPA (2-sided 95% CI) |
| --- | --- | --- | --- | --- |
| Overall (SNVs and indels) | Predicate Device (NGS) | All | 92.44% (91.45%, 93.33%) | 99.99% (99.99%, 99.99%) |
| | | SNVs | 91.53% (90.39%, 92.55%) | 99.99% (99.99%, 99.99%) |
| | | Insertions | 96.64% (91.68%,98.69%) | 99.99% (99.99%, 99.99%) |
| | | Deletions | 97.31% (95.12%,98.53%) | 99.99% (99.99%, 99.99%) |
| Variants with Evidence of Clinical Significance | Predicate Device (NGS) | All | 96.53% (92.64%, 98.4%) | 99.98% (99.97%, 99.98%) |
| | | SNVs | 96.15% (91.86%, 98.23%) | 99.98% (99.97%, 99.98%) |
| | | Insertions | 100% (51.01%, 100%) | 99.99% (99.96%, 99.99%) |
| | | Deletions | 100% (77.19%, 100%) | 99.97% (99.96%, 99.98%) |
| Variants with Potential of Clinical Significance | Predicate Device (NGS) | All | 92.19% (91.15%, 93.12%) | 99.99% (99.99%, 99.99%) |
| | | SNVs | 91.23% (90.03%, 92.30%) | 99.99% (99.99%, 99.99%) |
| | | Insertions | 96.52% (111/115) (91.40%, 98.64%) | 99.99% (99.99%, 99.99%) |
| | | Deletions | 97.21% (349/359) (94.95%, 98.48%) | 99.99% (99.99%, 99.99%) |
| Hotspot Variants | Predicate Device (NGS) | All | 98.08% (512/522) (96.51%, 98.96%) | 99.98% (99.97%, 99.98%) |
| | | SNVs | 97.89% (96.16%, 98.85%) | 99.98% (99.97%, 99.98%) |
| | | Insertions | 100% (79.61%, 100%) | 99.99% (99.96%, 99.99%) |
| | | Deletions | 100% (89.57%, 100%) | 99.97% (99.96%, 99.98%) |
| Non-Hotspot Variants | Predicate Device (NGS) | All | 91.27% (90.10%, 92.31%) | 99.99% (99.99%, 99.99%) |
| | | SNVs | 90.08% (88.72%, 91.29%) | 99.99% (99.99%, 99.99%) |
| | | Insertions | 96.15% (90.53%, 98.49%) | 99.99% (99.99%, 99.99%) |
| | | Deletions | 97.05% (94.65%, 98.39%) | 99.99% (99.99%, 99.99%) |
| ERBB2 | FISH | Amplification | 93.75% (83.16%, 97.85%) | 100% (88.30%, 100%) |

{25}

| ALK | FISH | Translocation | 88.89% (56.50%, 99.43%) | 90.91% (62.26%, 99.53%) |
| --- | --- | --- | --- | --- |
| RET | FISH | Translocation | 100% (56.55%, 100%) | 66.67% (41.71%, 84.82%) |
| ROS1 | FISH | Translocation | 100% (67.56%, 100%) | 100% (72.24%, 100%) |
| NTRK1 | NGS Comparator Assay | Translocation | 100% (43.85%, 100%) | 99.76% (98.66%, 99.99%) |
| MSI | IHC/dMMR | All Tumor Types | 97.50% (87.12%,99.87%) | 90.38% (79.39%, 95.82%) |
| MSI | IHC/dMMR | CRC or Endometrial | 100% (89.85%, 100%) | 96.88% (84.26%, 99.84%) |

