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, Software as a Medical Device, Real-World Evidence

Real-World Evidence

Submission	Device	Sponsor	RWD Sources	RWE Use Summary	Key Tags
K250003 · Aug 29, 2025	GENESEEQPRIME NGS Tumor Profiling Assay (FFPE) (GS6005)	Geneseeq Technology, Inc.	Retrospective clinical FFPE tumor tissue samples	Retrospective clinical samples were used to evaluate analytical performance (pass rates) and to establish clinical accuracy by comparing the device results against orthogonal methods (NGS, FISH, IHC, WES).	Retrospective cohort; Clinical accuracy; Analytical performance; FFPE tumor tissue

Clinical Evidence

Study Design	Population	Comparator	Key Endpoints
Tumor Pass Rate Analysis; Retrospective analysis of clinical cases	Patients with >40 tumor types; Sample Size: 11,642	Not applicable for this study	Assay pass rates
Clinical Accuracy Study; Retrospective method comparison	Patients with a variety of tumor types (n=40); Sample Size: 503	Validated NGS, FISH, IHC, and WES	PPA and NPA

AI Performance

Output	Algorithm	Acceptance	Observed	Dev DS	Dev Readers	Test DS	Test Readers
Somatic single nucleotide variants (SNVs)	Automated bioinformatics pipeline for variant calling	—	PPA 91.53% (95% CI: 90.39%, 92.55%); NPA 99.99% (95% CI: 99.99%, 99.99%)	3,189 matched pairs for training; 4,781 matched pairs for testing	—	503 clinical FFPE samples	—
Insertions and deletions (Indels)	Automated bioinformatics pipeline for variant calling	—	PPA 96.64% (Insertions) / 97.31% (Deletions); NPA 99.99% (95% CI: 99.99%, 99.99%)	3,189 matched pairs for training; 4,781 matched pairs for testing	—	503 clinical FFPE samples	—
ERBB2 amplification	Normalized coverage comparison against normal controls	Fold change ≥ 1.8	PPA 93.75% (95% CI: 83.16%, 97.85%); NPA 100% (95% CI: 88.30%, 100%)	46 FFPE ERBB2-negative samples for threshold establishment	—	77 FFPE samples for concordance with FISH	—
ALK translocation	Fusion read count analysis	Fusion reads ≥ 6 (canonical) or ≥ 12 (non-canonical)	PPA 88.89% (95% CI: 56.50%, 99.43%); NPA 90.91% (95% CI: 62.26%, 99.53%)	39 FFPE samples without targeted translocations for baseline establishment	—	20 FFPE samples for concordance with FISH	—
RET translocation	Fusion read count analysis	Fusion reads ≥ 6 (canonical) or ≥ 12 (non-canonical)	PPA 100% (95% CI: 56.55%, 100%); NPA 66.67% (95% CI: 41.71%, 84.82%)	39 FFPE samples without targeted translocations for baseline establishment	—	20 FFPE samples for concordance with FISH	—
ROS1 translocation	Fusion read count analysis	Fusion reads ≥ 6 (canonical) or ≥ 12 (non-canonical)	PPA 100% (95% CI: 67.56%, 100%); NPA 100% (95% CI: 72.24%, 100%)	39 FFPE samples without targeted translocations for baseline establishment	—	18 FFPE samples for concordance with FISH	—
NTRK1 translocation	Fusion read count analysis	Fusion reads ≥ 6 (canonical) or ≥ 12 (non-canonical)	PPA 100% (95% CI: 43.85%, 100%); NPA 99.76% (95% CI: 98.66%, 99.99%)	39 FFPE samples without targeted translocations for baseline establishment	—	423 samples for concordance with NGS comparator	—
Microsatellite instability (MSI)	Mutation status analysis of 61 microsatellite sites	MSI-H: MSI Score ≥ 16; MSS: MSI Score < 16	PPA 97.50% (95% CI: 87.12%, 99.87%); NPA 90.38% (95% CI: 79.39%, 95.82%)	81 FFPE and 19 standard samples for threshold determination	—	92 cases for concordance with IHC	—
Tumor mutation burden (TMB)	Normalized variant count per megabase of coding region	—	Pearson correlation coefficient 0.9408 (vs predicate); 0.9298 (vs WES)	—	—	423 samples (vs predicate); 208 matched FFPE specimens (vs WES)	—

Indications for 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

Analytical accuracy was evaluated using 503 clinical FFPE samples across 40 tumor types, compared against orthogonal methods (NGS, FISH, IHC, WES). Overall PPA for SNVs/indels was 92.44% (95% CI: 91.45%-93.33%). ERBB2 amplification concordance with FISH was 93.75% PPA. MSI status concordance with IHC was 97.50% PPA. TMB scores showed a Pearson correlation of 0.9408 with the predicate and 0.9298 with WES. No clinical data was required for this 510(k) submission.

Technological Characteristics

Hybrid capture-based NGS assay. Materials: biotinylated DNA probes, magnetic streptavidin beads. Energy source: Illumina NextSeq 550Dx sequencer. Connectivity: standalone software (GENESIS) for analysis and reporting. Software: ML-based variant calling (ToSeq module). Sterilization: N/A (reagents). Form factor: kit-based assay.

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)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

## I. Background Information

### A. 510(k) Number

K250003

### B. Applicant

Genesee Technology Inc.

### C. Proprietary and Established Names

GENESEEQPRIME NGS Tumor Profiling Assay (FFPE)

### D. Regulatory Information

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

## II. Submission/Device Overview

### A. Purpose for submission

New device

### B. Measurand

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. A complete list of genes and their corresponding regions of interest covered by the assay can be found in Appendix A.

### C. Type of Test:

Next-Generation Sequencing Tumor Profiling Test

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### III. Intended Use/Indications for Use

#### A. Intended Use(s)

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.

#### B. Indication(s) for Use:

Same as above

#### C. Special Conditions for Use Statement(s)

Rx - For prescription use
For in vitro diagnostic use

#### D. Special Instrument Requirements

Illumina NextSeq® 550Dx Sequencer

### IV. Device/System Characteristics

#### A. Device Description

##### 1. Reagents

The GENESEEQPRIME NGS Tumor Profiling Assay (FFPE) (hereafter referred to as “GENESEEQPRIME”) is for use as part of a test system with the Illumina NextSeq 550Dx Sequencer and reagents. The included components of the GENESEEQPRIME assay are listed in Table 1. Geneseeq provided components include reagent kits and software for data analysis. The assay contains reagents for two full sequencing runs (i.e. 28 samples plus two external controls). Materials required but not provide are described below. A detailed list of required instruments, software reagents, consumables, and storage conditions are described in the product labeling (GENESEEQPRIME NGS Tumor Profiling Assay User Manual).

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Table 1. GENESEEQPRIME Kit: Reagents 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 |
| 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 |

## 2. Material Required but Not Provided

A list of materials required for upstream preparation of samples for sequencing, but not provided, is included as part of the GENESEEQPRIME assay is shown in Table 2. For a detailed list of reagents and consumables refer to the product labeling (GENESEEQPRIME NGS Tumor Profiling Assay User Manual).

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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 |
| 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, 20024908/20024905 or 20028871 |
| Disposable Pipet Basin | User's choice |
| Aerosol Barrier, Nuclease-free, Low Retention Sterile Pipette Tips (1000ul, 200ul, 20ul, 10ul) | User's choice |
| Nuclease-free, microcentrifuge tubes for preparing master mixes | User's choice |
| dsDNA quantification assay tubes | User's choice |
| Appropriate PPE | User's choice |

### 3. GENESIS Software

The GENESIS by GENESEEQ 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

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sequencing data. The software saves sample information and reports only and does not provide backup of raw sequencing data.

GENESIS Software is designed to prevent the use of unqualified instruments and reagent kits. Improper use will result in no report being generated.

# 4. Instrument

The GENESEEQPRIME is validated for use on the Illumina NextSeq 550Dx sequencing platform.

Other required equipment and specifications for the specific equipment not included in the GENESEEQPRIME assay are described in Table 3.

Table 3. Other Required Equipment, 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. |
| 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. |

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| Equipment | Notes |
| --- | --- |
| 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. |

### 5. Sample Preparation

The GENESEEQPRIME assay requires genomic DNA isolated from formalin- fixed paraffin-embedded (FFPE) tissue specimens using a validated commercially available DNA extraction method (column-based or beads-based). The GENESEEQPRIME assay has been validated with FFPE sample stored at room temperature (15 – 25°C) for up to 5 years. The recommended number of FFPE sections of each sample for DNA extraction are shown in Table 4.

Table 4. Tissue Sample Size and FFPE Section Specifications 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 |

The tumor volume and minimum tumor content needed to obtain sufficient DNA for testing to achieve stated performance is at least 20% (Table 5). If the specimen contains less than 20% tumor content, the tissue should be macro-dissected to select as much viable tumor as possible to minimize the amount of adjacent non-tumor tissue.

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Table 5. Specimen Handling and Processing for Validated Specimen Types

| Tissue Type | Volume | Minimum Tumor Proportion | Macrodissection Requirements (Based on tumor proportion) | 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 |

### 6. DNA Extraction

The GENESEEQPRIME assay requires genomic DNA isolated from FFPE tissue using an appropriate commercially available DNA extraction method (magnetic bead-based and spin column-based). DNA extraction kits should be able to yield 50 ng of DNA with a minimum concentration of 1 ng/μL. The concentration of the extracted genomic DNA can be measured by a fluorescence quantification method. The recommended DNA input for GENESEEQPRIME is 100 ng of total DNA recovered from tissue with a minimum 20% viable tumor nuclei. While recommended DNA input for the assay is 100 ng, results can be obtained with DNA inputs down to 50 ng. The assay has been validated with extracted DNA stored at -20°C – 15°C for up to 12 months.

### 7. Library Preparation

Illumina compatible DNA adaptors need to be added onto the end of fragmented DNA. Each adaptor contains unique index composed of eight nucleotides to distinguish one sample from another, which enables multiplexing and sequencing multiple libraries in one sequencing run.

