← Product Code PQP · P210011

# xT CDx (P210011)

_Tempus Labs, Inc. · PQP · Apr 28, 2023 · Pathology · APPR_

**Canonical URL:** https://fda.innolitics.com/device/P210011

## Device Facts

- **Applicant:** Tempus Labs, Inc.
- **Product Code:** PQP
- **Decision Date:** Apr 28, 2023
- **Decision:** APPR
- **Device Class:** Class 3
- **Review Panel:** Pathology
- **Attributes:** AI/ML

## Intended Use

xT CDx is a qualitative Next Generation Sequencing (NGS)-based in vitro diagnostic device intended for use in the detection of substitutions (single nucleotide variants (SNVs) and multi-nucleotide variants (MNVs)) and insertion and deletion alterations (INDELs) in 648 genes, as well as microsatellite instability (MSI) status, using DNA isolated from formalin-fixed paraffin embedded (FFPE) tumor tissue specimens, and DNA isolated from matched normal blood or saliva specimens, from previously diagnosed cancer patients with solid malignant neoplasms. The test is intended as a companion diagnostic (CDx) to identify patients who may benefit from treatment with the targeted therapies listed in the Companion Diagnostic Indications table in accordance with the approved therapeutic product labeling. Additionally, xT CDx is intended to provide tumor mutation profiling to be used by qualified health care professionals in accordance with professional guidelines in oncology for patients with previously diagnosed solid malignant neoplasms. Genomic findings other than those listed in the Companion Diagnostic Indications table are not prescriptive or conclusive for labeled use of any specific therapeutic product. xT CDx is a single-site assay performed at Tempus Labs, Inc., Chicago, IL.

## Device Story

xT CDx is a single-site NGS assay performed at Tempus Labs. It processes DNA from FFPE tumor tissue and matched normal blood or saliva. The workflow includes DNA extraction, whole-genome shotgun library construction, and hybridization-based capture of 648 cancer-related genes and 239 MSI loci. Sequencing is performed on the Illumina NovaSeq 6000. Sequence data is processed via a custom bioinformatics pipeline for alignment, variant calling (SNVs, MNVs, INDELs), and MSI classification. Results are curated by automated software using databases like MSK OncoKB. The output provides mutation profiling and CDx status for specific therapies. Healthcare professionals use these results to guide treatment decisions for patients with solid malignant neoplasms. The device benefits patients by identifying potential eligibility for targeted therapies and providing comprehensive genomic profiling.

## Clinical Evidence

Clinical validity established using 412 CRC samples. Concordance evaluated against FDA-approved Praxis and therascreen assays. Overall concordance with Praxis was 100% (190/190) and with therascreen was 99.6% (249/250). Analytical accuracy for tumor profiling was assessed against an orthogonal NGS method using 416 samples across 31 cancer types, showing 99.1% PPA and 100% NPA. MSI status accuracy was assessed against IHC in 316 samples, showing 94% PPA and 98% NPA.

## Technological Characteristics

NGS-based assay using Illumina NovaSeq 6000 (sequencing-by-synthesis). Hybridization-based capture of 648 genes and 239 MSI loci. DNA input: 50ng minimum. FFPE tumor and matched normal blood/saliva specimens. Bioinformatics pipeline aligns to GRCh37. MSI classification uses a univariate logistic regression classifier. Quality control metrics include on-target rates, unique read counts, and fingerprint scores.

## Regulatory Identification

A next generation sequencing (NGS) oncology panel is a device used for the qualitative detection of germline or somatic variants in one or more cancer-related genes. The device is intended to be used on DNA or RNA isolated from human clinical specimens.

## Reference Devices

- Praxis Extended Ras Panel ([P160038](/device/P160038.md))
- Qiagen therascreen KRAS RGQ PCR Kit ([P110027](/device/P110027.md))
- FoundationOne CDx
- ONCO/Reveal Dx Lung and Colon Cancer Assay (O/RDx-LCCA)

## Submission Summary (Full Text)

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>
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SUMMARY OF SAFETY AND EFFECTIVENESS DATA (SSED)

I. GENERAL INFORMATION

Device Generic Name: Next Generation Sequencing Oncology Panel, Somatic Variant Detection System

Device Trade Name: xT CDx

Device Procode: PQP

Applicant's Name and Address: Tempus Labs, Inc. (Tempus)
600 W. Chicago Ave
Chicago, IL 60654

Date(s) of Panel Recommendation: None

Premarket Approval Application (PMA) Number: P210011

Date of FDA Notice of Approval: April 28, 2023

II. INDICATIONS FOR USE

xT CDx is a qualitative Next Generation Sequencing (NGS)-based in vitro diagnostic device intended for use in the detection of substitutions (single nucleotide variants (SNVs) and multi-nucleotide variants (MNVs)) and insertion and deletion alterations (INDELs) in 648 genes, as well as microsatellite instability (MSI) status, using DNA isolated from formalin-fixed paraffin embedded (FFPE) tumor tissue specimens, and DNA isolated from matched normal blood or saliva specimens, from previously diagnosed cancer patients with solid malignant neoplasms.

The test is intended as a companion diagnostic (CDx) to identify patients who may benefit from treatment with the targeted therapies listed in the Companion Diagnostic Indications table in accordance with the approved therapeutic product labeling.

Additionally, xT CDx is intended to provide tumor mutation profiling to be used by qualified health care professionals in accordance with professional guidelines in oncology for patients with previously diagnosed solid malignant neoplasms. Genomic findings other than those listed in the Companion Diagnostic Indications table are not prescriptive or conclusive for labeled use of any specific therapeutic product.

xT CDx is a single-site assay performed at Tempus Labs, Inc., Chicago, IL.

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# Companion diagnostic indications

|  Tumor Type | Biomarker(s) Detected | Therapy  |
| --- | --- | --- |
|  Colorectal cancer (CRC) | KRAS wild type (absence of mutations in codons 12 or 13) | Erbitux (cetuximab)  |
|  Colorectal cancer (CRC) | KRAS wild type (absence of mutations in exons 2, 3, or 4) and NRAS wild type (absence of mutations in exons 2, 3, or 4) | Vectibix (panitumumab)  |

# III. CONTRAINDICATIONS

There are no known contraindications.

# IV. WARNINGS AND PRECAUTIONS

The warnings and precautions can be found in the xT CDx labeling.

# V. DEVICE DESCRIPTION

xT CDx is a single-site assay performed at Tempus Labs, Inc. located at 600 West Chicago Avenue Suite 510, Chicago, IL 60654. The assay includes reagents, software, instruments (qualified by Tempus), and procedures for testing DNA extracted from FFPE tumor specimens and matched normal saliva or blood specimens.

Extracted DNA undergoes whole-genome shotgun library construction and hybridization-based capture of specified regions from 648 cancer-related genes (including intronic overhang and selected promoter regions) (Appendix 1), and 239 loci for MSI. Using the Illumina NovaSeq 6000 platform, hybrid-capture-selected libraries are sequenced to highly uniform depth (targeting ♦ 500x median coverage of tumor samples, with ♦ 95% of exons at ♦ 150x coverage and ♦ 98% of exons at ♦ 100x coverage). Sequence data is processed using a customized analysis pipeline designed to detect substitutions (SNVs and MNVs) and INDELs in coding and noncoding genomic regions targeted by the assay. Additionally, MSI status is reported by an MSI classification algorithm to classify tumors into three categories: Microsatellite Instability High (MSI-H), Microsatellite Stable (MSS) and Equivocal (MSI cannot be determined).

# Test Output

The output of the test is generated via curation by automated software using information from several databases including information derived from the FDA-recognized content

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of Memorial Sloan Kettering's Precision Oncology Knowledge Base (MSK OncoKB, https://www.oncokb.org), and consists of results representing three categories:

Level 1: CDx claims for KRAS and NRAS as noted in the Intended Use

Level 2: Cancer Mutations with Evidence of Clinical Significance

Level 3: Cancer Mutations with Potential Clinical Significance

Genomic findings other than those listed in the Companion Diagnostic Indications table of the intended use statement (i.e., Levels 2 and 3) are not prescriptive or conclusive for labeled use of any specific therapeutic product.

## Test Kit Contents

xT CDx includes a specimen collection and shipping kit (the Specimen Kit). The Specimen Kit contains the following components (Table 1).

Table 1. Specimen Kit contents

|  Blood Kit | Saliva Kit | Tissue Kit*  |
| --- | --- | --- |
|  • Informational Brochures (2)
• Document Checklist
• Specimen ID Label
• Seal Sticker
• Cold Pack
• Prelabeled FedEx Clinical Shipping Pak
• 3x5 Biohazard Bag w/ Absorbent Sheet
• Blood Collection tubes | • Informational Brochures (2)
• Document Checklist
• Specimen ID Label
• Seal Sticker
• Prelabeled FedEx Clinical Shipping Pak
• 3x5 Biohazard Bag w/ Absorbent Sheet
• Saliva Collection tube | • Informational Brochure w/ Specimen Requirements
• Seal Sticker
• 12x15 Biohazard bag
• Prelabeled FedEx Clinical Shipping Pak
• Microscope Slide cases (2)  |

* For tissue collection, Tempus Labs, Inc. (Tempus) receives either previously prepared slides or blocks.

## Instruments

xT CDx uses the Illumina NovaSeq 6000 Sequencer, a high throughput sequencing system employing sequencing-by-synthesis chemistry. xT CDx is intended to be performed with serial number-controlled instruments. All instruments are qualified by Tempus Labs, Inc. (Tempus) under Tempus' Quality System.

## Principles of Operation

All assay reagents included in the xT CDx assay process are qualified by Tempus Labs, Inc. and are compliant with the medical device Quality System (QS) regulation.

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1. Specimen Collection and Preparation

FFPE tumor specimens received either as unstained slides or as an FFPE block, and matched normal specimens, are collected using materials supplied in the Specimen Kit and prepared following standard pathology practices. Preparation and review of a Hematoxylin and Eosin (H&E) slide is performed as a laboratory service in accordance with standard laboratory practices, prior to initiation of the xT CDx assay. H&E stained slides are reviewed by a board-certified pathologist to ensure that adequate tissue, tumor content and sufficient nucleated cells are present to satisfy minimum tumor content (tumor purity). Specifically, the minimum recommended tumor purity for detection of variants by xT CDx is 20%, with macrodissection required for specimens with tumor purity lower than 20%.

2. DNA Extraction

Genomic DNA is extracted from tumor tissue specimens and patient-matched normal specimens. Specimen specific extraction methods are used for each sample type. The recovered DNA is quantified and qualified. The DNA is then mechanically sheared prior to library preparation. The minimum amount of DNA required to perform the test is 50 ng.

3. Library Preparation

Library preparation is performed using the Illumina KAPA DNA Hyper Prep Kit with unique dual indices. First, end repair and A-tailing occurs to create end-repaired, 5'-phosphorylated, 3'-dA-tailed dsDNA fragments on the sheared DNA. Then DNA pair adapters with 3'-dTMP overhangs are ligated to 3'-dA-tailed molecules. The libraries are amplified with high fidelity, low-bias PCR. After bead-based purification of libraries, library yield and quality are assessed.