# i. Germline mutation filtering pipeline

The objective of this analysis is to compare the efficacy of GENESEEQPRIME at filtering out germline mutations in comparison to using databases alone. For comparison, we also obtained data from an orthogonal NGS method. Samples used in the method comparison study were filtered for matching normal sample (buffy coat of adjacent FFPE). 11 FFPE samples with matching normal samples were selected to undergo this analysis. Samples originate from 6 different cancer types and includes colorectal cancer, gastric cancer, head and neck cancer, non-small cell lung cancer, breast cancer, and endometrial cancer (Table 10). The mutation results of each tumor-normal sample pair are compared to identify the reference list of germline and somatic mutations. In these 11 samples, 292 somatic variants were reported in total from the GENESEEQPRIME assay. 8 variants were found to be germline variants based on comparison to the normal control sample (2.74% false positive rate). The orthogonal device reported 276 somatic mutations that were also covered on the GENESEEQPRIME panel. Of those, 38 were determined to be germline variants (13.77% false positive rate). For each sample, 2 to 7 variants were reported by the orthogonal device and were detected but not reported by the GENESEEQPRIME assay. These variants were all found to be present in the germline control sample, indicating that the GENESEEQPRIME assay successfully filtered out germline variants using only tumor samples. Based on the findings from this analysis, we determine that the GENESEEQPRIME is efficient at filtering out germline variants and has germline variants reporting rate 2.74%. In the samples tested, 100% of variants reported by the orthogonal device and filtered out by the GENESEEQPRIME were germline mutations.

{26}

Table 10. Summary of germline variants found in each tumor or germline control sample using GENESEEQPRIME.

| Sample ID | Germline control type | Cancer type | GENESEEQ PRIME reported variants | Orthogonal reported variants on GENESEE Q PRIME targetable range | Number of variants found in germline control |
| --- | --- | --- | --- | --- | --- |
| Sample 1 | Buffy Coat | Colorectal Cancer | 38 | 29 | 3 |
| Sample 2 | Buffy Coat | Gastric Cancer | 6 | 11 | 7 |
| Sample 3 | Buffy Coat | Head and Neck Cancer | 8 | 12 | 3 |
| Sample 4 | Tumor adjacent tissue | Colorectal Cancer | 150 | 137 | 2 |
| Sample 5 | Tumor adjacent tissue | Non-Small Cell Lung Cancer | 27 | 19 | 3 |
| Sample 6 | Buffy Coat | Breast Cancer | 4 | 6 | 2 |
| Sample 7 | Tumor adjacent tissue | Breast Cancer | 5 | 6 | 2 |
| Sample 8 | Buffy Coat | Endometrial Cancer | 37 | 33 | 3 |
| Sample 9 | Tumor adjacent tissue | Colorectal Cancer | 2 | 5 | 3 |
| Sample 10 | Buffy Coat | Breast Cancer | 8 | 12 | 2 |
| Sample 11 | Tumor adjacent tissue | Non-Small Cell Lung Cancer | 7 | 6 | 2 |

# ii. Accuracy - SNVs and Indels

Accuracy of variant calls (variants with evidence of clinical significance, hotspot SNVs, non-hotspot SNVs, insertions, deletions) was obtained using a comparison between the reported results of the GENESEEQPRIME and the predicate (NGS based assay) and was determined using positive percent agreement (PPA) and negative prevent agreement (NPA) at a variant level. The study included a total of 4789 variants including 4015 SNVs, 196 insertions, and 570 deletions. Out of 503 FFPE tumor specimens, 423 had

{27}

both predicate and GENESEEQPRIME results. Overall, the GENESEEQPRIME assay yielded concordance analytical performance for variant calls across the SNVs and Indels with a PPA $\geq 91.53\%$ and an NPA $\geq 99.00\%$ . Table 11 shows the concordance between detected mutations for each category of variants (Variants with evidence of clinical significance, variants with potential clinical significance, hotspot, non-hotspot, and overall).

Table 11. Concordance for different categories of variants between GENESEEQPRIME and the predicate device