The GENESEEQPRIME assay workflow begins with genomic DNA. Genomic DNA is quantified using a fluorometer. DNA molecules are mechanically sheard to a target size of ~300 bp and subjected to a magnetic bead purification step to remove smaller fragments and perform an exchange of buffer. Fragmented DNA is end-repaired, phosphorylated, and adenylated. Index adaptors are then ligated to the A-tailed DNA molecules. Unincorporated adaptors and reagents are removed by magnetic bead purification. Adaptor-ligated DNA is enriched by PCR amplification. Primer dimers and residual reagents are removed by magnetic bead purification. Sample libraries must be ≥ 10 ng/μL (≥ 200 ng total amount) prior to proceeding to hybridization / target enrichment.

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# 8. Hybrid Capture NGS

The adapter-ligated libraries are pooled together, denatured by heating, and subjected to hybridization with biotinylated probes (single-stranded DNA oligonucleotides) in a length of ~120 bp. Targeted regions are captured using magnetic streptavidin coated beads. Captured libraries are enriched by PCR amplification. Primer dimers and residual reagents are removed by magnetic bead purification. Final library quality is assessed using a library quantification kit the prior to sequencing. A total output volume of > 9 ng at a concentration of ≥ 0.5 ng/μL indicates a successful targeted enrichment.

# 9. Sequencing

Sequencing libraries prepared from this kit are sequenced on Illumina NextSeq 550Dx sequencing platforms. Sample libraries are quantified and normalized into a sequencing pool of up to eight samples and the controls. A maximum of 28 samples with one positive control and one negative control can be run in one sequencing batch. Pooled sample libraries are quantified using a qPCR method, loaded on a sequencing flow cell, and sequenced.

# 10. Data Analysis

a) Data Management System (DMS): Sequencing data is automatically 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 and FASTQ Generation: Demultiplexing software generates FASTQ files containing sequence reads and base quality score information. The FASTQ formatted files are used for subsequent processing of samples.
c) Run QC Check: Cluster density and the Q30 read proportion are used to determine the quality control for each sequencing run. Cluster density measures the number of clusters on a flow cell, with a sequencer cluster density threshold of ≥ 135. The proportion of total reads with a Q score of at least 30 (Q30) must be ≥ 80% for each run.
d) Read Alignment and BAM Generation: Genomic alignment is performed to map sequence reads for each sample to the human reference genome (hg19/GRCh37). Alignments are saved as Binary Alignment Map (BAM) formatted files, which contain read placement information relative to the reference genome with quality scores. Aligned BAM files are further processed in a pipeline to identify genomic alterations.
e) Sample QC Checks: Samples are checked for potential contamination through a bioinformatic analysis of genome haplotypes, based on analysis of pre-defined SNP sites that are representative of populations and individuals. Samples containing multiple haplotypes are considered potentially contaminated. Samples with ≥ 4% contamination are flagged as failed. Sequence coverage is evaluated across the panel requiring ≥ 90% of targeted regions have a minimum overage > 100x.

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f) Mutation Calling: A fully automated pipeline for bioinformation analysis is used to identify genomic alterations, including SNVs, indels, select amplification and translocations, MSI, and TMB.

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 SNV and indel, at least 2% allele frequency and five mutant reads is required. For a hotspot variant, at least a 1% variant allele frequency (VAF) and four supporting mutant reads is required.

ii) Amplifications: The assay is validated to detect only ERBB2 amplifications. ERBB2 amplifications are reported when a > 1.8 change is observed.

iii) Translocations: The assay is validated to report four translocations only, ALK, RET, ROS1, and NTRK1. At least six fusion supporting reads are required for translocation with a hotspot gene and its canonical partner; and 12 fusion supporting reads are required for translocations with a hotspot gene and its non-canonical partner.

iv) Microsatellite Status: Microsatellite instability is assessed from the mutation status of 61 microsatellite sites from within the region 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. A sample is deemed to be microsatellite stable (MSS) when < 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 a sequence mutation is performed to exclude low mutant allele fraction mutations (< 2% VAF, non-hotspot variants, < 1% VAF, hotspot variants, < 0.4% VAF clinically significant variants), common somatic driver mutations, and common germline mutations. Both synonymous and non-synonymous alterations are considered for the mutation load. TMB is reported as the number of mutations per megabase (Muts/Mb).

## 11. Controls

i) Negative Control: An external control that is provided in the GENESEEQPRIME assay reagent kit consists of non-cancerous cell line (NA18535) derived DNA with no variants of interest. The external control is processed from library preparation through sequencing to serve as an end-to-end control to validate the quality of the sequencing run. Failure of either external control to meet the quality control thresholds will result in all test samples on the run being reported as invalid.

ii) Positive Control: An external control that is provided in the GENESEEQPRIME assay reagent kit consists of cell line derived-DNA with multiple verified sequence mutations (BRAF V600E, EGFR L858R, EGFR Exon 19 Deletion, KRAS G13D, TPM3~NTRK1 translocation, CD74~ROS1 translocation, and ERBB2 amplification). The external control is processed from library preparation through sequencing to serve as an end-to-end control to demonstrate assay performance. All seven alterations must be detected for a sequencing run to pass quality control. Failure of either external control to meet the quality control thresholds will result in all test samples on the run being reported as invalid.

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## 12. Result Reporting

The GENESEEQPRIME assay reports SNV and indels in protein coding regions across all genes in the panel. In addition, amplifications are reported for *ERBB2* as well as translocations for *ALK*, *RET*, *ROS1*, and *NTRK1*. Germline mutations, including common polymorphisms, in the population present in 1000g (version 201508), ExAC (version 0.3nontcga), and gnomAD (version r2.0.1), are filtered and excluded from the final report. The assay also reports on two genomic signatures, MSI and TMB.

Variants are reported in one of two levels of evidence¹: Variants with Evidence of Clinical Significance and Variants with Potential Clinical Significance. Variants reporting as having evidence of clinical significance are defined by AMP/ASCO/CAP guidelines (Li et al., 2017)². The variants listed in the section Variants with Evidence of Clinical Significance are determined based on the selected tumor type. Only variants clinically associated with the tested tumor type will appear in the Variants with Evidence of Clinical Significance section. Any remaining detected variants will appear as the Variants with Potential Clinical Significance. A list of all 425 genes is provided in Appendix A; and a list of excluded regions in the genes or genes with variants that are excluded due to challenging regions (e.g., low complexity/repeats) is provided in Appendix B and Appendix C, respectively.

## 13. 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 |

¹ Refer to https://www.fda.gov/media/109050/download

² Li MM et al. Standards and Guidelines for the Interpretation and Reporting of Sequence Variants in Cancer: A Joint Consensus Recommendation of the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists. J Mol Diagn. 2017 Jan;19(1):4-23.

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| Quality Metric | Level of Qualification | Passing Criteria |
| --- | --- | --- |
| Q30 Reads | Batch-Level | %Q30 (Total) ≥ 80% |
| External Control | Batch-Level | Seven 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% ≥ 50x Dedup Depth |
| Hotspot SNVs and Indels | Analyte-Level | Mutant reads ≥ 4 VAF ≥ 1% ≥ 50x Dedup Depth |
| Non-hotspot SNVs and Indels | Analyte-Level | Mutant reads ≥ 5 VAF ≥ 2% ≥ 100x Dedup Depth |
| MSI Detection | Analyte-Level | MSI-H: MSI Score ≥ 16 MSS: 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 |

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## B. Principle of Operation

The GENESEEQPRIME assay kit is an in vitro diagnostic assay that uses targeted next generation sequencing to detect tumor gene alterations in genomic DNA isolated from formalin-fixed paraffin-embedded (FFPE) tumor tissue in a 425 gene panel. GENESEEQPRIME targets cancer-associated genes that are enriched from genomic libraries using a hybrid capture-based chemistry. Genomic libraries are prepared and captured. Samples are pooled for sequencing. After sequencing, automated software executes a bioinformatics analytics pipeline to identify genomic alterations in sequence data. The GENESEEQPRIME assay workflow does not use a patient-matched normal sample but filters polymorphisms using databases. A summary of the alterations found, including a PDF report, are reported in output files and provided in a user interface as part of the GENESIS software.

## C. Determination of Assay Thresholds

### 1. Requirements on Exon Coverage

A power analysis was conducted to determine the minimum sequence coverage necessary to detect mutations with true underlying VAFs as low as 2% (Figure 1). Statistical power was estimated based on a requirement of four mutant observations to make a positive call. Sequence coverage of > 400x coverage provided 95% statistical power for detection of true mutations at 2% VAF (95% CI: 1.02%-3.90%). For mutations with > 5% underlaying VAF, sequence coverage of > 300x provides 95% statistical power for detection (mean VAF = 17.0%, 95% CI: 3.05%-8.10%).


Figure 1. Power Analysis for Mutations Detected based on Exon Coverage. Mutations were separated into four 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.

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Summary statistics were calculated for individual exons across a cohort of samples to identify exons with consistent below-target coverage. These specific regions were removed from the GENESEEQPRIME assay and are not included in variant analysis or reporting. The excluded regions are listed in Appendix B and Appendix C. No Variants with Evidence of Clinical Significance or somatic hotspot mutations are masked from the report.

Sequence coverage was evaluated in the remaining regions across a cohort of 200 FFPE samples, and 90% of targeted regions (4,156 of 5,286 regions) were sequenced to a depth of 100x or greater with 90% of regions of interest achieved a sequencing depth of 200x or greater (Figure 2).


**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% of regions. The leftmost

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vertical dotted line indicates the sample level average deduplicated depth of 200x. The rightmost vertical dotted line indicates the sample level average deduplicated depth of 400x.

## 2. Requirements on Sample Coverage

Sample coverage was evaluated across a range of FFPE samples (n=200 across 10 different tumor types) to obtain sample summary statistics. Overall sample coverage was high in targeted regions of interest. The mean coverage across all targeted regions for the FFPE samples was 978x (SD = ± 245x).