4. Hybrid Capture

Resultant libraries are enriched by hybridization to a set of custom probes that are subsequently immobilized onto magnetic beads and unbound fragments are then washed away. To reduce non-specific binding of untargeted regions, blockers are included in the hybridization step. The enriched DNA targets are amplified. The amplified libraries are assessed and pooled for sequencing.

5. Sequencing

The amplified target-captured tumor and normal libraries are sequenced to a median depth of 500x and 150x, respectively, on an Illumina NovaSeq 6000 System using patterned flow cell technology. Each tumor sample is required to have unique reads above 8,000,000. Samples with insufficient coverage are failed. A matched normal

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specimen is sequenced in order to subtract germline variants from the tumor sequencing results to more accurately report somatic variants.

## 6. Data Analysis

### A. Read Alignment and BAM Generation

FASTQ files are aligned to the Genome Reference Consortium Human Build 37 (GRCh37). During this process, remaining adapter sequences are trimmed. A Sequence Alignment Map (SAM) output file is generated which is then converted to a Binary Alignment Map (BAM) file. This file is then sorted by chromosome, indexed, and PCR duplicate FASTQ entries are marked.

### B. Variant Calling – SNVs, MNVs and INDELs

The analysis pipeline identifies SNVs, MNVs and INDELs. SNVs and MNVs reflect changes in the identity of a nucleotide (or nucleotides) at a specific position (or positions) without any change to the total number of nucleotides detected. SNVs are single nucleotide variants, in which the identity of only a single nucleotide is changed. MNVs are considered to be two or more adjacent or nearby SNVs detected on the same haplotype in an individual. INDELs refer to both insertions and deletions, in which the total number of nucleotides is altered relative to wild type (i.e., additional nucleotides are present, or nucleotides are absent). Paired sample variant calling is performed on tumor samples and their respective matched normal samples. Filtering is performed to remove low quality sequence data and sources of sequencing artifacts.

### C. Variant Annotation

Predicted functional effect and clinical interpretation for each mutation is curated by automated software using information from several databases and clinical evidence, including information derived from the FDA-recognized content (https://www.fda.gov/media/152847/download) of Memorial Sloan Kettering’s Precision Oncology Knowledge Base (MSK OncoKB, https://www.oncokb.org) and Tempus’ custom proprietary database using criteria that include known evolutionary models, functional data, clinical data, known gene-disease relationships, hotspot regions within genes, internal and external somatic databases, primary literature, and the unique combination of data from having paired tumor/normal DNA.

To determine that variant calling can be performed with high levels of confidence, there are four quality control steps performed as part of xT CDx sequence data analyses:

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- Alignment and Coverage Quality Control: This first process computes a series of statistics on the output of the reference genome alignment process such as read mapping rates, on target rates, and PCR duplication rates. The second process computes the depth and uniformity of coverage for all bases analyzed by the assay. The goal is to confirm that all bases within the target regions are sequenced to sufficient depth so that if a variant were present, it would be called successfully down to the lower Limit of Detection (LoD) of the assay.
- Sequence Data Quality Control: This includes per base quality scores for both forward and reverse reads which are output from the sequencer.
- Variant Quality Control: This includes sequencing of control samples and analysis of results against specifications.
- Sample Provenance Quality Control: In order to confirm that the specimen is free from exogenous DNA which may result in false positive results, the tumor sample is analyzed for common germline polymorphisms at low variant allele frequency (VAF). If the ratio of known germline polymorphisms to all mutations exceeds a pre-specified cutoff, the sample will be flagged as potentially contaminated by another human specimen. Concomitantly, the tumor and normal sample are compared against one another along a pre-specified set of common germline polymorphisms in order to confirm that both the normal and tumor specimens were derived from the same individual. If the discordance in common germline polymorphism minor allele frequency between the tumor specimen and the normal specimen exceeds a pre-specified threshold, the analysis will be flagged for further review.

## D. MSI Status Calling

xT CDx analyzes 239 microsatellite loci that are frequently unstable in tumors with mismatch repair deficiencies to determine the frequency of DNA slippage events. The information detected is used by the MSI classification algorithm to classify tumors into three categories: Microsatellite Instability High (MSI-H), Microsatellite Stable (MSS) and Equivocal (MSI cannot be determined).

To be an MSI locus mapping read, the read must be mapped to the MSI locus during the alignment step in the xT CDx bioinformatics pipeline. The mapping read must also contain the 5 base pairs in both the front and rear flank of the microsatellite, with any number of the expected repeating units in between. The minimum required number of reads must map to a microsatellite in both the normal sample and the tumor sample for it to be included in the test. A prespecified threshold of microsatellite loci on the panel must be included for the algorithm to be run. If these thresholds are not met, no MSI status is reported.

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Each microsatellite is tested for instability, as measured by changes in the number of repeat units in the tumor sample compared to the normal sample using the Kolmogorov-Smirnov test. If p :S 0.05, the locus is considered unstable and used to predict the binary MSI-H status based on the proportion of unstable microsatellites. The proportion of unstable microsatellites is entered into a univariate logistic regression classifier.

The classifier groups samples into three categories, MSI-H, MSS and equivocal, based on where this probability falls relative to two probability thresholds, a high confidence MSS threshold and a high confidence MSI-H threshold. If the probability that a sample is MSI-H exceeds the high confidence MSI-H threshold, the sample is classified as MSI-H. If the probability that a sample is MSI-H falls below the high confidence MSS threshold, the sample is considered MSS. For samples where the probability falls between the high confidence MSS and high confidence MSI-H threshold, the MSI status cannot be called with confidence and the xT CDx report will not include a definitive status, mark the MSI result as "equivocal" and will recommend further testing. In addition, if the tumor proportion falls below the established LoD for the MSI test (30% tumor purity), the xT CDx report will not include an MSI status (result not determined) and will recommend further testing.

## 7. Controls

### A. Matched-Normal Control

DNA is extracted from a patient-matched normal specimen, for use as a matched normal control from either blood or saliva. One matched normal control is required for each patient.

### B. Positive Control

A positive control sample containing known variants at defined allele frequencies is included with each sequencing run. Positive controls are rotated in order to evaluate variants from different genes and at different frequencies. The positive control is a contrived material with synthetically derived variants. Data generated from the positive control sample is analyzed using the same data analysis pipeline as patient samples, and frequencies of the detected mutations are reviewed to determine if (1) the known mutations are among those called, and (2) the observed frequencies for the known mutations match their pre-defined acceptable range, which is determined as three standard deviations from the average of the observed values. If known mutations in the positive control sample are not called, and the observed frequency of any mutation differs from expected value by more than the established boundary this results in a quality control (QC) failure.

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# C. No Template Control (NTC)

A NTC is included at nucleic acid extraction, library preparation, and hybridization/capture The NTCs have predefined acceptability criteria in each step.

# 8. Quality Metrics

Reporting takes in account the quality metrics outlined in Table 2. Quality metrics are assessed across the following categories:

- Batch-level: Metrics that are quantified per sequencing run; if the external control fails these criteria, no results are reported for the entire batch of samples.
- Sample-level: Metrics that are quantified per sample; no device results are generated for samples failing these metrics.
- Paired-sample level: Assessed to confirm that given tumor and matched normal samples are derived from the same individual.
- Analyte-level: Metrics that are quantified for individual alteration types and positions. Variants passing analyte-level metrics are reported.

Table 2. Summary of xT CDx post-sequencing key quality control metrics

|  Metric | Batch/Sample/Analyte | Required Value  |
| --- | --- | --- |
|  Positive Control | Batch level | Known sequence mutations are detected and pass acceptance criteria  |
|  On Target Rate (Normal) | Sample level | ◇ 50%  |
|  Tumor On Target Rate (Tumor) | Sample level | ◇ 50%  |
|  Total Unique Reads (Normal) | Sample level | ◇ 5,000,000  |
|  Total Unique Reads (Tumor) | Sample level | ◇ 8,000,000  |
|  Total Reads (Normal) | Sample level | ◇ 10,000,000  |
|  Total Reads (Tumor) | Sample level | ◇ 10,000,000  |
|  PCR Duplication Rate (Normal) | Sample level | < 85%  |
|  PCR Duplication Rate (Tumor) | Sample level | < 85%  |

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|  Metric | Batch/Sample/Analyte | Required Value  |
| --- | --- | --- |
|  Fingerprint Score | Paired-sample level | >0.65 to demonstrate that there are similar common SNVs between the tumor and normal samples  |
|  Hotspot substitutions | Analyte level | VAF◆3%  |
|  Non-hotspot substitutions | Analyte level | VAF◆5%  |
|  Hotspot INDELs | Analyte level | VAF◆5%  |
|  Non-hotspot INDELs | Analyte level | VAF◆10%  |

# VI. ALTERNATIVE PRACTICES AND PROCEDURES

There are FDA-approved CDx alternatives for the detection of NRAS and KRAS biomarkers to direct the use of specific therapies using FFPE CRC specimens. The approved CDx tests are listed in Table 3, below.

Table 3. Alternative FDA-approved CDx assays for CDx biomarkers identified by xT CDx

|  Biomarker | Device | Company | Technology | Therapy  |
| --- | --- | --- | --- | --- |
|  NRAS Variants | Praxis Extended Ras Panel | Illumina | NGS1 | Vectibix (panitumumab)  |
|   |  FoundationOne CDx | Foundation Medicine, Inc. | NGS1 | Vectibix (panitumumab)  |
|  KRAS Variants | Cobas KRAS Mutation Test | Roche Molecular Systems, Inc. | PCR2 | Erbitux (cetuximab) Vectibix (panitumumab)  |
|   |  therascreen KRAS RGQ PCR Kit | QIAGEN | PCR2 | Erbitux (cetuximab) Vectibix (panitumumab)  |
|   |  Praxis Extended Ras Panel | Illumina | NGS1 | Erbitux (cetuximab) Vectibix (panitumumab)  |

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|  Biomarker | Device | Company | Technology | Therapy  |
| --- | --- | --- | --- | --- |
|   | FoundationOne CDx | Foundation Medicine, Inc. | NGS^{1} | Erbitux (cetuximab)
Vectibix (panitumumab)  |
|   |  ONCO/Reveal Dx Lung and Colon Cancer Assay (O/RDx-LCCA) | Pillar Biosciences, Inc. | NGS^{1} | Erbitux (cetuximab)
Vectibix (panitumumab)  |

1. NGS = Next Generation Sequencing
2. PCR = Polymerase Chain Reaction

For additional details see the FDA List of Cleared or Approved Companion Diagnostic Devices at: https://www.fda.gov/medical-devices/vitro-diagnostics/list-cleared-or-approved-companion-diagnostic-devices-vitro-and-imaging-tools

## VII. MARKETING HISTORY

xT CDx has not been marketed in the United States or any foreign country.

Tempus initially designed and developed the Tempus xT Laboratory-Developed Test (LDT), and the first commercial sample was tested in 2017. The Tempus xT LDT has been used to detect the presence of genomic alterations in FFPE tumor tissue specimens by sequencing of patient-matched tumor and normal tissue. Tempus xT LDT is not FDA-cleared or approved.