| Variant category | Analysis category | PPA (n/N) (95%CI) | NPA (n/N) (95%CI) |
| --- | --- | --- | --- |
| Variants with evidence of clinical significance | All | 96.53% (167/173) (92.64%, 98.4%) | 99.94% (21811/21823) (99.90%, 99.97%) |
| | SNVs | 96.15% (150/156) (91.86%, 98.23%) | 99.95% (17601/17610) (99.90%, 99.97%) |
| | Insertions | 100% (4/4) (51.01%, 100%) | 99.94% (1687/1688) (99.67%, 99.99%) |
| | Deletions | 100% (13/13) (77.19%, 100%) | 99.92% (2523/2525) (99.71%, 99.98%) |
| Hotspot variants | All | 98.08% (512/522) (96.51%, 98.96%) | 99.98% (780573/780758) (99.97%, 99.98%) |
| | SNVs | 97.89% (464/474) (96.16%, 98.85%) | 99.98% (706191/706359) (99.97%, 99.98%) |
| | Insertions | 100% (15/15) (79.61%, 100%) | 99.99% (20286/20289) (99.96%, 99.99%) |
| | Deletions | 100% (33/33) (89.57%, 100%) | 99.97% (54096/54110) (99.96%, 99.98%) |
| Non-hotspot variants | All | 91.27% (2300/2520) (90.10%, 92.31%) | 99.99% (1121666711/1121668264) (99.99%, 99.99%) |
| | SNVs | 90.08% (1871/2077) (88.72%, 91.29%) | 99.99% (373440485/373441781) (99.99%, 99.99%) |
| | Insertions | 96.15% (100/104) (90.53%, 98.49%) | 99.99% (374130206/374130280) (99.99%, 99.99%) |
| | Deletions | 97.05% (329/339) (94.65%, 98.39%) | 99.99% (374096020/374096203) (99.99%, 99.99%) |
| Variants with potential clinical significance | All | 92.19% (2645/2869) (91.15%, 93.12%) | 99.99% (1122425051/1122426777) (99.99%, 99.99%) |
| | SNVs | 91.23% (2185/2395) (90.03%, 92.30%) | 99.99% (374129075/374130530) (99.99%, 99.99%) |
| | Insertions | 96.52% (111/115) (91.40%, 98.64%) | 99.99% (374148805/374148881) (99.99%, 99.99%) |

{28}

| | Deletions | 97.21% (349/359) (94.95%, 98.48%) | 99.99% (374147171/374147368) (99.99%, 99.99%) |
| --- | --- | --- | --- |
| Overall | All | 92.44% (2812/3042) (91.45%, 93.33%) | 99.99% (1120688450/1120690189) (99.99%, 99.99%) |
| | SNVs | 91.53% (2335/2551) (90.39%, 92.55%) | 99.99% (372648833/372650297) (99.99%, 99.99%) |
| | Insertions | 96.64% (115/119) (91.68%,98.69%) | 99.99% (374079851/374079928) (99.99%, 99.99%) |
| | Deletions | 97.31% (362/372) (95.12%,98.53%) | 99.99% (373959766/373959964) (99.99%, 99.99%) |
| Insertion | 1~5bp | 96.43% (108/112) (91.18%,98.60%) | 99.99% (374079866/374079935) (99.99%, 99.99%) |
| | 6~10bp | 100% (5/5) (56.55%,100%) | 99.99% (374080038/374080042) (99.99%, 99.99%) |
| | 11~20bp | 100% (2/2) (34.24%,100%) | 99.99% (374080044/374080045) (99.99%, 99.99%) |
| | 21~30bp | N/A% (0/0) (N/A, N/A) | 99.99% (374080044/374080047) (99.99%, 99.99%) |
| Deletion | 1~5bp | 97.60% (325/333) (95.33%,98.78%) | 99.98% (373959827/373960003) (99.97%, 99.98%) |
| | 6~10bp | 90.91% (10/11) (62.26%,99.53%) | 99.99% (373960319/373960325) (99.99%, 99.99%) |
| | 11~20bp | 95.83% (23/24) (79.76%,99.79%) | 99.99% (373960301/373960312) (99.99%, 99.99%) |
| | 21~30bp | 100% (4/4) (51.01%,100%) | 99.99% (373960327/373960332) (99.99%, 99.99%) |