Sequence coverage was further evaluated to establish the minimum requirements and reporting of variants. Based on a power analysis, a minimum sequence coverage of 100x is essential to call mutations with true underlaying mutation frequency of 2% or higher. The number of exons for all individual samples meeting this coverage threshold was evaluated to establish a per sample threshold. The samples evaluated included a range of DNA quality estimates. Of the 200 samples evaluated, >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 high coverage supports tolerance of occasional low coverage regions that may be seen with varying sample quality. A threshold of 90% of evaluated regions with at least 100x coverage was selected and is used to determine if a sample sequence to sufficient depth for analysis and reporting.


Figure 3. Distribution of mean coverage per sample in GENESEEQPRIME across 200 FFPE Samples.

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Figure 4. Distribution of mean coverage values per sample (x-axis) and percent of regions with more than 100x coverage (y-axis). Dotted line indicates the cut off of 90% regions with more than 100x coverage.

### 3. Requirements on Mutation Coverage, Allele Depth, and Frequency

Variant calling parameters were established at multiple cut offs for each variant type using 30 non-cancerous FFPE samples. An average of 8 mutations were detected per sample and 273 (209 non-hotspot, 58 hotspot, and 6 clinically significant variants) false positive variants were identified using a less stringent cut off. After inclusion of the filter criteria the rejection rate of false positive variants was 100% (Table 7).

Table 7. Sample Error Correction by Allele Depth and Allele Frequency

| Variant Category: | Non-hotspot Variants | Hotspot Variants | Clinically Significant Variants |
| --- | --- | --- | --- |
| Filter Criteria: | AD ≥ 5, AF ≥ 2% | AD ≥ 4, AF ≥ 1% | AD ≥ 4, AF ≥ 0.4% |
| Pre-Filter Variant Count | 209 | 58 | 6 |
| Post-Filter Variant Count | 0 | 0 | 0 |
| Rejection Rate (%) | 100 | 100 | 100 |

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# a) Variants with Evidence of Clinical Significance

The GENESEEQPRIME assay reports variants with Evidence of Clinical Significance and Variants with Potential Clinical Significance. A database was utilized for clinical evidence curation according to a three-tiered approach for reporting biomarkers in tumor profiling NGS tests. Variants classified as Level 2 were grouped as Variants with Evidence of Clinical Significance, and variants classified as Level 3 were grouped as Variants with Potential Evidence of Clinical Significance. Variants belonging to the Level 2 category are detected by the GENESEEQPRIME assay if the variant allele frequency is ≥ 0.4% and has at least four supporting reads.

# b) Somatic Mutation Detection by Tumor Only Sequencing (ToSeq) Module

GENESIS software includes a machine learning module developed by Geneseeq called “ToSeq” to distinguish germline SNVs and indels form somatic SNV and indels without the need for a matched normal sample. A set of 848 fixed features are used to evaluate variants. Features fall into one of the following categories: 1) location of variant, 2) variant allele frequency, 3) pathogenicity based on the InvterVar classification, 4) identity of the annotated reference and alternate alleles, 5) variant function classification, 6) sample copy number status, 7) frequency of occurrence within publicly available germline variant databases, and 8) internally established mutational hotspot database. The culmination of distinguishing features will correspond to a binary output status for each variant as either “Somatic” or “Germline”.

The performance of the somatic mutation calling was evaluated using tumor-normal sample pairs (n = 7,970). Matched pairs were analyzed using Geneseeq’s variant calling algorithm targeting regions covered by the GENESEEQPRIME assay. The machine learning model was trained using a set of 3,189 matched pairs and contained 53,664 SNVs and 4,338 indels; and independently tested using 4,781 matched pairs containing 79,070 SNVs and 6,748 indels. The reference list of germline and somatic mutations was identified by comparing the results from each matched pair. Out of the three training models used, the Distributed Random Forest model produced the best performance for ToSeq SNV and INDEL models (Figure 5) with a sensitivity of 95.59% (41,027/42,918, 95% CI: 95.40% - 95.78%) and a PPV of 95.63% (41,027/42,902, 95% CI: 95.43% - 95.82%).

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Figure 5. ROC Curve Analysis of ToSeq Performance. The curve is graphed using the best performance model of one algorithm on training and test datasets. The AUC and associated 95% CI is provided in the bottom right corner of each graph. Red line – Distributed forest (DRF) model. Green line – Generalized Linear Model. Blue line – Deep Learning Model

### c) ERBB2 Copy Number Variation Detection Cut Off

The GENESEEQPRIME assay cut off value for reporting ERBB2 copy number variation was established using 46 FFPE ERBB2-negative samples. Amplification status for was determined by comparing the normalized coverage of the region of interest against a panel of normal controls. A threshold at four times the standard deviation from the mean of the copy number was chosen for the assay. Amplification of the ERBB2

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gene is reported when a fold change (in relation to the diploid state) of over 1.8x is detected.

# d) MSI-H Cut Off Determination

Microsatellite instability is evaluated in terms of frequency of somatic sites out of the total number of tracked sites. The threshold for separating MSS from MSI-H samples was determined using 81 FFPE and 19 standard samples (obtained from the National Institutes for Food and Drug Control (NIFDC) in China). A reference MSI status was provided by the supplier for each sample and served as the true MSI status. Variant calls from GENESEEQPRIME assay were compared to the true MSI status (Figure 6). The greatest AUROC was observed when somatic sites occupied between 15% to 17% of the total sites. The MSI score cut off for the GENESEEQPRIME assay is set to 16%.


Figure 6. Receiver Operator Characteristic Curve for Microsatellite Score Determined by GENESEEQPRIME.

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# e) Structural Variant Cut Off Determination

The baseline fusion read count of gene translocations was established using a total of 39 FFPE samples without the targeted translocations (ALK, n = 10; RET, n = 8; ROS1, n = 10; and NTRK1, n = 11). Structural variants with canonical and non-canonical gene partners were used in the analysis. Gene translocation cut off values with a canonical and non-canonical gene partner was determined to be 6 and 12, respectively and are shown in Table 8.

Table 8. Detection Limits for Gene Translocation Negative Samples

| Major Gene | Partner Gene | Range of Supporting Reads for Negative Samples |
| --- | --- | --- |
| ALK^{1} | Canonical (n = 4) | 2 – 5 |
| | Non-Canonical (n = 6) | 6 – 11 |
| RET | Canonical (n = 3) | 2 – 4 |
| | Non-Canonical (n = 5) | 6 – 11 |
| ROS1 | Canonical (n = 5) | 2 – 5 |
| | Non-Canonical (n = 5) | 3 – 11 |
| NTRK1 | Canonical (n = 5) | 4 – 5 |
| | Non-Canonical (n = 6) | 2 – 11 |

# D. Substantial Equivalence Information:

1. Predicate Device Name(s)

PGDx elio Tissue Complete

2. Predicate 510(k) Number(s)

K192063

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# 3. Comparison with Predicate(s)

| Characteristics | Predicate device: PGDx elio tissue complete | Subject Device: GENESEEQPRIME NGS Tumor Profiling Assay (FFPE) |
| --- | --- | --- |
| **Similarities** | | |
| **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 |

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| Characteristics | Predicate device: PGDx elio tissue complete | Subject Device: GENESEEQPRIME FFPE tissue tumor profiling assay |
| --- | --- | --- |
| Similarities | | |
| 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 | 425 |
| Variant Types | Somatic Variants including point mutations, small insertions, and small deletions, ERBB2 amplification, 4 gene translocations (, RETNTRK2ALKRET, and NTRK3), MSI and TMB information. | Somatic Variants including point mutations, small insertions, and small deletions, ERBB2 amplification, 4 gene translocations (ROS1, ALK, RET, and NTRK1), 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%). |

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| Characteristics | Predicate device: PGDx elio tissue complete | Subject Device: GENESEEQPRIME FFPE tissue tumor profiling assay |
| --- | --- | --- |
| Differences | | |
| **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 sponsor-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 |

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| Characteristics | Predicate device: PGDx elio tissue complete | Subject Device: GENESEEQPRIME FFPE tissue tumor profiling assay |
| --- | --- | --- |
| Differences | | |
| **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. | Variant calls are organized into Variant with Evidence of Clinical Significance or 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. Standards/Guidance Documents Referenced

The following FDA guidance documents were consulted:

1. 1. FDA Fact Sheet - CDRH's Approach to Tumor Profiling Next Generation Sequencing Tests (2017),
2. 2. General Principles of Software Validation; Final Guidance for Industry and FDA Staff (January 11, 2002),
3. 3. Guidance for Industry Cybersecurity for Networked Medical Devices Containing Off-the-Shelf (OTS) Software (January 14, 2005),
4. 4. Guidance for Industry and FDA Staff Statistical Guidance on Reporting Results from Studies Evaluating Diagnostic Tests (March 13, 2007),
5. 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. 6. Postmarket Management of Cybersecurity in Medical Devices Guidance for Industry and Food and Drug Administration Staff (December 28, 2016),
7. 7. Content of Premarket Submissions for Device Software Functions Guidance for Industry and Food and Drug Administration Staff (June 14, 2023), and

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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. Analytical Performance - General**

The GENESEEQPRIME kit is a targeted NGS panel with 425 genes. The targeted regions of interest in GENESEEQPRIME are designed to detect SNVs, small indels < 30 bp in length in the coding exons of the targeted genes, as well as *ERBB2* amplifications, *ALK*, *RET*, *ROS*, and *NTRK1* translocations, MSI, and TMB. For SNVs and indels, a representative approach to validation of the targeted genes in the panel was submitted with data representing variant types for SNVs and indels, and at the gene levels for amplifications and translocations indicated with this assay. In addition, the assay was evaluated for performance regarding the panel-wide quality metrics.

# **a) Invalid Rates**

Performance throughout verification and validation of the device was tracked and a summary of the rates for first pass (no repeat) is presented below. The data shown represents the first pass rate of each sample. Repeat testing was not conducted for this analysis. Data were aggregated for clinical cases from > 40 tumor types. Resulting pass rates by tumor type across the workflow are shown in Table 9.