## VIII. POTENTIAL ADVERSE EFFECTS OF THE DEVICE ON HEALTH

Failure of the device to perform as expected or failure to correctly interpret test results may lead to incorrect test results, and subsequently, inappropriate patient management decisions. Patients with false results may be inappropriately treated with one of the therapies listed in the above intended use statement and may experience adverse reactions associated with inappropriate therapy. Patients with false negative results may not be considered for treatment with the indicated therapy. There is also a risk of delayed results, which may lead to delay of treatment with indicated therapy.

For the specific adverse events related to the approved therapeutics, please see approved FDA therapeutic product labeling.

## IX. SUMMARY OF NONCLINICAL STUDIES

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# A. Laboratory Studies

## 1. Sample Coverage

The sequencing read depth of the device was evaluated by sequencing duplicate libraries from 10 normal diploid samples using worst-case run conditions for detection of somatic alterations. The mean coverage (read depth) for all targeted regions across all samples ranged from 508x to 1218x (with an overall mean of 905x). All sequenced libraries had more than 98% of exons sequenced with a read depth ⇄ 150x. The interlibrary median mean coverage for all targeted hotspots ranged from 564.1x to 1557.4x (mean of 1041.6x). The coverage of target regions enables calling of variants at the xT CDx reporting thresholds, which are 3% VAF for substitutions and 5% for INDELs at hotspots, and 5% for substitutions and 10% for INDELs at non-hotspots.

## 2. Accuracy – Comparison to an Orthogonal Method

### i. CDx Accuracy – KRAS and NRAS mutation detection in CRC samples

To support the use of xT CDx as a companion diagnostic for cetuximab and panitumumab, a total of 349 banked CRC samples were tested by xT CDx and compared to results obtained with the original CDx assays; the FDA approved QIAGEN therascreen KRAS RGQ PCR Kit (therascreen) and the Illumina Praxis Extended RAS Panel (Praxis), respectively. Concordance between xT CDx results and these approved CDx devices is presented in Section X. Summary of Primary Clinical Studies.

In short, the agreement study was conducted using the comparison of xT CDx to two (2) orthogonal methods (FDA approved Praxis and therascreen assays). A total set of 190 patient-matched tumor and normal CRC samples was sequenced with xT CDx and Praxis, and a total set of 250 patient-matched tumor and normal CRC samples was sequenced with xT CDx and therascreen. The reported mutations from xT CDx were compared with results of Praxis and therascreen assays and used to calculate the PPA and NPA. Of the 84 CDx variants positively identified by Praxis, 83 were identified by xT CDx yielding a PPA of 98.8% (95% CI: 93.54-99.97%); of the 88 CDx variants positively identified by therascreen, 87 were identified by xT CDx, yielding a PPA of 98.86% (95% CI: 93.83-99.97%). Of the 8400 CDx variants identified as negative by Praxis, 8400 were identified as negative by xT CDx, yielding a NPA of 100.0% (95% CI: 99.96-100.0%); of the 1134 CDx variants identified as negative by therascreen, 1134 were identified as negative by xT CDx, yielding a NPA of 100.0% (95% CI: 99.68-100%). These results are shown in Section X. Summary of Primary Clinical Studies (Tables 58-70).

In addition to concordance with FDA approved Praxis and therascreen assays using clinical study samples, analytical accuracy of detecting KRAS and NRAS mutations by xT CDx was also evaluated by comparison with results obtained using a validated NGS-based orthogonal method (OM1), using 69 patient-matched tumor and normal CRC

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samples. Summary of these CDx analytical accuracy results, as well as a breakdown by KRAS and NRAS, are provided below in Table 4 and Table 5, respectively. Note that CDx variants are presented simply as variants, which are defined as the list of mutations detected for clinical use (i.e., by the Praxis and therascreen assays), which includes no insertions or deletions, and does not distinguish between SNVs and MNVs. For comparison with OM1, negative CDx variant positions were defined as those positions at which a unique CDx variant was detected in at least one tested sample, by OM1. (For comparison with the Praxis and therascreen assays that is presented in Section X, negative CDx variant positions were defined as all positions at which those assays could detect a CDx variant.) The reported variants from xT CDx were compared with results of OM1 and used to calculate the PPA and NPA. Of the 31 CDx variants positively identified by OM1, 31 were identified by xT CDx, yielding a PPA of  $100\%$  (95% CI: 88.8-100.0%). Of the 649 CDx variants identified as negative by OM1, 648 were identified as negative by xT CDx, yielding a NPA of  $99.8\%$  (95% CI: 99.1-100.0%).

# CDx Accuracy (KRAS)

The detection of KRAS mutations by xT CDx was compared with results obtained using an externally validated NGS assay as an orthogonal method (OM1), using a total set of 69 patient-matched tumor and normal CRC samples, sequenced with xT CDx. The reported KRAS CDx variants from xT CDx were compared to results of OM1 and used to calculate the PPA and NPA. Of the 30 CDx variants in KRAS that were positively identified by OM1, 30 were identified by xT CDx, yielding a PPA of  $100\%$  (95% CI: 88.43-100.0%). Of the 591 CDx variants in KRAS identified as negative by OM1, 590 were identified as negative by xT CDx, yielding a NPA of  $99.83\%$  (95% CI: 99.06-100.0%).

Table 4. Positive and negative percent agreement of KRAS mutation results in CRC samples from the xT CDx Tumor Profiling Accuracy Study (n = 69) compared to OM1

|  Location | Mutation | TP | FN | FP | TN | Total | PPA (95% CI)* | NPA (95%CI)*  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  KRAS Exon 2 | c.34G>T | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|   |  c.34G>A | 2 | 0 | 0 | 67 | 69 | 100.0 (15.81-100) | 100.0 (94.64-100)  |
|   |  c.34G>C | 1 | 0 | 0 | 68 | 69 | 100.0 (2.50-100) | 100.0 (94.72-100)  |
|   |  c.34_35GG>TT | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |

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|  Location | Mutation | TP | FN | FP | TN | Total | PPA (95% CI)* | NPA (95%CI)*  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   | c.34_35GG>AA | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|   |  c.34_36GGT>T GG | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|   |  c.35G>A | 12 | 0 | 1 | 56 | 69 | 100.0 (73.54-100) | 98.2 (90.61-100)  |
|   |  c.35G>T | 5 | 0 | 0 | 64 | 69 | 100.0 (47.82-100) | 100.0 (94.40-100)  |
|   |  c.35G>C | 2 | 0 | 0 | 67 | 69 | 100.0 (15.81-100) | 100.0 (94.64-100)  |
|   |  c.37G>T | 1 | 0 | 0 | 68 | 69 | 100.0 (2.50-100) | 100.0 (94.72-100)  |
|   |  c.37G>C | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|   |  c.38G>A | 3 | 0 | 0 | 66 | 69 | 100.0 (29.24-100) | 100.0 (94.56-100)  |
|   |  c.38_39GC>TT | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|   |  c.38_39GC>AA | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|   |  c.38_39GC>AT | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|  KRAS Exon 3 | c.175G>A | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|   |  c.176C>G | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|   |  c.181C>A | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|   |  c.181C>G | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|   |  c.182A>T | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|   |  c.182A>G | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |

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|  Location | Mutation | TP | FN | FP | TN | Total | PPA (95% CI)* | NPA (95%CI)*  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   | c.183A>C | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|   |  c.183A>T | 3 | 0 | 0 | 66 | 69 | 100.0 (29.24-100) | 100.0 (94.56-100)  |
|  KRAS Exon 4 | c.351A>C | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|   |  c.351A>T | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|   |  c.436G>A | 1 | 0 | 0 | 68 | 69 | 100.0 (2.50-100) | 100.0 (94.72-100)  |
|   |  c.436G>C | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|   |  c.437C>T | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |

NE = not estimable
*CI = Confidence Interval, calculated using the Clopper-Pearson Exact Method

In addition, the detection of KRAS mutations by xT CDx was also assessed by comparison with results obtained using 2 additional validated orthogonal methods (FDA approved Praxis and therascreen assays). A total set of 190 patient-matched tumor and normal CRC samples was sequenced with xT CDx and Praxis, and a total set of 250 patient-matched tumor and normal CRC samples was sequenced with xT CDx and therascreen. The reported KRAS CDx variants from xT CDx were compared to results of Praxis and therascreen and used to calculate the PPA and NPA. Of the 75 CDx variants in KRAS that were positively identified by Praxis, 74 were identified by xT CDx, yielding a PPA of 98.67% (95% CI: 92.79-99.97%); of the 88 CDx variants in KRAS positively identified by therascreen, 87 were identified by xT CDx, yielding a PPA of 98.86% (95% CI: 93.83-99.97%). Of the 3276 CDx variants in KRAS identified as negative by Praxis, 3276 were identified as negative by xT CDx, yielding a NPA of 100.0% (95% CI: 99.89-100%); of the 1134 CDx variants in KRAS identified as negative by therascreen, 1134 were identified as negative by xT CDx, yielding a NPA of 100.0% (95% CI: 99.68-100%). These data are summarized and presented in the tables for the CDx concordance captured in the CDx Clinical Validation presented in Section X. Summary of Primary Clinical Studies, Table 63 and Table 69.

## CDx Accuracy (NRAS)

The detection of NRAS mutations by xT CDx was compared to results obtained using an externally validated NGS assay as an orthogonal method (OM1). A total set of 69 patient-

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matched tumor and normal CRC samples was sequenced with OM1 and xT CDx. The reported NRAS CDx variants from xT CDx were compared to results of OM1 and used to calculate the PPA and NPA. Of the 1 CDx variant in NRAS that was positively identified by OM1, 1 was identified by xT CDx, yielding a PPA of  $100\%$  (95% CI: 2.5-100.0%). Of the 68 CDx variants in NRAS identified as negative by the OM1, 68 were identified as negative by xT CDx, yielding a NPA of  $100\%$  (95% CI: 94.72-100.0%).

The following CDx accuracy Table 5 reflects results from CRC samples that were also tested as a part of the tumor profiling accuracy study compared to OM1, and summarizes the results for NRAS, showing all CDx variants by exon and variant.

Table 5. Positive and negative percent agreement of NRAS mutation results in CRC samples from the xT CDx Tumor Profiling Accuracy Study (n = 69) compared to OM1

|  Location | Mutation | TP | FN | FP | TN | Total | PPA (95% CI)* | NPA (95%CI)*  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  NRAS Exon 2 | c.34G>T | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|   |  c.34G>A | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|   |  c.34G>C | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|   |  c.34_35GG>TT | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|   |  c.34_35GG>AA | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|   |  c.34_36GGT>T GG | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|   |  c.35G>A | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|   |  c.35G>T | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|   |  c.35G>C | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|   |  c.37G>T | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|   |  c.37G>C | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |

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|   | c.38G>A | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |  c.38_39GC>TT | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|   |  c.38_39GC>AA | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|   |  c.38_39GC>AT | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|  NRAS Exon 3 | c.175G>A | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|   |  c.176C>G | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|   |  c.181C>A | 1 | 0 | 0 | 68 | 69 | 100.0 (2.50-100) | 100.0 (94.72-100)  |
|   |  c.181C>G | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|   |  c.182A>T | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|   |  c.182A>G | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|   |  c.183A>C | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|   |  c.183A>T | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|  NRAS Exon 4 | c.351A>C | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|   |  c.351A>T | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|   |  c.436G>A | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
|   |  c.436G>C | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |

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|   | c.437C>T | 0 | 0 | 0 | 69 | 69 | NE | 100.0 (94.79-100)  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |

NE = not estimable
*CI = Confidence Interval, calculated using the Clopper-Pearson Exact Method

The detection of NRAS mutations by xT CDx was also assessed by comparison to results obtained using an additional validated orthogonal method (FDA approved Praxis). A total set of 190 patient-matched tumor and normal CRC samples was sequenced with xT CDx and Praxis. The reported NRAS variants from xT CDx were compared to results of Praxis and used to calculate the PPA and NPA. Of the 9 CDx variants in NRAS that were positively identified by Praxis, 9 were identified by xT CDx, yielding a PPA of  $100.0\%$  (95% CI: 66.37-100%). Of the 5124 CDx variants in NRAS identified as negative by Praxis, 5124 were identified as negative by xT CDx, yielding a NPA of  $100.0\%$  (95% CI: 99.93-100%). These data are summarized and presented in the tables for the CDx concordance captured in the CDx Clinical Validation presented in Section X. Summary of Primary Clinical Studies, Table 63.