# iii. Accuracy - ERBB2 amplification

In total, 79 different FFPE samples representing eight different tumor types, including breast cancer, colorectal cancer, endometrial cancer, stomach cancer, ovarian cancer, non-small cell lung cancer, esophagus cancer, and biliary tract cancer, were analyzed for concordance between FISH status and GENESEEQPRIME ERBB2 status. Two samples did not pass the FISH quality controls due to high background noise and were removed from the analysis. Three samples had a positive ERBB2 FISH results but were negative for ERBB2 in the GENESEEQPRIME assay. These samples were also found to be negative for ERBB2 when tested with an orthogonal NGS method. The PPA and

{29}

NPA values for ERBB2 amplification reflect the totals across borderline and nonborderline samples (Table 12). In non-borderline cases (excluding all cases of a FISH ratio 1.5 - 2.5), a PPA of $95.56\%$ (95% CI: 85.14%, 98.77%) and an NPA, PPV, and NPV at or above $91.67\%$ (95%CI: 74.15%, 97.68%) (Table 13) was observed.

When borderline FISH values (1.5-2.5) were excluded, the PPA slightly improved to $95.56\%$ , with NPA remaining at $100\%$ . Notably, in cases limited to borderline FISH values (1.5-2.5), the PPA decreased $(66.66\%)$ , while NPA stayed at $100\%$ , indicating the assay performs more reliably outside borderline ranges.

Table 12. Summary of concordance between GENESEEQPRIME assay and ERBB2 FISH assay results.

| ERBB2 amplification* | FISH | | | | |
| --- | --- | --- | --- | --- | --- |
| | | | Detected | Not detected | Total |
| GENESEEQPRIME | Detected | 45 | 0 | 45 | |
| | Not detected | 3 | 29 | 32 | |
| | Total | 48 | 29 | 77 | |
| PPA | | | 93.75% (83.16%, 97.85%) | | |
| NPA | | | 100% (88.30%, 100%) | | |
| OPA | | | 96.10% (89.16%, 98.67%) | | |

*Cancer types include breast cancer (n=48), colorectal cancer (n=12), endometrial cancer (n=4), stomach cancer (n=4), ovary cancer (n=3), NSCLC (n=3), esophagus cancer (n=2), and biliary tract cancer (n=1).

Table 13. Summary of concordance between GENESEEQPRIME assay and ERBB2 FISH borderline assay results.

| Category | Total cases | TP | FP | FN | TN | PPA (95% CI) | NPA (95% CI) | PPV (95% CI) | NPV (95% CI) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| All Cases | 77 | 45 | 0 | 3 | 29 | 93.75% (83.16%, 97.85%) | 100% (88.30%, 100%) | 100% (92.13%, 100%) | 90.63% (75.78%, 96.76%) |
| Excluding FISH 1.5-2.5 | 67 | 43 | 0 | 2 | 22 | 95.56% (85.17%, 98.77%) | 100% (85.13%, 100%) | 100% (91.80%, 100%) | 91.67% (74.15%, 97.68%) |
| Excluding FISH 1.8-2.2 | 75 | 45 | 0 | 3 | 27 | 93.75% (83.16%, 97.85%) | 100% (87.54%, 100%) | 100% (92.13%, 100%) | 90% (74.38%, 96.54%) |
| Only FISH 1.5-2.5 | 10 | 2 | 0 | 1 | 7 | 66.66% (20.77%, 98.29%) | 100% (64.57%, 100%) | 100% (32.23%, 100%) | 87.5% (52.91%, 99.36%) |
| Only FISH 1.8-2.2 | 2 | 0 | 0 | 0 | 2 | NA (NA) | 100% (32.24%, 100%) | NA (NA) | 100% (32.24%, 100%) |

TP - True Positive, FP - False Positive, FN - False Negative, TN - True Negative

{30}

In addition to comparing the analytical performance of the GENESEEQPRIME assay to results of the medically established method, the results were also compared to the predicate device results. A total of 34 ERBB2 positive samples representing seven different tumor types including, breast cancer, colorectal cancer, endometrial cancer, ovarian cancer, esophagus cancer, non-small cell lung cancer, and gallbladder cancer, were used in this concordance study to detect ERBB2 amplifications. ERBB2 amplifications were detected in 32 out of 34 samples in both the GENESEEQPRIME assay and the orthogonal device. Two samples with ERBB2 amplifications were detected but were not reported by the orthogonal device due to a difference in reporting thresholds between the two devices. Out of a total of 423 samples, 421 samples $(99.53\%)$ showed an agreement between the GENESEEQPRIME assay and the orthogonal device. The concordance between GENESEEQPRIME assay and comparator NGS-based assay in detected ERBB2 amplifications is shown in Table 14 below.