**Table 9. Comparability of Tumor Pass Rates for the GENESEEQPRIME Assay**

| Tumor Type | Total Samples | Total Failures | Total Passes | Failed Pre-Sequencing QC | Failed Post-Sequencing QC | Pass Rate (%) |
| --- | --- | --- | --- | --- | --- | --- |
| Bladder cancer | 55 | 6 | 49 | 4 | 2 | 89.09 |
| Breast cancer | 564 | 52 | 512 | 28 | 24 | 90.78 |
| Triple negative breast cancer | 96 | 8 | 88 | 4 | 4 | 91.67 |
| Adenocarcinoma | 23 | 2 | 21 | 1 | 1 | 91.30 |
| Cervical cancer | 119 | 9 | 110 | 0 | 9 | 92.44 |
| Squamous | 20 | 1 | 19 | 0 | 1 | 95.00 |
| Colon cancer | 1389 | 36 | 1353 | 19 | 17 | 97.41 |
| Rectum cancer | 1061 | 27 | 1034 | 15 | 12 | 97.46 |
| Colorectal cancer, NOS* | 76 | 2 | 74 | 1 | 1 | 97.37 |
| Endometrial cancer | 133 | 9 | 124 | 0 | 9 | 93.23 |

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| Tumor Type | Total Samples | Total Failures | Total Passes | Failed Pre-Sequencing QC | Failed Post-Sequencing QC | Pass Rate (%) |
| --- | --- | --- | --- | --- | --- | --- |
| Adenocarcinoma | 21 | 4 | 17 | 0 | 4 | 80.95 |
| Squamous | 118 | 15 | 103 | 0 | 15 | 87.29 |
| Esophageal cancer, NOS* | 10 | 1 | 9 | 0 | 1 | 90.00 |
| Adenocarcinoma | 303 | 27 | 276 | 10 | 17 | 91.09 |
| Gastric cancer | 402 | 30 | 372 | 14 | 16 | 92.54 |
| Gastrointestinal stromal tumor (GIST) | 94 | 10 | 84 | 7 | 3 | 89.36 |
| Brain glioma | 67 | 8 | 59 | 5 | 3 | 88.06 |
| Nasopharyngeal carcinoma | 27 | 3 | 24 | 2 | 1 | 88.89 |
| Head and neck cancer, Other | 121 | 14 | 107 | 8 | 6 | 88.43 |
| Cholangiocarcinoma | 119 | 4 | 115 | 4 | 0 | 96.64 |
| Liver cancer | 429 | 13 | 416 | 7 | 6 | 96.97 |
| Lung adenocarcinoma | 3964 | 317 | 3647 | 178 | 139 | 92.00 |
| Lung squamous cell carcinoma | 427 | 25 | 402 | 10 | 15 | 94.15 |
| Non-small cell lung cancer | 197 | 15 | 182 | 10 | 5 | 92.39 |
| Small cell lung cancer | 80 | 4 | 76 | 0 | 4 | 95.00 |
| Mediastinal | 10 | 0 | 10 | 0 | 0 | 100.00 |
| Melanoma | 167 | 19 | 148 | 7 | 12 | 88.62 |
| Neuroendocrine | 49 | 2 | 47 | 0 | 2 | 95.92 |
| High Grade Serous | 378 | 20 | 358 | 4 | 16 | 94.71 |
| Ovarian cancer | 138 | 8 | 130 | 2 | 6 | 94.20 |
| Pancreatic cancer | 311 | 13 | 298 | 8 | 5 | 95.82 |
| Prostate cancer | 116 | 9 | 107 | 9 | 0 | 92.24 |
| Renal cancer | 294 | 13 | 281 | 6 | 7 | 95.58 |
| Soft tissue sarcoma | 140 | 13 | 127 | 2 | 11 | 90.71 |

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| Tumor Type | Total Samples | Total Failures | Total Passes | Failed Pre-Sequencing QC | Failed Post-Sequencing QC | Pass Rate (%) |
| --- | --- | --- | --- | --- | --- | --- |
| Thyroid cancer | 46 | 3 | 43 | 1 | 2 | 93.48 |
| Bone cancer | 17 | 1 | 16 | 0 | 1 | 94.12 |
| Parathyroid | 9 | 1 | 8 | 0 | 1 | 88.89 |
| Skin cancer | 17 | 3 | 14 | 0 | 3 | 82.35 |
| Urinary tract | 6 | 0 | 6 | 0 | 0 | 100.00 |
| Uterine | 29 | 2 | 27 | 0 | 2 | 93.10 |
| Sum | 11642 | 749 | 10893 | 366 | 383 | 93.57 |

*NOS – Not otherwise specified; samples that was not categorized into any of the other subtypes.

## 2. Precision/Reproducibility

### a) Interlaboratory Reproducibly

Interlaboratory reproducibility of the GENESEEQPRIME assay was assessed across three different sites, using DNA extracted from 28 FFPE tissue specimens. The samples represented a range of SNVs, indels, ERBB2 amplifications, ALK, ROS1, RET, and NTRK1 translocations, MSI, and TMB. Each of the 28 samples were tested in duplicate by two different operators on 12 sequencing runs across three non-consecutive days at each of the three independent laboratory sites using a single kit lot (36 total sequencing runs and 1,008 total replicates). Allele frequencies for the variants in the specimens spanned all ranges. Each replicate began with the workflow post-DNA extraction.

Reproducibility was assessed by: 1) the average positive and negative agreement between all possible replicates within each test condition listed above were used to analyze sources of variance; 2) coefficient of variance of TMB score between all replicates within each test condition listed above were used to analyze sources of variance; 3) concordance of MSI status calls between all replicates of each samples was used; 4) positive call rate for variants that were detected in over 50% of replicates for each sample (Modal PCR) were used to determine per specimen reproducibility; 5) modal positive call rate (PCR) and negative call rate (NCR) of variants stratified by allele frequency and variant type (SNV, INS, DEL) were determined to show the reproducibility of GENESEEQPRIME assay performance across all levels and types of variants.

The sample used in the multi-site reproducibility study, along with their expected variants, are presented in Table 10 below. In terms of invalid rate, the first pass rate was 94.35% (951/1008).

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**Table 10. Samples used in the Multi-Site Reproducibility Study**

| Tissue Type | Expected SNVs with Evidence of Clinical Significance | Number of Variants with Protanal Clinical Significance | Translocation (Trans) or Amplification (Amp) | Mean TMB Score (Muts/Mb) | MSI-H Status |
| --- | --- | --- | --- | --- | --- |
| Colorectal Cancer | 0 | 39 | *NTRK1* (Trans) | 46.0 | Yes |
| Lung Adenocarcinoma | 0 | 8 | *ALK* (Trans) | 1.4 | |
| Thyroid Cancer | *BRAF* V600E | 7 | | 0.3 | |
| Melanoma | *BRAF* V600E | 31 | | 16.6 | |
| Endometrial Cancer | 0 | 50 | | 44.5 | Yes |
| Breast Cancer | *PIK3CA* H1047R & *PIK3CA* E545K | 7 | *ERBB2* (Amp) | 2.3 | |
| Lung Adenocarcinoma | 0 | 16 | | 13.7 | |
| Colorectal Cancer | 0 | 43 | *ERBB2* (Amp) | 10.6 | |
| Lung Adenocarcinoma | 0 | 35 | | 29.5 | |
| Head and Neck Cancer | 0 | 17 | | 3.5 | |
| Esophageal Cancer | 0 | 13 | *ERBB2* (Amp) | 9.8 | |
| Lung Adenocarcinoma | 0 | 16 | *ROS1* (Trans) | 3.5 | |
| Colorectal Cancer | *BRAF* V600E | 71 | | 75.3 | Yes |

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| Tissue Type | Expected SNVs with Evidence of Clinical Significance | Number of Variants with Protonal Clinical Significance | Translocation (Trans) or Amplification (Amp) | Mean TMB Score (Muts/Mb) | MSI-H Status |
| --- | --- | --- | --- | --- | --- |
| Lung Adenocarcinoma | *EGFR* L858R | 9 | | 7.2 | |
| Lung Adenocarcinoma | 0 | 34 | | 5.9 | |
| Prostate Cancer | 0 | 8 | | 1.0 | |
| Prostate Cancer | 0 | 4 | | 2.3 | |
| Breast Cancer | *PIK3CA* H1047R | 17 | *ERBB2* (Amp) | 7.7 | |
| Lung Adenocarcinoma | *EGFR* e19del | 9 | | 7.1 | |
| Endometrial Cancer | 0 | 14 | | 11.7 | |
| Ovarian Cancer | 0 | 13 | | 9.6 | |
| Gastric Cancer | 0 | 98 | | 109.5 | Yes |
| Endometrial Cancer | 0 | 49 | | 54.1 | Yes |
| Bladder Cancer | 0 | 37 | | 18.8 | |
| Melanoma | *BRAF* V600K | 43 | | 56.8 | |
| Colorectal Cancer | 0 | 19 | *ERBB2* (Amp) | 10.7 | |
| Lung Adenocarcinoma | *EGFR* e20dup | 30 | | 11.0 | |

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| Tissue Type | Expected SNVs with Evidence of Clinical Significance | Number of Variants with Protonal Clinical Significance | Translocation (Trans) or Amplification (Amp) | Mean TMB Score (Muts/Mb) | MSI-H Status |
| --- | --- | --- | --- | --- | --- |
| Endometrial Cancer | 0 | 68 | | 74.4 | Yes |

# b) *Panel-wide Reproducibility*

Reproducibility was accessed for each variant across all 36 replicates. The modal positive and negative call rates were calculated along with the two-sided 95% confidence interval.

Table 11 summarizes the PCR and NCR stratified by mutation type (SNV, insertions, and deletions) and variant allele frequency (VAF). An overall modal PCR of 97.46% across all samples and replicates (16137/16558, 95% CI: 97.21%, 97.69%, average VAF range: 0.4% - 77.06%), with an increase in PCR at higher VAFs observed, and an overall NCR of 94.27% (10506/11144, 95% CI: 93.83%, 94.69%). Repeat testing for the failed samples was not performed.