# ii. Tumor Profiling Accuracy

The detection of mutations by xT CDx was assessed by comparison with results obtained using an externally validated NGS assay as an orthogonal method (OM1). A total set of 416 patient-matched tumor and normal samples representing 31 cancer types was sequenced with xT CDx (Table 6). The reported variants from xT CDx were compared with results of OM1 and used to calculate the PPA and NPA. Of the 1232 variants positively identified by OM1, 971, 18, 58, and 174 were identified as SNVs, MNVs, insertions and deletions respectively by xT CDx, observed in 373 exons across 84 genes, yielding an overall PPA of  $99.1\%$  (95% CI: 98.4-99.6%). Of the 415,000 variants identified as negative by OM1, 19, 3, 17 and 41 were identified as SNV, MNV, insertion and deletion variants respectively by xT CDx, yielding an overall NPA of  $100\%$  (95% CI: 100-100%).

Table 6. Distribution of cancer type among samples

|  Cancer Type | Count  |
| --- | --- |
|  Colorectal Cancer | 69  |
|  Breast Cancer | 44  |
|  Ovarian Cancer | 38  |
|  Glioblastoma | 34  |
|  Non-Small Cell Lung Cancer | 29  |
|  Endometrial Cancer | 26  |
|  Clear Cell Renal Cell Carcinoma | 22  |

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|  Cancer Type | Count  |
| --- | --- |
|  Bladder Cancer | 18  |
|  Melanoma | 17  |
|  Pancreatic Cancer | 14  |
|  Thyroid Cancer | 12  |
|  Low Grade Glioma | 12  |
|  Sarcoma | 10  |
|  Tumor of Unknown Origin | 8  |
|  Meningioma | 7  |
|  Prostate Cancer | 7  |
|  Gastrointestinal Stromal Tumor | 7  |
|  Endocrine Tumor | 6  |
|  Gastric Cancer | 5  |
|  Head and Neck Squamous Cell Carcinoma | 4  |
|  Kidney Cancer | 3  |
|  Brain Cancer | 3  |
|  Small Cell Lung Cancer | 3  |
|  Biliary Cancer | 3  |
|  Cervical Cancer | 3  |
|  Esophageal Cancer | 3  |
|  Oropharyngeal Cancer | 2  |
|  Liver Cancer | 2  |
|  Head and Neck Cancer | 2  |
|  Mesothelioma | 2  |
|  Adrenal Cancer | 1  |

The xT CDx tumor profiling analytical studies included insertions and deletions up to and greater than 25 bps in size. In clinical use, xT CDx will report insertions and deletions up to 25 bps. Concordance of variants was evaluated in both hotspot and non-hotspot regions. Differences in the number of reportable variants between the two assays were expected as a result of pipeline-specific variant filtering or germline variant classifications. In particular, the orthogonal method evaluates tumor samples followed by germline mutation filtering, whereas xT CDx sequences tumor and patient-matched

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normal samples to allow personalized subtraction of germline variants from tumor sequencing results. Across all samples evaluated, a total of 148 variants reported as somatic by the orthogonal method were identified in germline samples by xT CDx (Table 7).

Table 7. Germline variants that would be subtracted by xT CDx but were classified as somatic by the orthogonal method

|  Type | Number of Variants  |
| --- | --- |
|  Substitutions | 139  |
|  INDELs | 9  |
|  All Short Variants | 148  |

These were included as an output of xT CDx for the purposes of this analytical concordance study. A summary of Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) is provided in Table 8, below, for SNVs, MNVs, insertions and deletions. Accuracy by gene and variant type is provided in Table 9.

Table 8. Concordance summary for short variants

|  Variant Type | Total Unique Variants | True Positives | False Positives | False Negatives | True Negatives | PPA [Exact 95% CI] | NPA [Exact 95% CI]  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  All Variants | 1028 | 1221 | 80 | 11 | 414920 | 99.1% [98.4%, 99.6%] | 100.0% [100.0%, 100.0%]  |
|  All SNVs | 736 | 971 | 19 | 8 | 297042 | 99.2% [98.4%, 99.6%] | 100.0% [100.0%, 100.0%]  |
|  All MNVs | 22 | 18 | 3 | 1 | 8881 | 94.7% [74.0%, 99.9%] | 100.0% [99.9%, 100.0%]  |
|  All Insertions | 71 | 58 | 17 | 2 | 28656 | 96.7% [88.5%, 99.6%] | 100.0% [100.0%, 100.0%]  |
|  All Deletions | 199 | 174 | 41 | 0 | 80341 | 100.0% [97.9%, 100.0%] | 100.0% [100.0%, 100.0%]  |

Table 9. Accuracy by gene and variant type

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|  Gene | Total Exons | SNVs |   |   | MNVs |   |   | Insertions |   |   | Deletions  |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |  # of SNVs | SNV PPA [95% CI] | SNV NPA [95% CI] | # of MNVs | MNV PPA [95% CI] | MNV NPA [95% CI] | # of Ins | Ins PPA [95% CI] | Ins NPA [95% CI] | # of Del | Del PPA [95% CI] | Del NPA [95% CI]  |
|  ABL1 | 2 | 2 | 100.0% [15.8%, 100.0%] | 100.0% [99.5%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  AKT1 | 1 | 5 | 100.0% [47.8%, 100.0%] | 100.0% [99.1%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  ALK | 1 | 1 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  APC | 10 | 67 | 100.0% [94.6%, 100.0%] | 100.0% [100.0%, 100.0%] | 0 | - | - | 4 | 75.0% [19.4%, 99.4%] | 100.0% [99.8%, 100.0%] | 24 | 100.0% [85.8%, 100.0%] | 100.0% [100.0%, 100.0%]  |
|  ATM | 24 | 33 | 100.0% [89.4%, 100.0%] | 100.0% [100.0%, 100.0%] | 1 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%] | 2 | 100.0% [15.8%, 100.0%] | 100.0% [99.5%, 100.0%] | 6 | 100.0% [54.1%, 100.0%] | 100.0% [99.8%, 100.0%]  |
|  BAP1 | 9 | 13 | 100.0% [75.3%, 100.0%] | 100.0% [99.9%, 100.0%] | 0 | - | - | 0 | - | - | 3 | 100.0% [29.2%, 100.0%] | 100.0% [99.7%, 100.0%]  |
|  BRAF | 2 | 37 | 100.0% [90.5%, 100.0%] | 100.0% [99.9%, 100.0%] | 2 | 100.0% [15.8%, 100.0%] | 100.0% [99.5%, 100.0%] | 1 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%] | 2 | 100.0% [15.8%, 100.0%] | 100.0% [99.5%, 100.0%]  |
|  BRCA1 | 4 | 7 | 100.0% [59.0%, 100.0%] | 100.0% [99.9%, 100.0%] | 0 | - | - | 0 | - | - | 5 | 100.0% [47.8%, 100.0%] | 100.0% [99.8%, 100.0%]  |
|  BRCA2 | 7 | 14 | 92.9% [66.1%, 99.8%] | 100.0% [99.9%, 100.0%] | 1 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%] | 0 | - | - | 1 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%]  |
|  CBL | 2 | 3 | 100.0% [29.2%, 100.0%] | 100.0% [99.7%, 100.0%] | 1 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%] | 0 | - | - | 0 | - | -  |
|  CCNE1 | 2 | 2 | 100.0% [15.8%, 100.0%] | 100.0% [99.1%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  CD274 | 1 | 2 | 100.0% [15.8%, 100.0%] | 100.0% [99.1%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  CDH1 | 10 | 10 | 100.0% [69.2%, 100.0%] | 100.0% [99.9%, 100.0%] | 0 | - | - | 5 | 100.0% [47.8%, 100.0%] | 100.0% [99.8%, 100.0%] | 3 | 100.0% [29.2%, 100.0%] | 100.0% [99.7%, 100.0%]  |

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|  Gene | Total Exons | SNVs |   |   | MNVs |   |   | Insertions |   |   | Deletions  |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |  # of SNVs | SNV PPA [95% CI] | SNV NPA [95% CI] | # of MNVs | MNV PPA [95% CI] | MNV NPA [95% CI] | # of Ins | Ins PPA [95% CI] | Ins NPA [95% CI] | # of Del | Del PPA [95% CI] | Del NPA [95% CI]  |
|  CDK4 | 1 | 1 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  CDKN2A | 2 | 11 | 72.7% [39.0%, 94.0%] | 100.0% [99.9%, 100.0%] | 0 | - | - | 1 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%] | 4 | 100.0% [39.8%, 100.0%] | 99.9% [99.7%, 100.0%]  |
|  CHEK2 | 1 | 0 | - | - | 0 | - | - | 0 | - | - | 2 | 100.0% [15.8%, 100.0%] | 100.0% [99.1%, 100.0%]  |
|  CTNNB1 | 2 | 16 | 100.0% [79.4%, 100.0%] | 100.0% [99.9%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  DDR2 | 2 | 2 | 100.0% [15.8%, 100.0%] | 100.0% [99.5%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  DNMT3A | 3 | 6 | 100.0% [54.1%, 100.0%] | 100.0% [99.8%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  EGFR | 9 | 9 | 100.0% [66.4%, 100.0%] | 100.0% [99.9%, 100.0%] | 0 | - | 99.8% [98.6%, 100.0%] | 1 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%] | 1 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%]  |
|  ERBB2 | 6 | 6 | 100.0% [54.1%, 100.0%] | 100.0% [99.8%, 100.0%] | 0 | - | - | 2 | 100.0% [15.8%, 100.0%] | 100.0% [99.1%, 100.0%] | 0 | - | -  |
|  ERBB3 | 1 | 1 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  ERBB4 | 2 | 2 | 100.0% [15.8%, 100.0%] | 100.0% [99.5%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  ESR1 | 3 | 7 | 100.0% [59.0%, 100.0%] | 100.0% [99.8%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  EZH2 | 1 | 2 | 100.0% [15.8%, 100.0%] | 100.0% [99.5%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  FBXW7 | 6 | 20 | 100.0% [83.2%, 100.0%] | 100.0% [100.0%, 100.0%] | 0 | - | - | 0 | - | - | 2 | 100.0% [15.8%, 100.0%] | 100.0% [99.5%, 100.0%]  |