Table 14. Comparison of ERBB2 amplification for GENESEEQPRIME and orthogonal NGS device

| ERBB2 Amplification* | Orthogonal device | | | |
| --- | --- | --- | --- | --- |
| | | ERBB2 (+) | ERBB2 (-) | Total |
| GENESEEQPRIME | ERBB2 (+) | 32 | 2 | 34 |
| | ERBB2 (-) | 0 | 389 | 389 |
| | Total | 32 | 391 | 423 |
| PPA (2-sided 95% CI) | | 100% (89.28%, 100%) | | |
| NPA (2-sided 95% CI) | | 99.49% (98.15%, 99.86%) | | |
| OPA (2-sided 95% CI) | | 99.53% (98.29%, 99.87%) | | |

*Cancer type for samples determined as positive for ERBB2 amplification by either assay includes breast (n=27), colorectal (n=2), endometrial (n=1), ovary (n=1), esophagus (n=1), lung-NSCLC (n=1), and gallbladder(n=1).

# iv. Accuracy - Gene translocation concordance (ALK)

A total of 20 FFPE samples from four tumor types, including non-small cell lung cancer, renal carcinoma, colorectal cancer, and lung adenocarcinoma, were included in the analysis. Out of nine ALK positive samples, eight were detected for ALK translocations using both FISH and the GENESEEQPRIME assay. One ALK positive sample by FISH was not reported as positive by the GENESEEQPRIME assay due to a low number of supporting reads. The concordance between GENSEEQPRIME assay and FISH is shown in Table 15.

Table 15. Summary of concordance between GENESEEQPRIME assay and ALK FISH assay results.

| ALK Translocation* | FISH | | | |
| --- | --- | --- | --- | --- |
| | | ALK (+) | ALK (-) | Total |
| | ALK (+) | 8 | 1 | 9 |
| | ALK (-) | 1 | 0 | 1 |
| | ALK (-) | 1 | 1 | 1 |

{31}

In addition, a total of 10 ALK positive samples representing three different tumor types including, non-small cell lung cancer, renal cancer, and colorectal cancer, were used to assess the concordance of the GENESEEQPRIME assay to NGS based predicate device to detect ALK translocations. ALK translocations were detected in nine out of 10 samples in both the GENESEEQPRIME and the orthogonal comparator. One sample had an ALK translocation that was unreported by GENESEEQPRIME due to assay cutoff below the 6 reads required for reporting in GENESEEQPRIME. Further investigation into the analysis pipeline shows that the translocation event was detected by GENESEEQPRIME at only 5 alternate reads. The concordance results are shown in Table 16.

Table 16. Comparison of ALK translocation for GENESEEQPRIME and orthogonal device

| ALK Translocation* | Orthogonal device | | | |
| --- | --- | --- | --- | --- |
| | | ALK (+) | ALK (-) | Total |
| GENESEEQPRIME | ALK (+) | 9 | 0 | 9 |
| | ALK (-) | 1 | 413 | 414 |
| | Total | 10 | 413 | 423 |
| PPA | | 90.00% (59.59%, 99.49%) | | |
| NPA | | 100% (99.08%, 100%) | | |
| OPA | | 99.76% (98.67%, 99.99%) | | |

*Cancer type for samples tested positive for gene translocations by either assay includes lung-NSCLC (n=8), renal (n=1), and colorectal (n=1).

# v. Accuracy - Gene translocation concordance (RET)

Twenty (20) FFPE samples from 4 tumor types (non-small cell lung cancer, thyroid cancer, colorectal cancer, and sarcoma) were selected for this analysis. All samples that were found to be positive for $RET$ rearrangement using the FISH assay were also detected by GENESEEQPRIME (5/5). NPA for the detection of $RET$ rearrangement was $66.67\%$ (10/15). Table 17 shows the results of the analysis in a 2 by 2 matrix.