The positive call rates for individual sequence mutations assessed in the Interlaboratory Reproducibility study, along with the VAF range, mean, SD, and CV per variant per specimen tested are presented in **Appendix D**. A total of 671 SNVs and 144 indels (23 insertions, 121 deletions) are provided. Variants are listed by specimen with each specimen separated by a gray line. Discordant cases are denoted in light grey.

**Table 11. Interlaboratory Reproducibility Positive (PCR) and Negative (NCR) Call Rates**

| Variant Type | VAF Level (%) | Unique Mutations | PCR (%) (n/N) | NCR (%) (n/N) | Mean Allele Frequency Range (%) | Mean Allele Depth Range | Mean Loci Depth Range |
| --- | --- | --- | --- | --- | --- | --- | --- |
| All | AF≥0 | 815 | 97.46 (16137/16558) | 94.27 (10506/11144) | 0.4-77.06 | 4-1527 | 113-13236 |
| | AF≥2.0 | 590 | 97.51 (16080/16490) | 90.36 (3224/3568) | 2-77.06 | 5-1527 | 113-13236 |
| | AF≥5.0 | 457 | 98.9 (14425/14586) | 81.58 (775/950) | 5.16-77.06 | 14-1527 | 113-11822 |
| | AF≥10.0 | 389 | 99.24 (12349/12444) | 81.15 (633/780) | 10.01-77.06 | 24-1527 | 113-2710 |
| | AF≥15.0 | 288 | 99.13 (8999/9078) | 83.43 (594/712) | 15.06-77.06 | 24-1527 | 113-2710 |

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| Variant Type | VAF Level (%) | Unique Mutations | PCR (%) (n/N) | NCR (%) (n/N) | Mean Allele Frequency Range (%) | Mean Allele Depth Range | Mean Loci Depth Range |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Variants with Evidence of Clinical Significance | AF≥0 | 26 | 100 (306/306) | 95.14 (548/576) | 0.4-52.65 | 4-1425 | 250-2710 |
| | AF≥2.0 | 9 | 100 (306/306) | - | 10.19-52.65 | 76-1425 | 453-2710 |
| | AF≥5.0 | 9 | 100 (306/306) | - | 10.19-52.65 | 76-1425 | 453-2710 |
| | AF≥10.0 | 9 | 100 (306/306) | - | 10.19-52.65 | 76-1425 | 453-2710 |
| | AF≥15.0 | 8 | 100 (272/272) | - | 16.03-52.65 | 81-1425 | 453-2710 |
| Hotspot Variants | AF≥0 | 266 | 99.23 (1417/1428) | 95.99 (7305/7610) | 0.4-75.84 | 4-1425 | 206-2710 |
| | AF≥2.0 | 41 | 100 (1360/1360) | 67.65 (23/34) | 3.23-75.84 | 17-1425 | 267-2710 |
| | AF≥5.0 | 39 | 100 (1292/1292) | 67.65 (23/34) | 6.1-75.84 | 52-1425 | 267-2710 |
| | AF≥10.0 | 37 | 100 (1224/1224) | 67.65 (23/34) | 10.19-75.84 | 52-1425 | 267-2710 |
| | AF≥15.0 | 32 | 100 (1054/1054) | 67.65^{1} (23/34) | 15.07-75.84 | 52-1425 | 267-2710 |
| Non-Hotspot Variants | AF≥0 | 549 | 97.29 (14720/15130) | 90.58 (3201/3534) | 2-77.06 | 5-1527 | 113-13236 |
| | AF≥2.0 | 549 | 97.29 (14720/15130) | 90.58 (3201/3534) | 2-77.06 | 5-1527 | 113-13236 |
| | AF≥5.0 | 418 | 98.79 (13133/13294) | 82.1 (752/916) | 5.16-77.06 | 14-1527 | 113-11822 |
| | AF≥10.0 | 352 | 99.15 (11125/11220) | 81.77 (610/746) | 10.01-77.06 | 24-1527 | 113-2437 |
| | AF≥15.0 | 256 | 99.02 (7945/8024) | 84.22 (571/678) | 15.06-77.06 | 24-1527 | 113-2345 |
| Single Nucleotide Variations | AF≥0 | 671 | 97.52 (11936/12240) | 94.65 (10001/10566) | 0.4-75.84 | 4-978 | 113-13236 |
| | AF≥2.0 | 454 | 97.59 (11879/12172) | 91.45 (2983/3262) | 2-75.84 | 5-978 | 113-13236 |
| | AF≥5.0 | 326 | 99.28 (10329/10404) | 82.89 (562/678) | 5.16-75.84 | 18-978 | 113-11822 |
| | AF≥10.0 | 287 | 99.29 (9014/9078) | 82.89 (562/678) | 10.19-75.84 | 24-978 | 113-2498 |
| | AF≥15.0 | 218 | 99.2 (6678/6732) | 82.89 (562/678) | 15.06-75.84 | 24-978 | 113-2498 |

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| Variant Type | VAF Level (%) | Unique Mutations | PCR (%) (n/N) | NCR (%) (n/N) | Mean Allele Frequency Range (%) | Mean Allele Depth Range | Mean Loci Depth Range |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Insertions | AF≥0 | 23 | 97.19 (760/782) | - | 3.6-77.06 | 20-1527 | 399-2026 |
| | AF≥2.0 | 23 | 97.19 (760/782) | - | 3.6-77.06 | 20-1527 | 399-2026 |
| | AF≥5.0 | 21 | 96.92 (692/714) | - | 5.24-77.06 | 27-1527 | 399-2026 |
| | AF≥10.0 | 16 | 96.88 (527/544) | - | 11.77-77.06 | 69-1527 | 399-2026 |
| | AF≥15.0 | 12 | 96.81 (395/408) | - | 15.99-77.06 | 69-1527 | 399-2026 |
| Deletions | AF≥0 | 121 | 97.31 (3441/3536) | 87.37 (505/578) | 1.11-52.65 | 4-1425 | 267-2710 |
| | AF≥2.0 | 113 | 97.31 (3441/3536) | 78.76 (241/306) | 2.11-52.65 | 12-1425 | 267-2710 |
| | AF≥5.0 | 110 | 98.15 (3404/3468) | 78.31 (213/272) | 5.18-52.65 | 14-1425 | 267-2710 |
| | AF≥10.0 | 86 | 99.5 (2808/2822) | 69.61 (71/102) | 10.01-52.65 | 37-1425 | 285-2710 |
| | AF≥15.0 | 58 | 99.38 (1926/1938) | 94.12 (32/34) | 15.13-52.65 | 48-1425 | 299-2710 |
| *ERBB2* Amplification | | 5 | 100 (170/170) | - | N/A | N/A | N/A |
| *ALK* Translocation | | 1 | 100 (33/33) | - | N/A | 67-269 | 403-1114 |
| *RET* Translocation | | 1 | 100 (34/34) | - | N/A | 14-285 | 896-1942 |
| *ROS1* Translocation | | 1 | 100 (34/34) | - | N/A | 422-1458 | 75-212 |
| *NTRK1* Translocation | | 1 | 100 (34/34) | - | N/A | 28-129 | 533-1270 |

$^{1}$For hotspot variants above 2% VAF, only one mutation in one specimen was found in fewer than 50% of its replicates. This variant is expected to be an outlier due to being deemed a germline mutation and is not shown in the final report due to lack of clinical evidence supporting targeted drug use. It is expected with a larger sample size and more mutations, the modal NCR for hotspot variants will approach that of the non-hotspot variants.

### c) *Per Specimen*

The modal positive and negative call rates for sequence mutations (SNVs and indels) in each specimen are summarized in Table 12. A total of 651 unique SNV, 23 unique insertions, 121 unique deletions, and 144 indels were identified. A modal analysis yielded a 97.46% (16137/16558, 95% CI: 92.94% - 100%) positive call rate among all positives and an NCR of 94.27% (10506/11144, 95% CI: 83.82% - 96.64%).

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One replicate for Sample 2 did not pass the QC cut-off and was removed from analysis. R02 and R16 were found to have no modal PCR due to all detected variants in the samples having allele frequencies below the limit of detection of the GENESEEQPRIME assay, thus resulting in inconsistent variant calls.

**Table 12. Interlaboratory Reproducibility Modal Call Rates per Specimen**

| Specimen | Total Unique Mutations* Detected Across All Replicates | Modal Positive Call Rate (n/N) (95% CI) | Modal Negative Call Rate (n/N) (95% CI) |
| --- | --- | --- | --- |
| 1 | 39 | 95.35% (1005/1054) (93.91%,96.47%) | 91.54% (249/272) (87.63%,94.30%) |
| 2 | 8 | - | 94.32% (249/264) (90.84%,96.53%) |
| 3 | 8 | 100% (34/34) (89.85%,100%) | 96.22% (229/238) (92.97%,98.00%) |
| 4 | 32 | 100% (374/374) (98.98%,100%) | 96.64% (690/714) (95.05%,97.73%) |
| 5 | 50 | 99.59% (1219/1224) (99.05%,99.83%) | 91.81% (437/476) (89.00%,93.95%) |
| 6 | 9 | 100% (68/68) (94.65%,100%) | 92.44% (220/238) (88.36%,95.16%) |
| 7 | 16 | 96.73% (296/306) (94.09%,98.22%) | 92.02% (219/238) (87.87%,94.83%) |
| 8 | 43 | 94.41% (321/340) (91.44%,96.39%) | 95.10% (1067/1122) (93.67%,96.21%) |
| 9 | 35 | 98.47% (837/850) (97.4%,99.1%) | 96.18% (327/340) (93.57%,97.75%) |
| 10 | 17 | 100% (68/68) (94.65%,100%) | 96.08% (490/510) (94.02%,97.45%) |
| 11 | 13 | 95.8% (228/238) (92.44%,97.7%) | 90.20% (184/204) (85.34%,93.56%) |
| 12 | 16 | 100% (102/102) (96.37%,100%) | 96.61% (427/442) (94.48%,97.93%) |
| 13 | 72 | 95.86% (1923/2006) (94.9%,96.65%) | 96.38% (426/442) (94.20%,97.76%) |
| 14 | 10 | 94.12% (160/170) (89.51%,96.77%) | 94.12% (160/170) (89.51%,96.77%) |
| 15 | 34 | 83.82% (57/68) (73.31%,90.72%) | 96.60% (1051/1088) (95.35%,97.52%) |
| 16 | 8 | - | 87.50% (238/272) (83.04%,90.92%) |
| 17 | 4 | 100% (68/68) (94.65%,100%) | 83.82% (57/68) (73.31%,90.72%) |