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|  Gene | Total Exons | SNVs |   |   | MNVs |   |   | Insertions |   |   | Deletions  |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |  # of SNVs | SNV PPA [95% CI] | SNV NPA [95% CI] | # of MNVs | MNV PPA [95% CI] | MNV NPA [95% CI] | # of Ins | Ins PPA [95% CI] | Ins NPA [95% CI] | # of Del | Del PPA [95% CI] | Del NPA [95% CI]  |
|  FGFR1 | 4 | 5 | 100.0% [47.8%, 100.0%] | 100.0% [99.8%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  FGFR2 | 2 | 4 | 100.0% [39.8%, 100.0%] | 100.0% [99.5%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  FGFR3 | 4 | 6 | 100.0% [54.1%, 100.0%] | 100.0% [99.8%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  FGFR4 | 5 | 7 | 100.0% [59.0%, 100.0%] | 100.0% [99.9%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  FLT3 | 2 | 2 | 100.0% [15.8%, 100.0%] | 100.0% [99.5%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  GATA2 | 2 | 3 | 100.0% [29.2%, 100.0%] | 100.0% [99.7%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  GATA3 | 5 | 6 | 100.0% [54.1%, 100.0%] | 100.0% [99.8%, 100.0%] | 0 | - | - | 5 | 100.0% [47.8%, 100.0%] | 100.0% [99.8%, 100.0%] | 2 | 100.0% [15.8%, 100.0%] | 100.0% [99.5%, 100.0%]  |
|  GNA11 | 1 | 1 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  GNAS | 1 | 3 | 100.0% [29.2%, 100.0%] | 100.0% [99.7%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  HRAS | 1 | 3 | 100.0% [29.2%, 100.0%] | 100.0% [99.7%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  IDH1 | 1 | 13 | 100.0% [75.3%, 100.0%] | 100.0% [99.8%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  IDH2 | 1 | 1 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  JAK1 | 1 | 1 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |

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|  Gene | Total Exons | SNVs |   |   | MNVs |   |   | Insertions |   |   | Deletions  |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |  # of SNVs | SNV PPA [95% CI] | SNV NPA [95% CI] | # of MNVs | MNV PPA [95% CI] | MNV NPA [95% CI] | # of Ins | Ins PPA [95% CI] | Ins NPA [95% CI] | # of Del | Del PPA [95% CI] | Del NPA [95% CI]  |
|  JAK2 | 1 | 1 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  JAK3 | 2 | 4 | 100.0% [39.8%, 100.0%] | 100.0% [99.5%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  KDR | 3 | 4 | 100.0% [39.8%, 100.0%] | 100.0% [99.8%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  KIT | 6 | 7 | 100.0% [59.0%, 100.0%] | 100.0% [99.9%, 100.0%] | 0 | - | - | 2 | 50.0% [1.3%, 98.7%] | 100.0% [99.5%, 100.0%] | 3 | 100.0% [29.2%, 100.0%] | 100.0% [99.7%, 100.0%]  |
|  KRAS | 3 | 73 | 100.0% [95.1%, 100.0%] | 100.0% [99.9%, 100.0%] | 1 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%] | 0 | - | - | 0 | - | -  |
|  MAP2K1 | 2 | 2 | 100.0% [15.8%, 100.0%] | 100.0% [99.5%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  MAX | 1 | 0 | - | - | 0 | - | - | 0 | - | - | 1 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%]  |
|  MDM2 | 1 | 1 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  MED12 | 2 | 2 | 100.0% [15.8%, 100.0%] | 100.0% [99.5%, 100.0%] | 1 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%] | 0 | - | - | 0 | - | -  |
|  MET | 3 | 4 | 100.0% [39.8%, 100.0%] | 100.0% [99.8%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  MSH2 | 5 | 6 | 100.0% [54.1%, 100.0%] | 100.0% [99.8%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  MTOR | 2 | 2 | 100.0% [15.8%, 100.0%] | 100.0% [99.5%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  MYCL | 1 | 0 | - | - | 1 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%] | 0 | - | - | 0 | - | -  |

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|  Gene | Total Exons | SNVs |   |   | MNVs |   |   | Insertions |   |   | Deletions  |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |  # of SNVs | SNV PPA [95% CI] | SNV NPA [95% CI] | # of MNVs | MNV PPA [95% CI] | MNV NPA [95% CI] | # of Ins | Ins PPA [95% CI] | Ins NPA [95% CI] | # of Del | Del PPA [95% CI] | Del NPA [95% CI]  |
|  MYD88 | 2 | 3 | 100.0% [29.2%, 100.0%] | 100.0% [99.5%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  NF1 | 32 | 30 | 100.0% [88.4%, 100.0%] | 100.0% [100.0%, 100.0%] | 1 | 0.0% [0.0%, 97.5%] | 100.0% [99.1%, 100.0%] | 1 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%] | 14 | 100.0% [76.8%, 100.0%] | 100.0% [99.9%, 100.0%]  |
|  NF2 | 7 | 8 | 100.0% [63.1%, 100.0%] | 100.0% [99.9%, 100.0%] | 0 | - | - | 1 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%] | 4 | 100.0% [39.8%, 100.0%] | 100.0% [99.8%, 100.0%]  |
|  NFE2L2 | 1 | 1 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%] | 0 | - | - | 0 | - | - | 1 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%]  |
|  NOTCH1 | 17 | 19 | 100.0% [82.4%, 100.0%] | 100.0% [99.9%, 100.0%] | 1 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%] | 0 | - | - | 5 | 100.0% [47.8%, 100.0%] | 100.0% [99.8%, 100.0%]  |
|  NRAS | 2 | 7 | 100.0% [59.0%, 100.0%] | 100.0% [99.8%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  PDGFRA | 5 | 5 | 100.0% [47.8%, 100.0%] | 100.0% [99.8%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  PIK3CA | 8 | 75 | 97.3% [90.7%, 99.7%] | 100.0% [99.9%, 100.0%] | 0 | - | - | 0 | - | - | 3 | 100.0% [29.2%, 100.0%] | 100.0% [99.7%, 100.0%]  |
|  PIK3R1 | 10 | 9 | 100.0% [66.4%, 100.0%] | 100.0% [99.9%, 100.0%] | 0 | - | - | 3 | 100.0% [29.2%, 100.0%] | 100.0% [99.7%, 100.0%] | 12 | 100.0% [73.5%, 100.0%] | 100.0% [99.9%, 100.0%]  |
|  PPP2R1A | 2 | 4 | 100.0% [39.8%, 100.0%] | 100.0% [99.5%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  PTCH1 | 9 | 13 | 100.0% [75.3%, 100.0%] | 100.0% [99.9%, 100.0%] | 1 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%] | 0 | - | - | 3 | 100.0% [29.2%, 100.0%] | 100.0% [99.7%, 100.0%]  |
|  PTEN | 9 | 46 | 100.0% [92.3%, 100.0%] | 100.0% [100.0%, 100.0%] | 0 | - | - | 6 | 100.0% [54.1%, 100.0%] | 100.0% [99.8%, 100.0%] | 19 | 100.0% [82.4%, 100.0%] | 100.0% [99.9%, 100.0%]  |
|  PTPN11 | 3 | 2 | 100.0% [15.8%, 100.0%] | 100.0% [99.5%, 100.0%] | 0 | - | - | 0 | - | - | 1 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%]  |

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|  Gene | Total Exons | SNVs |   |   | MNVs |   |   | Insertions |   |   | Deletions  |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |  # of SNVs | SNV PPA [95% CI] | SNV NPA [95% CI] | # of MNVs | MNV PPA [95% CI] | MNV NPA [95% CI] | # of Ins | Ins PPA [95% CI] | Ins NPA [95% CI] | # of Del | Del PPA [95% CI] | Del NPA [95% CI]  |
|  RAF1 | 1 | 2 | 100.0% [15.8%, 100.0%] | 100.0% [99.5%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  RB1 | 16 | 15 | 100.0% [78.2%, 100.0%] | 100.0% [99.9%, 100.0%] | 0 | - | - | 4 | 100.0% [39.8%, 100.0%] | 100.0% [99.8%, 100.0%] | 9 | 100.0% [66.4%, 100.0%] | 100.0% [99.9%, 100.0%]  |
|  RPS6KB1 | 1 | 1 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  SF3B1 | 1 | 1 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  SMAD4 | 7 | 18 | 100.0% [81.5%, 100.0%] | 100.0% [99.9%, 100.0%] | 0 | - | - | 3 | 100.0% [29.2%, 100.0%] | 100.0% [99.7%, 100.0%] | 2 | 100.0% [15.8%, 100.0%] | 100.0% [99.5%, 100.0%]  |
|  SMARCB1 | 2 | 4 | 100.0% [39.8%, 100.0%] | 100.0% [99.7%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  SMO | 3 | 4 | 100.0% [39.8%, 100.0%] | 100.0% [99.8%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  SPOP | 1 | 1 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  STK11 | 7 | 8 | 87.5% [47.3%, 99.7%] | 100.0% [99.9%, 100.0%] | 0 | - | - | 3 | 100.0% [29.2%, 100.0%] | 100.0% [99.7%, 100.0%] | 1 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%]  |
|  TERT | 1 | 2 | 100.0% [15.8%, 100.0%] | 100.0% [99.5%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  TET2 | 6 | 20 | 100.0% [83.2%, 100.0%] | 100.0% [100.0%, 100.0%] | 0 | - | - | 1 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%] | 3 | 100.0% [29.2%, 100.0%] | 100.0% [99.7%, 100.0%]  |
|  TP53 | 8 | 205 | 99.5% [97.3%, 100.0%] | 100.0% [100.0%, 100.0%] | 8 | 100.0% [63.1%, 100.0%] | 100.0% [99.9%, 100.0%] | 9 | 100.0% [66.4%, 100.0%] | 100.0% [99.9%, 100.0%] | 24 | 100.0% [85.8%, 100.0%] | 100.0% [100.0%, 100.0%]  |
|  TSC1 | 8 | 8 | 100.0% [63.1%, 100.0%] | 100.0% [99.9%, 100.0%] | 0 | - | - | 1 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%] | 0 | - | -  |

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|  Gene | Total Exons | SNVs |   |   | MNVs |   |   | Insertions |   |   | Deletions  |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |  # of SNVs | SNV PPA [95% CI] | SNV NPA [95% CI] | # of MNVs | MNV PPA [95% CI] | MNV NPA [95% CI] | # of Ins | Ins PPA [95% CI] | Ins NPA [95% CI] | # of Del | Del PPA [95% CI] | Del NPA [95% CI]  |
|  TSC2 | 11 | 12 | 100.0% [73.5%, 100.0%] | 100.0% [99.9%, 100.0%] | 0 | - | - | 0 | - | - | 2 | 100.0% [15.8%, 100.0%] | 100.0% [99.5%, 100.0%]  |
|  U2AF1 | 1 | 1 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |
|  VHL | 3 | 9 | 100.0% [66.4%, 100.0%] | 100.0% [99.9%, 100.0%] | 0 | - | - | 4 | 100.0% [39.8%, 100.0%] | 100.0% [99.8%, 100.0%] | 12 | 100.0% [73.5%, 100.0%] | 100.0% [99.9%, 100.0%]  |
|  WT1 | 4 | 4 | 100.0% [39.8%, 100.0%] | 100.0% [99.8%, 100.0%] | 0 | - | - | 1 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%] | 0 | - | -  |
|  ZNF217 | 2 | 2 | 100.0% [15.8%, 100.0%] | 100.0% [99.5%, 100.0%] | 0 | - | - | 0 | - | - | 0 | - | -  |

# Accuracy - SNVs, MNVs, and INDELs

A breakdown of the Accuracy for SNVs, MNVs, and INDEL results by variant type, level and insertion/deletion size is provided in Table 10.