It is likely that the false positive detection rate for $RET$ translocation is due to the difference in sensitivity in the assays; 3 of the 5 false positive $RET$ translocations were detected using three separate NGS methods (GENESEEQPRIME, orthogonal NGS

{32}

method, biosample repository report) (Table 18). One discordant sample was detected by both GEENSEEQPRIME and the biosample repository, but failed QC for the orthogonal method. Another discordant sample was detected by both GENESEEQPRIME and FISH but was not reported as positive by the biosample repository method. These additional results suggest that the false positive rate for $RET$ translocation might be due to the differences in sensitivity between the GENESEEQPRIME assay and FISH, with fewer supporting reads compared to traditional FISH assays.

Table 17. Summary of concordance between GENESEEQPRIME assay and RET FISH assay results.

| RET Translocation* | FISH | | | |
| --- | --- | --- | --- | --- |
| | | RET (+) | RET (-) | Total |
| GENESEEQPRIME | RET (+) | 5 | 5 | 10 |
| | RET (-) | 0 | 10 | 10 |
| | Total | 5 | 15 | 20 |
| PPA | | 100% (56.55%, 100%) | | |
| NPA | | 66.67% (41.71%, 84.82%) | | |
| OPA | | 75.00% (53.13%, 88.81%) | | |

*Cancer types include NSCLC (n=11), thyroid cancer (n=6), colorectal cancer (n=2), and sarcoma (n=1).

Table 18. Detection status of RET translocations in 5 discordant FFPE samples using 3 NGS methods

| Sample ID | Cancer Type | RET FISH Test Result | GENESEEQPRIME gene translocation Call | Orthogonal method gene translocation Call | Biorepository gene translocation call |
| --- | --- | --- | --- | --- | --- |
| Sample 1 | Lung-NSCLC | Negative | Detected RET: exon6~RET: exon12 (23 supporting reads) | Detected RET-RET (21 supporting reads) | Not detected |
| Sample 2 | Lung-NSCLC | Negative | Detected CCDC6: exon1~RET: exon12 (84 supporting reads) | Failed QC | Detected CCDC6:exon1 ~RET:exon12 |
| Sample 3 | Thyroid | Negative | Detected NCOA4: exon7~RET: exon12 (239 supporting reads) | Detected NCOA4-RET (186 supporting reads) | Detected NCOA4:exon7 ~RET:exon12 |

{33}

In addition, a total of 10 RET positive samples representing five different tumor types including, non-small cell lung cancer, thyroid cancer, colorectal cancer, and neuroendocrine tumor, were used to compare the results of the GENESEEQPRIME assay to the results of the validated orthogonal NGS assay (predicate device) to detect RET translocations. The GENESEEQPRIME detected RET translocations in seven out of seven samples that were positive by the orthogonal NGS comparator (Table 19). Three samples with RET translocations reported by GENESEEQPRIME were negative for RET gene translocation in the orthogonal device report.

Table 19. Comparison of RET translocation for GENESEEQPRIME and orthogonal device

| RET Translocation* | Orthogonal device | | | |
| --- | --- | --- | --- | --- |
| | | Gene translocation | No gene translocation | Total |
| GENESEEQPRIME | Gene translocation | 7 | 3 | 10 |
| | No gene translocation | 0 | 413 | 413 |
| | Total | 7 | 416 | 423 |
| PPA | | 100% (64.57%, 100%) | | |
| NPA | | 99.28% (97.90%, 99.75%) | | |
| OPA | | 99.29% (97.94%, 99.76%) | | |

*Cancer type for samples tested positive for gene translocations by either assay includes lung-NSCLC (n =4), thyroid (n =2), colorectal (n =2), neuroendocrine tumor (n =1), and sarcoma (n =1).