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| Specimen | Total Unique Mutations* Detected Across All Replicates | Modal Positive Call Rate (n/N) (95% CI) | Modal Negative Call Rate (n/N) (95% CI) |
| --- | --- | --- | --- |
| 18 | 18 | 99.51% (203/204) (97.28%,99.97%) | 96.57% (394/408) (94.32%,97.95%) |
| 19 | 10 | 95.59% (195/204) (91.83%,97.66%) | 95.59% (130/136) (90.71%,97.96%) |
| 20 | 14 | 100% (306/306) (98.76%,100%) | 95.29% (162/170) (90.99%,97.60%) |
| 21 | 13 | 92.94% (158/170) (88.07%,95.92%) | 95.96% (261/272) (92.90%,97.73%) |
| 22 | 98 | 97.63% (2888/2958) (97.02%,98.12%) | 89.57% (335/374) (86.06%,92.28%) |
| 23 | 49 | 98.67% (1409/1428) (97.93%,99.15%) | 94.12% (224/238) (90.37%,96.46%) |
| 24 | 37 | 99.26% (540/544) (98.12%,99.71%) | 95.94% (685/714) (94.23%,97.16%) |
| 25 | 44 | 99.92% (1223/1224) (99.54%,100%) | 89.71% (244/272) (85.52%,92.78%) |
| 26 | 19 | 100% (340/340) (98.88%,100%) | 95.75% (293/306) (92.87%,97.50%) |
| 27 | 31 | 98.04% (200/204) (95.07%,99.23%) | 93.88% (798/850) (92.07%,95.30%) |
| 28 | 68 | 95.46% (1915/2006) (94.46%,96.29%) | 84.97% (260/306) (80.53%,88.54%) |

\*'Unique mut' indicates the union of all unique mutations from all repeats

# d) *Analysis of Source of Variance*

The Average Positive Agreement (APA) and Average Negative Agreement (ANA) was assessed to analyze the imprecision caused by different sources of variance across all 3 sites. Data analysis is presented stratified by variant type and presented for 1) overall, 2) site to site, 3) operator to operator, 4) day to day), and 5) within run concordance.

TMB was assessed using %CV of the TMB score across test sample replicates for samples. The results are shown in Table 13.

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**Table 13. Interlaboratory Reproducibility of GENESEEQPRIME Assay**

| Alteration Type | Metric | Overall (95% CI) | Inter-Site (95% CI) | Inter-Operator (95% CI) | Inter-Day (95% CI) | Repeatability (Within-Run) (95% CI) |
| --- | --- | --- | --- | --- | --- | --- |
| Variants with Evidence of Clinical Significance | APA | 92.49% (91.98%, 92.96%) | 92.43% (91.81%, 93.01%) | 92.28% (91.00%, 93.40%) | 93.00% (91.46%, 94.29%) | 92.99% (89.62%, 95.33%) |
| | ANA | 99.99% (99.99%, 99.99%) | 99.99% (99.99%, 99.99%) | 99.99% (99.99%, 99.99%) | 99.99% (99.99%, 99.99%) | 99.99% (99.99%, 99.99%) |
| Hotspot Variants | APA | 82.90% (82.59%, 83.21%) | 82.86% (82.48%, 83.23%) | 83.00% (82.25%, 83.73%) | 82.98% (82.05%, 83.88%) | 83.14% (81.24%, 84.88%) |
| | ANA | 99.98% (99.98%, 99.98%) | 99.98% (99.98%, 99.98%) | 99.98% (99.98%, 99.98%) | 99.98% (99.98%, 99.98%) | 99.98% (99.98%, 99.99%) |
| Non-Hotspot Variants | APA | 96.25% (96.20%, 96.31%) | 96.23% (96.17%, 96.29%) | 96.30% (96.17%, 96.42%) | 96.32% (96.17%, 96.48%) | 96.26% (95.93%, 96.56%) |
| | ANA | 99.99% (99.99%, 99.99%) | 99.99% (99.99%, 99.99%) | 99.99% (99.99%, 99.99%) | 99.99% (99.99%, 99.99%) | 99.99% (99.99%, 99.99%) |
| SNVs | APA | 94.22% (94.15%, 94.29%) | 94.19% (94.1%, 94.28%) | 94.25% (94.08%, 94.42%) | 94.33% (94.12%, 94.54%) | 94.28% (93.84%, 94.68%) |
| | ANA | 99.99% (99.99%, 99.99%) | 99.99% (99.99%, 99.99%) | 99.99% (99.99%, 99.99%) | 99.99% (99.99%, 99.99%) | 99.99% (99.99%, 99.99%) |
| Insertions (All) | APA | 97.38% (97.17%, 97.57%) | 97.40% (97.15%, 97.63%) | 97.42% (96.9%, 97.85%) | 97.17% (96.49%, 97.71%) | 97.34% (95.89%, 98.29%) |
| | ANA | 99.99% (99.99%, 99.99%) | 99.99% (99.99%, 99.99%) | 99.99% (99.99%, 99.99%) | 99.99% (99.99%, 99.99%) | 99.99% (99.99%, 99.99%) |
| Insertions 1 – 5 bp | APA | 97.12% (96.89%, 97.33%) | 97.15% (96.87%, 97.4%) | 97.16% (96.6%, 97.64%) | 96.89% (96.15%, 97.49%) | 97.08% (95.49%, 98.12%) |
| | ANA | 99.99% (99.99%, 99.99%) | 99.99% (99.99%, 99.99%) | 99.99% (99.99%, 99.99%) | 99.99% (99.99%, 99.99%) | 99.99% (99.99%, 99.99%) |
| Insertions 5 – 10 bp | APA | 100% (99.66%, 100%) | 100% (99.5%, 100%) | 100% (98.06%, 100%) | 100% (97.09%, 100%) | 100% (89.28%, 100%) |
| | ANA | 100% (100%, 100%) | 100% (100%, 100%) | 100% (100%, 100%) | 100% (100%, 100%) | 100% (100%,100%) |

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| Alteration Type | Metric | Overall (95% CI) | Inter-Site (95% CI) | Inter-Operator (95% CI) | Inter-Day (95% CI) | Repeatability (Within-Run) (95% CI) |
| --- | --- | --- | --- | --- | --- | --- |
| Insertions 21 – 30 bp | APA | 100% (99.66%, 100%) | 100% (99.5%, 100%) | 100% (98.06%, 100%) | 100% (97.09%, 100%) | 100% (89.28%, 100%) |
| | ANA | 100% (100%, 100%) | 100% (100%, 100%) | 100% (100%, 100%) | 100% (100%, 100%) | 100% (100%, 100%) |
| Deletions (All) | APA | 96.70% (96.60%, 96.81%) | 96.68% (96.55%, 96.80%) | 96.83% (96.57%, 97.06%) | 96.68% (96.36%, 96.97%) | 96.64% (95.97%, 97.2%) |
| | ANA | 99.99% (99.99%, 99.99%) | 99.99% (99.99%, 99.99%) | 99.99% (99.99%, 99.99%) | 99.99% (99.99%, 99.99%) | 99.99% (99.99%, 99.99%) |
| Deletions 1 – 5 bp | APA | 96.61% (96.5%, 96.71%) | 96.58% (96.45%, 96.71%) | 96.73% (96.47%, 96.97%) | 96.58% (96.25%, 96.88%) | 96.54% (95.85%, 97.12%) |
| | ANA | 99.99% (99.99%, 99.99%) | 99.99% (99.99%, 99.99%) | 99.99% (99.99%, 99.99%) | 99.99% (99.99%, 99.99%) | 99.99% (99.99%, 99.99%) |
| Deletions 5 – 10 bp | APA | 100% (99.66%, 100%) | 100% (99.5%, 100%) | 100% (98.06%, 100%) | 100% (97.09%, 100%) | 100% (89.28%, 100%) |
| | ANA | 100% (100%, 100%) | 100% (100%, 100%) | 100% (100%, 100%) | 100% (100%, 100%) | 100% (100%, 100%) |
| Deletions 11 – 20 bp | APA | 100% (99.83%, 100%) | 100% (99.75%, 100%) | 100% (99.02%, 100%) | 100% (98.52%, 100%) | 100% (94.34%, 100%) |
| | ANA | 100% (100%, 100%) | 100% (100%, 100%) | 100% (100%, 100%) | 100% (100%, 100%) | 100% (100%, 100%) |
| MSI | APA | 100% (99.94%, 100%) | 100% (99.92%, 100%) | 100% (99.67%, 100%) | 100% (99.75%, 100%) | 100% (98.04%, 100%) |
| | ANA | 100% (99.99%, 100%) | 100% (99.98%, 100%) | 100% (99.91%, 100%) | 100% (99.93%, 100%) | 100% (99.46%, 100%) |
| *ERBB2* Amplification | APA | 100% (99.93%, 100%) | 100% (99.90%, 100%) | 100% (99.61%, 100%) | 100% (99.70%, 100%) | 100% (97.66%, 100%) |
| | ANA | 100% (99.99%, 100%) | 100% (99.98%, 100%) | 100% (99.91%, 100%) | 100% (99.94%, 100%) | 100% (99.48%, 100%) |