Table 10. Concordance breakdown for short variants inclusive of substitutions and INDELs

|  Variant Type | Total Unique Variants | True Positives | False Positives | False Negatives | True Negatives | PPA [Exact 95% CI] | NPA [Exact 95% CI]  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  All Samples | 1028 | 1221 | 80 | 11 | 414920 | 99.1% [98.4%, 99.6%] | 100.0% [100.0%, 100.0%]  |
|  All SNVs | 736 | 971 | 19 | 8 | 297042 | 99.2% [98.4%, 99.6%] | 100.0% [100.0%, 100.0%]  |
|  Level 1 SNVs | 10 | 31 | 1 | 0 | 648 | 100.0% [88.8%, 100.0%] | 99.8% [99.1%, 100.0%]  |
|  Level 2 SNVs | 128 | 209 | 1 | 1 | 27649 | 98.1% [95.3%, 99.5%] | 100.0% [100.0%, 100.0%]  |

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|  Variant Type | Total Unique Variants | True Positives | False Positives | False Negatives | True Negatives | PPA [Exact 95% CI] | NPA [Exact 95% CI]  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  Level 3 SNVs | 336 | 419 | 17 | 2 | 144384 | 99.5% [98.3%, 99.9%] | 100.0% [100.0%, 100.0%]  |
|  All MNVs | 22 | 18 | 3 | 1 | 8881 | 94.7% [74.0%, 99.9%] | 100.0% [99.9%, 100.0%]  |
|  Level 1 MNVs | 0 | 0 | 0 | 0 | 0 | N/A | N/A  |
|  Level 2 MNVs | 6 | 4 | 2 | 0 | 1508 | 100.0% [39.8%, 100.0%] | 99.9% [99.5%, 100.0%]  |
|  Level 3 MNVs | 11 | 9 | 1 | 1 | 5353 | 90.0% [55.5%, 99.7%] | 100.0% [99.9%, 100.0%]  |
|  All Insertions | 71 | 58 | 17 | 2 | 28656 | 96.7% [88.5%, 99.6%] | 99.9% [99.9%, 100.0%]  |
|  Level 2 Insertions | 20 | 19 | 2 | 0 | 2902 | 100.0% [82.4%, 100.0%] | 99.9% [99.8%, 100.0%]  |
|  Level 3 Insertions | 51 | 37 | 15 | 2 | 24946 | 94.9% [82.7%, 99.4%] | 99.9% [99.9%, 100.0%]  |
|  Insertions , 1-5bp | 60 | 47 | 16 | 2 | 24214 | 95.9% [86.0%, 99.5%] | 99.9% [99.9%, 100.0%]  |
|  Insertions , 6-10bp | 5 | 5 | 0 | 0 | 2020 | 100.0% [47.8%, 100.0%] | 100.0% [99.8%, 100.0%]  |
|  Insertions , 11-15bp | 3 | 4 | 0 | 0 | 1211 | 100.0% [39.8%, 100.0%] | 100.0% [99.7%, 100.0%]  |
|  Insertions , 16-20bp | 1 | 1 | 0 | 0 | 404 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%]  |
|  Insertions , 21-25bp | 0 | 0 | 0 | 0 | 0 | N/A | N/A  |
|  Insertions , >25bp | 2 | 1 | 1 | 0 | 807 | 100.0% [2.5%, 100.0%] | 99.9% [99.3%, 100.0%]  |
|  All Deletions | 199 | 174 | 41 | 0 | 80341 | 100.0% [97.9%, 100.0%] | 99.9% [99.9%, 100.0%]  |

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|  Variant Type | Total Unique Variants | True Positives | False Positives | False Negatives | True Negatives | PPA [Exact 95% CI] | NPA [Exact 95% CI]  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|   |  |  |  |  |  | 100.0%] |   |
|  Level 2 Deletions | 23 | 22 | 2 | 0 | 5558 | 100.0% [84.6%, 100.0%] | 100.0% [99.9%, 100.0%]  |
|  Level 3 Deletions | 154 | 128 | 39 | 0 | 65087 | 100.0% [97.2%, 100.0%] | 99.9% [99.9%, 100.0%]  |
|  Deletions, 1-5bp | 155 | 134 | 35 | 0 | 62573 | 100.0% [97.3%, 100.0%] | 99.9% [99.9%, 100.0%]  |
|  Deletions, 6-10bp | 18 | 19 | 1 | 0 | 7269 | 100.0% [82.4%, 100.0%] | 100.0% [99.9%, 100.0%]  |
|  Deletions, 11-15bp | 6 | 5 | 1 | 0 | 2423 | 100.0% [47.8%, 100.0%] | 100.0% [99.8%, 100.0%]  |
|  Deletions, 16-20bp | 6 | 6 | 0 | 0 | 2424 | 100.0% [54.1%, 100.0%] | 100.0% [99.8%, 100.0%]  |
|  Deletions, 21-25bp | 3 | 3 | 0 | 0 | 1212 | 100.0% [29.2%, 100.0%] | 100.0% [99.7%, 100.0%]  |
|  Deletions, >25bp | 11 | 7 | 4 | 0 | 4440 | 100.0% [59.0%, 100.0%] | 99.9% [99.8%, 100.0%]  |

# Accuracy - Hotspot Concordance

For hotspot concordance analysis with the orthogonal method, reported variants in hotspot regions overlapping with orthogonal method targeted regions were analyzed. From the 416 analyzed study samples, 164 samples had at least 1 reported variant in an overlapping hotspot region. The intersection of the defined hotspot regions of xT CDx and the orthogonal method targeted regions included 214 total base pairs, in which 192 reported variants were evaluated, including 50 unique SNVs, 3 unique MNVs, 2 unique insertions and 3 unique deletions. A summary of Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) is provided in Table 11, below, for SNVs, MNVs, insertions and deletions.

Table 11. Concordance summary for short variants in hotspot regions

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|  Variant Type | Total Unique Variants | True Positives | False Positives | False Negatives | True Negatives | PPA [Exact 95% CI] | NPA [Exact 95% CI]  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  All Variants | 58 | 188 | 2 | 2 | 23298 | 98.9% [96.2%, 99.9%] | 100.0% [100.0%, 100.0%]  |
|  All SNVs | 50 | 180 | 2 | 2 | 20066 | 98.9% [96.1%, 99.9%] | 100.0% [100.0%, 100.0%]  |
|  All MNVs | 3 | 3 | 0 | 0 | 1212 | 100.0% [29.2%, 100.0%] | 100.0% [99.7%, 100.0%]  |
|  All Insertions | 2 | 2 | 0 | 0 | 808 | 100.0% [15.8%, 100.0%] | 100.0% [99.5%, 100.0%]  |
|  All Deletions | 3 | 3 | 0 | 0 | 1212 | 100.0% [29.2%, 100.0%] | 100.0% [99.7%, 100.0%]  |

A breakdown of the Accuracy for SNVs, MNVs, and INDELs in hotspot regions by variant type, level (i.e., variants with evidence of clinical significance/Level 2, and variants with potential clinical significance/Level 3) and insertion/deletion size is provided in Table 12.

Table 12. Concordance breakdown for short variants inclusive of substitutions and INDELs relative to orthogonal method for Level 2 and Level 3 variants

|  Variant Type | Total Unique Variants | True Positives | False Positives | False Negatives | True Negatives | PPA [Exact 95% CI] | NPA [Exact 95% CI]  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  All Variants | 58 | 188 | 2 | 2 | 23298 | 98.9% [96.2%, 99.9%] | 100.0% [100.0%, 100.0%]  |
|  All SNVs | 50 | 180 | 2 | 2 | 20066 | 98.9% [96.1%, 99.9%] | 100.0% [100.0%, 100.0%]  |
|  Level 1 SNVs | 10 | 31 | 1 | 0 | 648 | 100.0% [88.8%, 100.0%] | 99.8% [99.1%, 100.0%]  |
|  Level 2 SNVs | 36 | 98 | 0 | 1 | 5468 | 99.0% [94.5%, 100.0%] | 100.0% [99.9%, 100.0%]  |
|  Level 3 SNVs | 26 | 51 | 1 | 1 | 13950 | 98.1% [89.7%, 100.0%] | 100.0% [100.0%, 100.0%]  |

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|  Variant Type | Total Unique Variants | True Positives | False Positives | False Negatives | True Negatives | PPA [Exact 95% CI] | NPA [Exact 95% CI]  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  All MNVs | 3 | 3 | 0 | 0 | 1212 | 100.0% [29.2%, 100.0%] | 100.0% [99.7%, 100.0%]  |
|  Level 1 MNVs | 0 | 0 | 0 | 0 | 0 | N/A | N/A  |
|  Level 2 MNVs | 2 | 2 | 0 | 0 | 293 | 100.0% [15.8%, 100.0%] | 100.0% [98.7%, 100.0%]  |
|  Level 3 MNVs | 1 | 1 | 0 | 0 | 919 | 100.0% [2.5%, 100.0%] | 100.0% [99.6%, 100.0%]  |
|  All Insertions | 2 | 2 | 0 | 0 | 808 | 100.0% [15.8%, 100.0%] | 100.0% [99.5%, 100.0%]  |
|  Level 2 Insertions | 0 | 0 | 0 | 0 | 126 | N/A | 100.0% [97.1%, 100.0%]  |
|  Level 3 Insertions | 1 | 1 | 0 | 0 | 278 | 100.0% [2.5%, 100.0%] | 100.0% [98.7%, 100.0%]  |
|  Insertion, 1-5bp | 1 | 1 | 0 | 0 | 404 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%]  |
|  Insertion, 6-10bp | 1 | 1 | 0 | 0 | 404 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%]  |
|  Insertion, 11-15bp | 0 | 0 | 0 | 0 | 0 | N/A | N/A  |
|  Insertion, 16-20bp | 0 | 0 | 0 | 0 | 0 | N/A | N/A  |
|  Insertion, 21-25bp | 0 | 0 | 0 | 0 | 0 | N/A | N/A  |
|  Insertion, >25bp | 0 | 0 | 0 | 0 | 0 | N/A | N/A  |
|  All Deletions | 3 | 3 | 0 | 0 | 1212 | 100.0% [29.2%, 100.0%] | 100.0% [99.7%, 100.0%]  |
|  Level 2 Deletions | 1 | 1 | 0 | 0 | 114 | 100.0% [2.5%, 100.0%] | 100.0% [96.8%, 100.0%]  |