# vi. Accuracy - Gene translocation concordance (ROS1)

The accuracy of the GENESEEQPRIME assay for calling ROS1 translocation of samples is evaluated in comparison to the orthogonal method (ROS1 FISH). Eight (8) out of 8 ROS1 positive samples and 10 out of 10 negative RET samples were concordant between the GENESEEQPRIME and FISH assay. No samples failed for either assay. PPA of ROS1 gene translocation is found to be $100\%$ (8/8). Negative variant call concordance is $100\%$ (10/10). Table 20 shows the results of the analysis in a 2 by 2 matrix. The results from the above assessment show that the GENESEEQPRIME is highly concordant to

{34}

FISH results for detecting ROS1 translocations.

Table 20. Summary of concordance between GENESEEQPRIME assay and ROS1 FISH assay results.

| ROS1 Translocation* | FISH | | | |
| --- | --- | --- | --- | --- |
| | | ROS1 (+) | ROS1 (-) | Total |
| GENESEEQPRIME | ROS1 (+) | 8 | 0 | 8 |
| | ROS1 (-) | 0 | 10 | 10 |
| | Total | 8 | 10 | 18 |
| PPA | | 100% (67.56%, 100%) | | |
| NPA | | 100% (72.24%, 100%) | | |
| OPA | | 100% (82.41%, 100%) | | |

*Cancer type include NSCLC (n=18).

In addition, ROS1 positive samples representing two different tumor types including, non-small lung cancer and breast cancer, were used to evaluate concordance between the GENESEEQPRIME assay and the orthogonal validated NGS comparator assay to detect ROS1 translocations. For ROS1 translocations, comparisons between the assays show a PPA of $100\%$ for ROS1 (6/6) translocations, as well as an NPA of $99.28\%$ (414/417) (Table 21). ROS1 positive FISH results were obtained for two of the three samples with discordant results.

Table 21. Comparison of ROS1 translocation for GENESEEQPRIME and orthogonal device

| ROS1 Translocation | Orthogonal NGS Comparator | | | |
| --- | --- | --- | --- | --- |
| | | Gene translocation | No gene translocation | Total |
| GENESEEQPRIME | Gene translocation | 6 | 3 | 9 |
| | No gene translocation | 0 | 414 | 414 |
| | Total | 6 | 417 | 423 |
| PPA | | 100% (60.97%, 100%) | | |
| NPA | | 99.28% (97.91%, 99.76%) | | |
| OPA | | 99.29% (97.94%, 99.76%) | | |

*Cancer type for samples tested positive for gene translocations by either assay includes lung-NSCLC (n=8) and breast cancer (n=1)

# vii. Gene translocation concordance (NTRK1)

A total of four (4) NTRK1 positive samples representing two different tumor types including, colorectal cancer, were used to assess the analytical accuracy of the

{35}

GENESEEQPRIME assay to detect NTRK1 translocations. GENESEEQPRIME assay results were compared to validated orthogonal NGS assay. For NTRK1 translocations, comparisons between the assays show a PPA of $100\%$ for NTRK1 (3/3) translocations, as well as an NPA $99.76\%$ (419/420) (Table 21). One sample was reported positive by GENESEEQPRIME that was reported negative for NTRK1 translocation by the orthogonal comparator. The concordance between the detected NTRK1 translocations is shown in Table 22.

Table 22. Comparison of NTRK1 translocation for GENESEEQPRIME and orthogonal device

| NTRK1 Translocation | Orthogonal NGS Comparator | | | |
| --- | --- | --- | --- | --- |
| | | Gene translocation | No gene translocation | Total |
| GENESEEQPRIME | Gene translocation | 3 | 1 | 4 |
| | No gene translocation | 0 | 419 | 419 |
| | Total | 3 | 420 | 423 |
| PPA | | 100% (43.85%, 100%) | | |
| NPA | | 99.76% (98.66%, 99.99%) | | |
| OPA | | 99.76% (98.67%, 99.99%) | | |

*Cancer type for samples tested positive for gene translocations by GENESEEQPRIME include colorectal cancer $(n = 3)$ and sarcoma $(n = 1)$ .