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| Alteration Type | Metric | Overall (95% CI) | Inter-Site (95% CI) | Inter-Operator (95% CI) | Inter-Day (95% CI) | Repeatability (Within-Run) (95% CI) |
| --- | --- | --- | --- | --- | --- | --- |
| ALK Translocation | APA | 100% (99.64%, 100%) | 100% (99.47%, 100%) | 100% (97.93%, 100%) | 100% (98.44%, 100%) | 100% (88.65%, 100%) |
| | ANA | 100% (99.99%, 100%) | 100% (99.98%, 100%) | 100% (99.93%, 100%) | 100% (99.94%, 100%) | 100% (99.56%, 100%) |
| RET Translocation | APA | 100% (99.66%, 100%) | 100% (99.50%, 100%) | 100% (98.06%, 100%) | 100% (98.53%, 100%) | 100% (89.28%, 100%) |
| | ANA | 100% (99.99%, 100%) | 100% (99.98%, 100%) | 100% (99.93%, 100%) | 100% (99.94%, 100%) | 100% (99.56%, 100%) |
| ROS1 Translocation | APA | 100% (99.66%, 100%) | 100% (99.50%, 100%) | 100% (98.06%, 100%) | 100% (98.53%, 100%) | 100% (89.28%, 100%) |
| | ANA | 100% (99.99%, 100%) | 100% (99.98%, 100%) | 100% (99.93%, 100%) | 100% (99.94%, 100%) | 100% (99.56%, 100%) |
| NTRK1 Translocation | ANA | 100% (99.66%, 100%) | 100% (99.50%, 100%) | 100% (98.06%, 100%) | 100% (98.53%, 100%) | 100% (89.28%, 100%) |
| | APA | 100% (99.99%, 100%) | 100% (99.98%, 100%) | 100% (99.93%, 100%) | 100% (99.94%, 100%) | 100% (99.56%, 100%) |
| TMB | CV | 8.18% | 1.16% | 0.54% | 0.17% | 7.17% |

# e) Precision for MSI

Precision for MSI was evaluated across 22 microsatellite stable (MSS) and six microsatellite instability-high (MSI-H) samples with a range of MSI scores. The mean MSI score, MSI range, SD, and %CV for the score along with positive call rates are provided in Table 14.

Table 14. MSI Performance in the Interlaboratory Reproducibility Study

| Case No. | Modal Status | Total Replicates | Mean MSI Score (%) | MSI Score Range (%) | SD | %CV | Positive Call Rate (%) (95% CI) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | MSI-H | 34 | 25.58 | (19.64, 29.51) | 2.60 | 10.17 | 100 (89.85, 100) |

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| Case No. | Modal Status | Total Replicates | Mean MSI Score (%) | MSI Score Range (%) | SD | %CV | Positive Call Rate (%) (95% CI) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 2 | MSS | 33 | 6.05 | (1.69, 10) | 1.91 | 31.62 | 100 (89.57, 100) |
| 3 | MSS | 34 | 5.35 | (1.64, 11.48) | 2.18 | 40.75 | 100 (89.85, 100) |
| 4 | MSS | 34 | 5.84 | (1.75, 10.00) | 2.17 | 37.15 | 100 (89.85, 100) |
| 5 | MSI-H | 34 | 56.96 | (51.67, 63.33) | 2.68 | 4.70 | 100 (89.85, 100) |
| 6 | MSS | 34 | 6.05 | (1.69, 11.67) | 2.53 | 41.85 | 100 (89.85, 100) |
| 7 | MSS | 34 | 7.03 | (3.28, 10.00) | 1.71 | 24.38 | 100 (89.85, 100) |
| 8 | MSS | 34 | 3.55 | (0, 8.62) | 2.02 | 56.84 | 100 (89.85, 100) |
| 9 | MSS | 34 | 4.04 | (0, 9.84) | 2.41 | 59.60 | 100 (89.85, 100) |
| 10 | MSS | 34 | 5.21 | (0, 8.77) | 2.06 | 39.47 | 100 (89.85, 100) |
| 11 | MSS | 34 | 4.56 | (1.67, 8.33) | 1.91 | 41.92 | 100 (89.85, 100) |
| 12 | MSS | 34 | 5.18 | (1.75, 8.93) | 1.94 | 37.37 | 100 (89.85, 100) |
| 13 | MSI-H | 34 | 77.10 | (66.67, 81.97) | 3.40 | 4.41 | 100 (89.85, 100) |
| 14 | MSS | 34 | 5.13 | (1.72, 10.17) | 2.00 | 38.91 | 100 (89.85, 100) |
| 15 | MSS | 34 | 6.80 | (3.51, 14.81) | 2.74 | 40.27 | 100 (89.85, 100) |
| 16 | MSS | 34 | 3.11 | (0, 6.90) | 1.85 | 59.47 | 100 (89.85, 100) |
| 17 | MSS | 34 | 4.50 | (1.67, 8.33) | 2.05 | 45.64 | 100 (89.85, 100) |
| 18 | MSS | 34 | 7.09 | (3.33, 13.79) | 2.59 | 36.55 | 100 (89.85, 100) |
| 19 | MSS | 34 | 4.56 | (0, 8.33) | 1.71 | 37.48 | 100 (89.85, 100) |
| 20 | MSS | 34 | 5.71 | (1.82, 10.00) | 1.85 | 32.38 | 100 (89.85, 100) |
| 21 | MSS | 34 | 9.11 | (5.17, 15.25) | 2.35 | 25.83 | 100 (89.85, 100) |

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| Case No. | Modal Status | Total Replicates | Mean MSI Score (%) | MSI Score Range (%) | SD | %CV | Positive Call Rate (%) (95% CI) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 22 | MSI-H | 34 | 76.40 | (70.49, 81.97) | 2.94 | 3.85 | 100 (89.85, 100) |
| 23 | MSI-H | 34 | 55.81 | (48.21, 62.30) | 3.15 | 5.64 | 100 (89.85, 100) |
| 24 | MSS | 34 | 6.35 | (1.79, 10.53) | 2.46 | 38.74 | 100 (89.85, 100) |
| 25 | MSS | 34 | 5.20 | (1.69, 9.84) | 2.16 | 41.64 | 100 (89.85, 100) |
| 26 | MSS | 34 | 5.22 | (1.72, 10.17) | 2.25 | 43.17 | 100 (89.85, 100) |
| 27 | MSS | 34 | 3.76 | (0, 8.77) | 2.00 | 53.31 | 100 (89.85, 100) |
| 28 | MSI-H | 34 | 75.09 | (68.85, 80.33) | 2.70 | 3.59 | 100 (89.85, 100) |

# *f) Precision for Tumor Mutational Burden (TMB)*

Precision of TMB was evaluated across 28 FFPE samples (with samples near the analytical borderline value of 1.1 Muts/Mb) using the coefficient of variance (%CV) to measure the degree of variation. The overall variance between the samples was 8.18% (Table 15). The data demonstrates the precision of TMB scores.

**Table 15. TMB Performance across all Repeats**

| Sample | Replicates | Range | Mean | SD | %CV |
| --- | --- | --- | --- | --- | --- |
| 1 | 34 | 43.4-48.6 | 45.97 | 1.24 | 2.70 |
| 2 | 33 | 0-3.2 | 1.44 | 0.72 | 50.10 |
| 3* | 34 | 0 – 2.1 | 0.31 | 0.67 | 212.10 |
| 4 | 34 | 15.9-19 | 16.62 | 1.02 | 6.16 |
| 5 | 34 | 42.3-47.6 | 44.52 | 1.43 | 3.22 |
| 6 | 34 | 1.1-4.2 | 2.29 | 0.68 | 29.56 |
| 7 | 34 | 11.6-15.9 | 13.72 | 1.15 | 8.35 |
| 8 | 34 | 8.5-13.7 | 10.63 | 1.28 | 12.01 |
| 9 | 34 | 28.6-31.7 | 29.47 | 0.92 | 3.14 |
| 10 | 34 | 3.2-5.3 | 3.50 | 0.63 | 18.07 |
| 11 | 34 | 8.5-10.6 | 9.77 | 0.78 | 8.00 |
| 12 | 34 | 3.2-5.3 | 3.51 | 0.75 | 21.29 |
| 13 | 34 | 70.9-78.3 | 75.32 | 1.68 | 2.24 |
| 14 | 34 | 4.2-9.5 | 7.24 | 1.33 | 18.42 |
| 15 | 34 | 4.2-9.5 | 5.93 | 1.35 | 22.73 |

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| Sample | Repeats | Range | Mean | SD | %CV |
| --- | --- | --- | --- | --- | --- |
| 16* | 34 | 0 - 3.2 | 0.95 | 0.82 | 85.65 |
| 17 | 34 | 2.1-3.2 | 2.26 | 0.48 | 21.31 |
| 18 | 34 | 7.4-8.5 | 7.69 | 0.56 | 7.32 |
| 19 | 34 | 5.3-8.5 | 7.15 | 0.70 | 9.77 |
| 20 | 34 | 10.6-13.7 | 11.73 | 0.59 | 5.03 |
| 21 | 34 | 7.4-11.6 | 9.59 | 1.12 | 11.70 |
| 22 | 34 | 106.8-113.1 | 109.54 | 1.93 | 1.76 |
| 23 | 34 | 48.6-57.1 | 54.08 | 1.95 | 3.61 |
| 24 | 34 | 16.9-22.2 | 18.78 | 1.29 | 6.85 |
| 25 | 34 | 55-60.3 | 56.78 | 1.33 | 2.35 |
| 26 | 34 | 10.6-11.6 | 10.72 | 0.35 | 3.30 |
| 27 | 34 | 8.5-23.3 | 10.99 | 2.70 | 24.62 |
| 28 | 34 | 70.9-79.3 | 74.38 | 2.04 | 2.74 |
| **TMB Median %CV (All Samples)** | | | | | **8.18** |
| **TMB Mean CV% (All Samples)** | | | | | **21.58** |
| **TMB Median %CV (All Samples with the Mean TMB greater than LoB)** | | | | | **7.66** |
| **TMB Mean %CV (All Samples with the Mean TMB greater than LoB)** | | | | | **11.78** |

*Sample 3 and Sample 16 have a mean TMB below the LoB (1.1Muts/Mb) of the GENESEEQPRIME Assay.