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|  Variant Type | Total Unique Variants | True Positives | False Positives | False Negatives | True Negatives | PPA [Exact 95% CI] | NPA [Exact 95% CI]  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|   |  |  |  |  |  | 100.0%] |   |
|  Level 3 Deletions | 0 | 0 | 0 | 0 | 290 | N/A | 100.0% [98.7%, 100.0%]  |
|  Deletion, 1-5bp | 2 | 2 | 0 | 0 | 808 | 100.0% [15.8%, 100.0%] | 100.0% [99.5%, 100.0%]  |
|  Deletion, 6-10bp | 0 | 0 | 0 | 0 | 0 | N/A | N/A  |
|  Deletion, 11-15bp | 0 | 0 | 0 | 0 | 0 | N/A | N/A  |
|  Deletion, 16-20bp | 0 | 0 | 0 | 0 | 0 | N/A | N/A  |
|  Deletion, 21-25bp | 0 | 0 | 0 | 0 | 0 | N/A | N/A  |
|  Deletion, >25bp | 1 | 1 | 0 | 0 | 404 | 100.0% [2.5%, 100.0%] | 100.0% [99.1%, 100.0%]  |

# Accuracy - MSI

The detection of MSI status by xT CDx was assessed by comparison with results obtained using a validated orthogonal method (IHC staining of MLH1, MSH2, MSH6 and PMS2). A total set of 316 patient-matched tumor and normal samples representing 30 cancer types were sequenced with xT CDx. The reported MSI status from xT CDx was compared with results of the IHC staining [MSI High (MSI-H)/deficient mismatch repair (dMMR) versus non-MSI-H/proficient mismatch repair] and used to calculate the PPA and NPA for MSI. Of the 117 samples identified as positive by IHC testing (Abnormal IHC, loss), 110 were identified as MSI-H by xT CDx, yielding a PPA of  $94.0\%$  (95% CI: 88-98%). Of the 199 samples identified as negative by IHC testing (Normal IHC, intact), 195 were identified as MSS by xT CDx, yielding a NPA of  $98\%$  (95% CI: 95-99%). Results of MSI concordance testing are provided in Table 13, below.

Table 13. Concordance summary for MSI status relative to IHC

|   | Normal IHC | Abnormal IHC  |
| --- | --- | --- |
|  xT CDx MSI Stable (MSS) | 195 | 7  |
|  xT CDx MSI High (MSI-H) | 4 | 110  |

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The distribution of the solid tumor specimens included in the study, separated by CRC/EC (colorectal cancer/endometrial cancer) and non-CRC/non-EC cancer types and MSI results is provided in Table 14.

Table 14. Distribution of solid tumor specimens, separated by CRC/EC and Non-CRC/Non-EC

|  Cancer Type | Number of samples | Abnormal IHC | Normal IHC  |
| --- | --- | --- | --- |
|  CRC/EC | 108 | 75 | 33  |
|  Non-CRC/EC | 208 | 42 | 166  |
|  Total | 316 | 117 | 199  |

The MSI accuracy cohort included 108 unique CRC/EC and 208 unique non-CRC and non-EC tumor-normal paired samples across 30 tumor types. Analysis of these specimens separated by CRC/EC and non-CRC/non-EC, as well as overall, is provided in Tables 15-18 below.

Table 15. MSI accuracy in CRC/EC specimens relative to the orthogonal method

|  IHC status | xT CDx MSI status  |   |   |
| --- | --- | --- | --- |
|   |  Stable | Equivocal | High  |
|  Normal | 32 | 0 | 1  |
|  Abnormal | 3 | 0 | 72  |

Table 16. MSI accuracy in non-CRC/non-EC specimens relative to the orthogonal method

|  IHC status | xT CDx MSI status  |   |   |
| --- | --- | --- | --- |
|   |  Stable | Equivocal | High  |
|  Normal | 163 | 0 | 3  |
|  Abnormal | 3 | 1 | 38  |

Table 17. MSI accuracy in all specimens relative to the orthogonal method

|  IHC status | xT CDx MSI status  |   |   |
| --- | --- | --- | --- |
|   |  Stable | Equivocal | High  |
|  Normal | 195 | 0 | 4  |
|  Abnormal | 6 | 1 | 110  |

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Table 18. Agreement for MSI status overall and by cohort relative to the orthogonal method

|  Cohort | OPA [Exact 95% CI] | PPA [Exact 95% CI] | NPA [Exact 95% CI]  |
| --- | --- | --- | --- |
|  All | 96.5% [94%, 98%] | 94.0% [88%, 98%] | 98.0% [95%, 99%]  |
|  CRC/EC | 96.3% [91%, 99%] | 96.0% [89%, 99%] | 97.0% [84%, 100%]  |
|  non-CRC/non-EC | 96.6% [93%, 99%] | 90.5.8% [77%, 97%] | 98.2% [95%, 100%]  |

Note: Equivocal results were considered to be discordant with the orthogonal method for calculation of agreement.

# Accuracy - QC pass rate for Accuracy of Short Variants

Table 19 details the pass rate at each QC step of the device for the specimens analyzed to characterize the accuracy for SNVs, MNVs, and INDELs, following exclusion of samples with insufficient extracted material. QC1 is not detailed here since QC1 is an extraction metric and these samples were previously extracted. A total of 429 samples were successfully sequenced and passed QC4; 13 samples failed QC4 due to the reasons detailed in Table 20.

Table 19. QC pass rate for Accuracy of Short Variants

|  Quality Control | FFPE Libraries | Blood Libraries | Saliva Libraries  |
| --- | --- | --- | --- |
|  QC2 | 442/445 passed | 361/361 passed | 84/84 passed  |
|  QC3 | 442/442 passed | 361/361 passed | 84/84 passed  |
|  Tumor-normal matched pairs passing QC3 | 442/442 passed  |   |   |
|  QC4* | 429/442 passed  |   |   |

*QC4 metric is derived from collection of data using FFPE-blood (n=359) or FFPE-saliva (n=83) matched samples, where the QC4 “pass” status is assigned to the tumor-normal matched pair.

Table 20. QC4 failure explanation

|  Failure Reason | Number of Samples  |
| --- | --- |
|  Low normal unique read count; low normal total read count | 1  |
|  Tumor contamination (somatic variants found in germline) | 9  |
|  High tumor PCR duplication rate | 2  |
|  Low tumor unique read count | 1  |

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Among the 429 samples that passed xT CDx QC, 6 samples failed OM1 QC. Six additional samples were removed because of unavailable results or inappropriate sample types, and one was removed because it had no variants called by either xT CDx or OM1. The final intersection of the QC-passing samples with OM1 and xT CDx resulted in a total of 416 samples, which were the set of samples analyzed for this study.

Data obtained from sequencing samples with xT CDx for the Accuracy of Short Variants study were re-analyzed for hotspot regions. Table 19 above has the summary of the QC pass rates for the samples processed with xT CDx. All 416 QC-passing samples evaluated in the Accuracy of Short Variants study were evaluated for hotspot regions.

# Accuracy - QC pass rate of MSI study

A total of 440 specimens were analyzed for this study. Data obtained from sequencing 396 samples for Accuracy for SNVs, MNVs, and INDELs were analyzed for this study. Data from 8 colorectal cancer specimens from the determination of LoD for MSI study were also analyzed to increase the number of colorectal cancer specimens included in the study. From these 404 samples, there were 293 samples that also had data for MSI status from the OM, which is an IHC method. Of these 293 samples, 282 passed QC4.

An additional 36 non-CRC/non-EC specimens were included based on MSI-H status as determined by IHC.

Table 21 details the pass rate at each QC step of xT CDx for the 36 additional MSI-H non-CRC/non-EC specimens specifically processed for this study. QC1 is not detailed here since these samples were previously extracted. With xT CDx, 34 samples were sequenced and passed QC4.

Table 21. QC pass rate for Accuracy for Additional non-CRC/non-EC specimens in the MSI study

|  Quality Control | FFPE Libraries | Blood Libraries | Saliva Libraries  |
| --- | --- | --- | --- |
|  QC2 | 35/36 passed | 29/30 passed | 6/6 passed  |
|  QC3 | 35/35 passed | 29/29 passed | 6/6 passed  |
|  Tumor-normal matched pairs passing QC3 | 34/34 passed  |   |   |
|  QC4* | 34/34 passed  |   |   |

*QC4 metric is derived from collection of data using FFPE-blood (n=29) or FFPE-saliva (n=6) matched samples, where the QC4 "pass" status is assigned to the tumor-normal matched pair.

Overall, the comparison between the MSI status determined by xT CDx and IHC included 316 samples from 30 different tumor types.

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# iii. Wild-Type Accuracy

The data originally generated in Accuracy of Short Variants was re-analyzed to assess wild type (WT) accuracy. 100 SNV positions and 100 INDEL positions were randomly selected (pre-specified) without knowledge of concordance, from the list of variants reported by the OM1 and in regions reportable by xT CDx, based on having at least one sample with a non-WT call in the OM1 dataset. Additionally, all 8 MNV positions that were present in both xT CDx and OM1 were selected. The resulting list of 208 positions represented 45 genes.

Each class of mutations (SNV, MNV, insertion, deletion) was evaluated within the specimens which had at least one selected mutation of that class. For example, for the 100 chosen SNVs, 165 samples contained at least one of the chosen SNVs and those were the 165 samples evaluated. In total, 224 specimens which produced results from xT CDx and OM1 and had at least one mutation (non-WT) in one of these 208 pre-specified positions were included in this analysis. See Table 22 for detailed information.

Table 22. WT accuracy by variant type; and by insertion and deletion size compared to OM1

|   | xT CDxWT/ OM1WT | xT CDx+/ OM1WT | xT CDxWT/ OM1+ | xT CDx+/ OM1+ | OPA [95% CI] | NPA [95% CI] | PPA [95% CI]  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  All Variants | 25537 | 3 | 3 | 325 | 100.0% [99.9%, 100.0%] | 100.0% [100.0%, 100.0%] | 99.1% [97.4%, 99.8%]  |
|  All SNVs | 19918 | 2 | 2 | 208 | 100.0% [99.9%, 100.0%] | 100.0% [100.0%, 100.0%] | 99.0% [96.6%, 99.9%]  |
|  SNVs - Level 2 | 3331 | 0 | 0 | 53 | 100.0% [99.9%, 100.0%] | 100.0% [99.9%, 100.0%] | 100.0% [93.3%, 100.0%]  |
|  SNVs - Level 3 | 16587 | 2 | 2 | 151 | 100.0% [99.9%, 100.0%] | 100.0% [100.0%, 100.0%] | 98.7% [95.4%, 99.8%]  |
|  All MNVs | 90 | 1 | 0 | 9 | 99.0% [94.6%, 100.0%] | 100.0% [96.0%, 100.0%] | 90.0% [55.5%, 99.7%]  |
|  All Insertions | 1329 | 0 | 1 | 38 | 99.9% [99.6%, 100.0%] | 99.9% [99.6%, 100.0%] | 100.0% [90.7%, 100.0%]  |
|  Insertions, 0-5bp | 1219 | 0 | 1 | 34 | 99.9% [99.6%, 100.0%] | 99.9% [99.5%, 100.0%] | 100.0% [89.7%, 100.0%]  |
|  Insertions, 5-10bp | 37 | 0 | 0 | 1 | 100.0% [90.7%, 100.0%] | 100.0% [90.5%, 100.0%] | 100.0% [2.5%, 100.0%]  |
|  Insertions, 10-15bp | 73 | 0 | 0 | 3 | 100.0% [95.3%, 100.0%] | 100.0% [95.1%, 100.0%] | 100.0% [29.2%, 100.0%]  |
|  Insertions, 15-20bp | 0 | 0 | 0 | 0 | N/A | N/A | N/A  |