# viii. Accuracy - MSI status concordance

The accuracy of the GENESEEQPRIME assay for calling MSI status of samples is evaluated in comparison to the orthogonal method, validated IHC test. A total of 92 samples were used for this analysis, of which 44 were colorectal, 22 of endometrial, 6 of stomach cancer, 2 of cervix, melanoma, skin, bladder, and thyroid, 1 of prostrate, kidney, pancreas, NSCLC, ovary, breast, hand and neck, and small intestine cancer. No samples failed QC for either assay. The positive agreement of GENESEEQPRIME in detecting microsatellite instability in comparison to IHC results is $97.50\%$ (39/40). Negative agreement for the two assays is $90.38\%$ (47/52). Table 23 shows the results of the analysis in a 2 by 2 matrix.

Furthermore, analysis was performed to examine the concordance to IHC of GENESEEQPRIME for specifically colorectal cancer and endometrial cancer samples, as these two tumor types tend to be over- represented in the MSI high population. Separating the samples based on cancer type reveals that CRC and endometrial cancer types had an over agreement of $98.48\%$ , while other cancer types had an overall agreement of $80.77\%$ . One case of false positive MSI was detected in the CRC/endometrial samples. This sample had a score of $19.64\%$ , which is close to the cutoff of $16\%$ .

{36}

Table 23. Summary of concordance between GENESEEQPRIME assay and IHC/dMMR assay results.

| MSI status* | IHC | | | |
| --- | --- | --- | --- | --- |
| | | dMMR | Not detected | Total |
| GENESEEQPRIME | MSI-H | 39 | 5 | 44 |
| | MSS | 1 | 47 | 48 |
| | Total | 40 | 52 | 92 |
| PPA | | 97.50% (87.12%,99.87%) | | |
| NPA | | 90.38% (79.39%, 95.82%) | | |
| OPA | | 93.48% (86.49%, 96.98%) | | |
| PPA (Only CRC +Endometrial) | | 100% (89.85%, 100%) | | |
| NPA (Only CRC +Endometrial) | | 96.88% (84.26%, 99.84%) | | |
| OPA (Only CRC +Endometrial) | | 98.48% (91.90%, 99.92%) | | |

*Cancer types include colorectal (n=44), endometrial (n=22), stomach cancer (n=6), sarcoma cancer (n=2), cervix cancer (n=2), melanoma cancer (n=2), skin cancer (n=2), bladder cancer (n=2), thyroid cancer (n=2), prostate cancer (n=1), kidney cancer (n=1), pancreas cancer (n=1), NSCLC (n=1), ovary cancer (n=1), breast cancer (n=1), hand and neck cancer (n=1), and small intestine cancer (n=1).

In addition, 423 samples representing 10 different tumor types including, colorectal cancer, endometrial cancer, cervical cancer, gastric cancer, skin cancer, thyroid cancer, bladder cancer, melanoma, and brain-glioma, were part of the study samples used to assess the concordance of the GENESEEQPRIME assay to the predicate device to detect MSI status. To ensure only samples with reliable MSI calls were included in this analysis, samples with an "Indeterminate" result for MSI status were removed. Comparing between the MSI status of all samples using GENESEEQPRIME and the orthogonal assay results showed a PPA of $96.77\%$ (30/31) and an NPA of $97.67\%$ (377/386) (Table 24).

Furthermore, the samples were characterized based on whether or not the tumor type was colorectal cancer (CRC) or endometrial cancer. Table 25 shows the concordance of MSI calls for GENESEEQPRIME and the orthogonal assay for all samples that belong to the CRC or endometrial tumor type. While the PPA achieved $96.15\%$ , 5 discordant cases were observed. Out of a total of 5 discordant samples, 4 of the samples (3 MSI-H and one MSS) had IHC results matching the GENESEEQPRIME MSI call. The remaining discordant sample had an MSI score of $19.64\%$ (near the threshold of $16\%$ ).

Table 24. Comparison of MSI status for GENESEEQPRIME and orthogonal device

| MSI | Orthogonal device | | | | |
| --- | --- | --- | --- | --- | --- |
| | | MSI-H | MSS | Indeterminate | Total |
| | MSI-H | 30 | 9 | 1 | 40 |
| | MSS | 1 | 377 | 1 | 379 |

{3…