#### g) *Lot-to-Lot Precision*

Performance of the GENESEEQPRIME assay was assessed across three unique kit lots by determining the concordance of variant calls in five FFPE tissue samples. The three unique kit lots were used to process five test cases in triplicate for a total of 45 observations. All batches were sequenced on the same instrument. Table 16. lists the APA and ANA used to assess lot to lot performance. The overall all panel-wide variants (SNVs, Insertions, and Deletions) APA is $\geq 98.11\%$ and the NPA did not fall below 99.99%. *ERBB2* amplification, *RET* translocation, and MSI status had a concordance of 100% across all comparisons. The %CV for TMB analyses is $< 2.46\%$.

**Table 16. Lot-to-Lot Precision of GENESEEQPRIME**

| Variant Type | Performance | Between Lot 1 & 2 | Between Lot 2 & 3 | Between Lot 1 & 3 |
| --- | --- | --- | --- | --- |
| Variants with Evidence of Clinical Significance | APA | 100% (93.36%, 100%) | 100% (93.36%, 100%) | 100% (93.36%, 100%) |
| | ANA | 100% (99.99%, 100%) | 100% (99.99%, 100%) | 100% (99.99%, 100%) |

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| Variant Type | Performance | Between Lot 1 & 2 | Between Lot 2 & 3 | Between Lot 1 & 3 |
| --- | --- | --- | --- | --- |
| Panel-Wide Variant (SNVs + Insertions+ Deletions) | APA | 98.48% (98.15%, 98.75%) | 98.77% (98.47%, 99.01%) | 98.11% (97.75%, 98.41%) |
| | ANA | 99.99% (99.99%, 99.99%) | 99.99% (99.99%, 99.99%) | 99.99% (99.99%, 99.99%) |
| Hotspot SNV (including Clinically Significant Variants) | APA | 99.03% (97.18%, 99.67%) | 100% (98.76%, 100%) | 99.10% (97.18%, 99.67%) |
| | ANA | 99.99% (99.99%, 99.99%) | 100% (99.99%, 100%) | 99.99% (99.99%, 99.99%) |
| Non-Hotspot SNVs (including Clinically Significant Variants) | APA | 98.35% (97.99%, 98.65%) | 98.63% (98.3%, 98.90%) | 97.94% (97.54%, 98.27%) |
| | ANA | 99.99% (99.99%, 99.99%) | 99.99% (99.99%, 99.99%) | 99.99% (99.99%, 99.99%) |
| MSI | APA | 100% (82.41%, 100%) | 100% (82.41%, 100%) | 100% (82.41%, 100%) |
| | ANA | 100% (94.93%, 100%) | 100% (94.93%, 100%) | 100% (94.93%, 100%) |
| SNVs (Hotspot + Non-Hotspot) | APA | 98.43% (98.09%, 98.72%) | 98.70% (98.38%, 98.95%) | 98.04% (97.66%, 98.36%) |
| | ANA | 99.99% (99.99%, 99.99%) | 99.99% (99.99%, 99.99%) | 99.99% (99.99%, 99.99%) |
| Insertions | APA | 100% (94.93%, 100%) | 100% (94.93%, 100%) | 100% (94.93%, 100%) |
| | ANA | 100% (99.99%, 100%) | 100% (99.99%, 100%) | 100% (99.99%, 100%) |
| Deletions | APA | 99.03% (97.18%, 99.67%) | 100% (98.76%, 100%) | 99.03% (97.18%, 99.67%) |
| | ANA | 99.99% (99.99%, 99.99%) | 100% (99.99%, 100%) | 99.99% (99.99%, 99.99%) |
| *ERBB2* Amplification | APA | 100% (82.41%, 100%) | 100% (82.41%, 100%) | 100% (82.41%, 100%) |
| | ANA | 100% (94.93%, 100%) | 100% (94.93%, 100%) | 100% (94.93%, 100%) |
| *RET* Translocation | APA | 100% (82.41%, 100%) | 100% (82.41%, 100%) | 100% (82.41%, 100%) |
| | ANA | 100% (94.93%, 100%) | 100% (94.93%, 100%) | 100% (94.93%, 100%) |
| TMB | %CV | 2.46% | 1.55% | 2.46% |

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### 3. Analytical Sensitivity – Limit of Detection (LoD)

The recommended DNA input for GENESEEQPRIME assay is 100 ng of total DNA recovered from tissue with a minimum 20% viable tumor nuclei. The LoD study was comprised of two steps: LoD establishment using cell lines and LoD confirmation with FFPE clinical tumor samples from clinical cases across a diverse set of cancers. Select specimens were used to evaluate specific mutations with evidence of clinical significance. Specimens were selected for allele frequencies near the claimed cut offs. Details of the data are discussed and shown below.

#### a) LoD – SNVs, Insertions, and Deletions

The LoD of the GENESEEQPRIME assay is defined as the lowest measured mean allele frequency at which 95% of replicates for a variant type are reliably detected. Target levels for detection were first established in a dilution series from cell lines with up to five targeted VAF levels. The analytical sensitivity and LoD was then confirmed in seven clinical FFPE specimens (six samples for SNVs, four for insertions, and four for deletions). The data was aggregated across two reagent kit lots. Cell lines were used to establish the LoD VAF range for 461 unique targeted and non-targeted variants (Hotspot SNVs, Non-Hotspot SNVs, SNVs, Insertions, and Deletions) across the panel (Table 17). A total of 300 observations were generated (three samples with 20 replicates at five dilution levels). Positive call status and VAF was evaluated from select variants identified in seven FFPE clinical specimens diluted with normal DNA derived from FFPE tissue. Each specimen was processed with two kit lot of GENESEEQPRIME across 20 replicates for a for a total of 140 observations.

The established analytical sensitivity ranges were confirmed at ≥ 95% call rate with FFPE clinical cases on a per variant level (Table 18) and using all somatic variants identified in FFPE clinical cases representing a range of VAFs for hotspot and non-hotspot positions (Table 18). The observed sequencing depth (DP), allele depth (AD), variant allele frequency (VAF), and average VAF are included. A summary of the established analytical sensitivity for the represented SNVs and Indels in FFPE tumor tissue is provided in Table 19.

Table 17. Established LoD VAF of Representative SNVs, Insertions, and Deletions

| Variant Type | Mean AF Range in Cell Lines (%) | Mean LoD in Cell Lines (%) | Unique Variants in Cell Lines | Mean LoD in FFPE Samples (%) | Unique Variants in FFPE in the Established Range |
| --- | --- | --- | --- | --- | --- |
| HS SNV | 1.15 - 6.73 | 2.33 | 26 | 2.14 | 22 |
| NHS SNV | 2.23 - 10.13 | 3.60 | 384 | 2.95 | 265 |
| INS | 0.93 - 6.37 | 2.82 | 9 | 2.93 | 4 |
| DEL | 0.89 - 15.18 | 5.32 | 42 | 5.44 | 9 |

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| Variant Type | Mean AF Range in Cell Lines (%) | Mean LoD in Cell Lines (%) | Unique Variants in Cell Lines | Mean LoD in FFPE Samples (%) | Unique Variants in FFPE in the Established Range |
| --- | --- | --- | --- | --- | --- |
| Indels at Homopolymer Context* | 2.70 - 15.18 | 6.98 | 25 | 7.94 | 5 |
| Indels at Non-Homopolymer Context | 0.89 - 6.36 | 3.15 | 26 | 2.62 | 8 |

*Homopolymer context - a region containing ≥ 5 consecutive identical nucleotides.

**Table 18. Analytical Sensitivity of Representative SNVs and Indels in FFPE Tumor Tissue**

| Variant Type | Gene | AA Chane | DP Range | AD Range | AF Range (%) | Mean AF (%) | Call Rate (%) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| INS | *ERBB2* | Y772_A775dup | 1515 - 1592 | 22 - 23 | 1.4 - 1.46 | 1.43 | 100 (20/20) |
| INS | KMT2B | P2259Sfs*44 | 530 - 552 | 29 - 29 | 5.25 - 5.47 | 5.33 | 100 (20/20) |
| INS | RAD51 | G45Wfs*20 | 688 - 738 | 17 - 20 | 2.34 - 2.71 | 2.45 | 100 (20/20) |
| INS | GATA | H333dup | 584-613 | 15 - 15 | 2.45-2.57 | 2.51 | 100 (20/20) |
| DEL | EGFR | E746_A750del | 858 - 935 | 7 - 7 | 0.75 - 0.82 | 0.78 | 100 (20/20) |
| DEL | CDH1 | T467Hfs*15 | 1188 - 1248 | 28 - 30 | 2.32 - 2.4 | 2.35 | 100 (20/20) |
| DEL | B2M | V69Wfs*34 | 975 - 1044 | 76 - 82 | 7.79 - 8.02 | 7.90 | 100 (20/20) |
| DEL | TP53 | R209Kfs*6 | 1362 - 1438 | 28 - 30 | 2 - 2.14 | 2.08 | 100 (20/20) |
| SNV | APC | R1450* | 672-721 | 16 - 17 | 2.32-2.45 | 2.38 | 100 (20/20) |
| SNV | EGFR | L858R | 973 - 1076 | 15 - 15 | 1.39 - 1.54 | 1.47 | 100 (20/20) |
| SNV | TP53 | Q104* | 971-1040 | 14 - 17 | 1.38-1.65 | 1.46 | 100 (20/20) |
| SNV | PTEN | Q245* | 852 - 901 | 13 - 16 | 1.49 - 1.8 | 1.63 | 100 (20/20) |
| SNV | NRAS | G12D | 771 - 823 | 18 - 19 | 2.21 - 2.33 | 2.29 | 100 (20/20) |
| SNV | KRAS | G12C | 1101 - 1177 | 18 - 19 | 1.55 - 1.63 | 1.60 | 100 (20/20) |

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**Table 19. Analytical…