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|   | xT CDxWT/ OM1WT | xT CDx+/ OM1WT | xT CDxWT/ OM1+ | xT CDx+/ OM1+ | OPA [95% CI] | NPA [95% CI] | PPA [95% CI]  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  Insertions, 20-25bp | 0 | 0 | 0 | 0 | N/A | N/A | N/A  |
|  All Deletions | 4200 | 0 | 0 | 70 | 100.0% [99.9%, 100.0%] | 100.0% [99.9%, 100.0%] | 100.0% [94.9%, 100.0%]  |
|  Deletions 0-5bp | 3600 | 0 | 0 | 60 | 100.0% [99.9%, 100.0%] | 100.0% [99.9%, 100.0%] | 100.0% [94.0%, 100.0%]  |
|  Deletions 5-10bp | 300 | 0 | 0 | 5 | 100.0% [98.8%, 100.0%] | 100.0% [98.8%, 100.0%] | 100.0% [47.8%, 100.0%]  |
|  Deletions 10-15bp | 60 | 0 | 0 | 1 | 100.0% [94.1%, 100.0%] | 100.0% [94.0%, 100.0%] | 100.0% [2.5%, 100.0%]  |
|  Deletions 15-10bp | 0 | 0 | 0 | 0 | N/A | N/A | N/A  |
|  Deletions 20-25bp | 0 | 0 | 0 | 0 | N/A | N/A | N/A  |

## 3. Precision

### a. Precision in Well-Characterized Material

The panel-wide precision/reproducibility of xT CDx was assessed for detecting variants in well-characterized reference material by repeated measurement of NA12878, a nucleic acid (NA) extracted from the GM12878 cell line. Precision was evaluated across 22 replicates which were processed over multiple library preparations and days (n=17), hybridization capture batches (n=8), and sequencing flow cells (n=8).

A total of 2673 variants were called across all 22 replicates, and 2624 of these variants were in the Genome in a Bottle (GIAB)¹ high confidence dataset. Table 23 shows the Coefficient of Variation (CV) distribution for all 2673 variants analyzed. In the study, 95.5% of samples had a CV below 10%. Across all samples, the mean CV of the variant allele frequency (VAF) was 3.7% +/- 3.9%. Mean %CV by zygosity of the variant, as declared in the GIAB variant call file (VCF) and type variant is presented in Table 23.

Table 23. Distribution of variants by %CV in well-characterized reference material

|   | CV < 10% | 10% ' .' CV < 15% | 15% ' .' CV < 20% | 20% < CV  |
| --- | --- | --- | --- | --- |

¹ Zook, J. M. et al. Extensive sequencing of seven human genomes to characterize benchmark reference materials. Sci. Data 3:160025 doi: 10.1038/sdata.2016.25 (2016)

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|  Number of Variants | 2552 | 73 | 24 | 24  |
| --- | --- | --- | --- | --- |
|  Percent of Variants | 95.5% | 2.70% | 0.90% | 0.90%  |

Table 24. Mean percent coefficient of variation (%CV) by zygosity declared in the GIAB VCF and type of variant for well-characterized reference material

|  Zygosity | SNVs and INDELs | SNVs Only | Insertions Only | Deletions Only  |
| --- | --- | --- | --- | --- |
|  All^{1} %CV) | 3.7% +/- 3.8% | 3.5% +/- 3.5% | 7.0% +/- 6.3% | 7.7% +/- 6.7%  |
|  Number of Total (All) Variants | 2673 | 2525 | 78 | 70  |
|  Homozygous Only (%CV) | 0.23% +/- 0.72% | 0.14% +/- 0.39% | 2.2% +/- 2.4% | 1.2% +/-1.6%  |
|  Number of Homozygous Variants | 938 | 889 | 30 | 19  |
|  Heterozygous Only (%CV) | 5.3% +/- 3.2% | 5.3% +/- 3.1% | 7.9% +/- 5.5% | 7.9% +/- 5.8%  |
|  Number Heterozygous of Variants | 1686 | 1635 | 22 | 29  |

1 Homozygous, Heterozygous, and missing (from GIAB VCF)

The VAF of each variant was measured in each replicate using xT CDx. For each variant, the %CV (standard deviation of VAFs / mean VAF) was calculated across replicates. Table 23 presents the distribution of the %CVs of the VAFs measured by xT CDx in the 22 replicates of the NA12878 sample.

The mean %CV for all variants of a given type SNVs and INDELs) based on zygosity was calculated as described for Table 23 (for example, for SNVs only: sum of %CVs for all SNV variants / total number of SNV variants), and is displayed in Table 24.

## b. Panel-wide Precision in Clinical Specimens

Panel-wide precision of xT CDx in clinical specimens was based on repeated measurement of 49 patient specimens representing 23 different tumor types (including

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melanoma, CRC, glioblastoma, and lung cancer). Replicates  $(n = 5 - 10)$  of each specimen were measured across 3 non-consecutive days, with multiple operators, reagent lots, instruments. A total of 317 replicates contributed to the evaluation of precision. The specimens and tumor types included in the study from 49 patients are shown in Table 25.

Table 25. Number of clinical specimens per tumor type in the panel-wide precision study

|  Tumor Type | Number of Specimens  |
| --- | --- |
|  Basal Cell Carcinoma | 1  |
|  Bladder Cancer | 6  |
|  Breast Cancer | 4  |
|  Colorectal Cancer | 5  |
|  Endocrine Tumor | 2  |
|  Endometrial Cancer | 4  |
|  Esophageal Cancer | 1  |
|  Gastric Cancer | 1  |
|  Head and Neck Cancer | 2  |
|  Liver Cancer | 1  |
|  Melanoma | 2  |
|  Meningioma | 1  |
|  Non-Small Cell Lung Cancer | 4  |
|  Ovarian Cancer | 1  |
|  Prostate Cancer | 1  |
|  Skin Cancer | 2  |
|  Tumor of Unknown Origin | 4  |
|  Adrenal Cancer | 1  |
|  Cervical Cancer | 1  |
|  Head and Neck Squamous Cell Carcinoma | 1  |

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Among the specimens evaluated, there were 289 total variants represented by 151 SNVs, 9 MNVs, 26 insertions and 103 deletions. The overall positive call rate across all precision conditions (days, operators, reagents lots, and instruments), for all specimens and replicates was 94.5%, and 97.0% for variants with a VAF ⇔ 15%. Results are shown in Table 26.

Table 26. Precision by variant type and VAF

|  Variant Type | VAF threshold (%) | Total variants | Mean VAF Range | Positive/Total Calls | Positive call rate (2-sided 95% CI)  |
| --- | --- | --- | --- | --- | --- |
|  SNV | ↓0 | 151 | 3.8-84.343 | 911/944 | 96.5% (95.1,97.6)  |
|   |  ↓5 | 150 | 5.388-84.343 | 907/939 | 96.6% (95.2,97.7)  |
|   |  ↓10 | 132 | 10.418-84.343 | 841/849 | 99.1% (98.2,99.6)  |
|   |  ↓15 | 110 | 15.067-84.343 | 718/726 | 98.9% (97.8,99.5)  |
|  MNV | ↓0 | 9 | 12.657-58.597 | 61/61 | 100.0% (94.1,100)  |
|   |  ↓5 | 9 | 12.657-58.597 | 61/61 | 100.0% (94.1,100)  |
|   |  ↓10 | 9 | 12.657-58.597 | 61/61 | 100.0% (94.1,100)  |
|   |  ↓15 | 6 | 15.124-58.597 | 35/35 | 100.0% (90.0,100)  |
|  Insertion | ↓0 | 26 | 11.25-61.114 | 153/165 | 92.7% (87.6,96.2)  |
|   |  ↓5 | 26 | 11.25-61.114 | 153/165 | 92.7% (87.6,96.2)  |
|   |  ↓10 | 26 | 11.25-61.114 | 153/165 | 92.7% (87.6,96.2)  |
|   |  ↓15 | 23 | 15.187-61.114 | 139/145 | 95.9% (91.2,98.5)  |
|  Deletion | ↓0 | 103 | 10.054-94.976 | 683/744 | 91.8% (89.6,93.7)  |
|   |  ↓5 | 103 | 10.054-94.976 | 683/744 | 91.8% (89.6,93.7)  |
|   |  ↓10 | 103 | 10.054-94.976 | 683/744 | 91.8% (89.6,93.7)  |

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The positive call rates for individual sequence mutations assessed for panel-wide precision, along with the VAF range, mean, median, SD, and %CV are presented in Table 27. Variants are listed by specimen, and variant type is identified in each case.

Table 27. Panel-wide precision by specimen and variant for all replicates

|  Sample | Gene Exon | Variant Type | Mutation (cDNA/Protein Changes) | VAF Range | VAF Mean | VAF Median | VAF (SD) | VAF (%CV) | Positive/Total Calls | Positive Call Rate (95% CI)  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  747 | ARID2 exon_15 | Deletion | c.3432delC p.Ile1145fs | 37.27-42.006 | 38.869 | 38.1 | 1.863 | 4.80% | 4/4 | 100.0% (39.8,100)  |
|   |  TP53 exon_4 | SNV | c.396G>T p.Lys132Asn | 34.351-37.248 | 35.587 | 35.375 | 1.147 | 3.20% | 4/4 | 100.0% (39.8,100)  |
|   |  NF1 exon_34 | Deletion | c.4487_4490delTTTC p.Leu1496fs | 22.36-30.667 | 27.215 | 27.917 | 3.125 | 11.50% | 4/4 | 100.0% (39.8,100)  |
|   |  RASA1 exon_1 | Deletion | c.124delC p.Leu42fs | 31.394-38.0 | 34.607 | 34.517 | 2.406 | 7.00% | 4/4 | 100.0% (39.8,100)  |
|  723 | GRIN2A exon_12 | SNV | c.3042G>A p.Trp1014* | 34.119-36.909 | 35.311 | 35.052 | 0.909 | 2.60% | 5/5 | 100.0% (47.8,100)  |
|   |  FANCA exon_33 | SNV | c.3208C>T p.Gln1070* | 34.48-36.863 | 35.316 | 35.292 | 0.849 | 2.40% | 5/5 | 100.0% (47.8,100)  |
|   |  TERT exon_1 | SNV | c.-124C>T . | 29.961-36.253 | 33.31 | 33.083 | 2.079 | 6.20% | 5/5 | 100.0% (47.8,100)  |
|   |  BRAF exon_15 | SNV | c.1799T>A p.Val600Glu | 48.048-52.455 | 49.439…

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**Source:** [https://fda.innolitics.com/device/P210011](https://fda.innolitics.com/device/P210011)

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