← Product Code [PQP](/productcode/PQP) · P200011S001

# oncoRevealTM CDx (P200011S001)

_Pillar Biosciences · PQP · Apr 18, 2024 · Medical Genetics · APPR_

**Canonical URL:** https://fda-staging.innolitics.com/device/P200011S001

## Device Facts

- **Applicant:** Pillar Biosciences
- **Product Code:** [PQP](/productcode/PQP.md)
- **Decision Date:** Apr 18, 2024
- **Decision:** APPR
- **Device Class:** Class 3
- **Review Panel:** Medical Genetics
- **Attributes:** Software as a Medical Device

## Indications for Use

The oncoReveal™ CDx is a qualitative next generation sequencing based in vitro diagnostic test that uses amplicon-based target enrichment technology for detection of single nucleotide variants (SNVs), insertions and deletions in 22 genes using DNA isolated from formalin-fixed paraffin-embedded (FFPE) tumor tissue specimens and using the Illumina MiSeqDx®. The test is intended as a companion diagnostic to identify patients who may benefit from treatment with the targeted therapies listed in Table 1 in accordance with the approved therapeutic product labeling. Additionally, oncoReveal™ CDx is intended to provide tumor mutation profiling to be used by qualified health care professionals in accordance with professional guidelines in oncology for previously diagnosed cancer patients with solid malignant neoplasms. Genomic findings other than those listed in Table 1 are not prescriptive or conclusive for labeled use of any specific therapeutic product.

## Device Story

oncoReveal CDx is an NGS-based IVD using amplicon-based target enrichment to detect SNVs, insertions, and deletions in 22 genes from FFPE tumor tissue. The device uses the Illumina MiSeqDx instrument. The workflow involves DNA extraction, library preparation (PCR amplification, barcoding), sequencing, and bioinformatic analysis via the PiVAT workstation. The system is used in clinical laboratories by trained personnel. The output is a report categorizing variants into Level 1 (CDx), Level 2 (Clinical Significance), and Level 3 (Potential Clinical Significance). The report assists healthcare professionals in oncology by providing tumor mutation profiles to guide treatment decisions in accordance with professional guidelines. The device benefits patients by identifying potential therapeutic targets and providing genomic information for solid tumors.

## Clinical Evidence

No new clinical studies were conducted for the tumor profiling indication. Analytical validation included accuracy studies comparing oncoReveal CDx to two externally validated NGS (evNGS) methods across 271 and 212 samples respectively, demonstrating high concordance (PPA >98%, NPA >99%). Precision, LoD, LoB, and stability studies were performed to support the expanded claim.

## Technological Characteristics

Amplicon-based NGS target enrichment; 22-gene panel; DNA input 30-80ng from FFPE; Illumina MiSeqDx platform; BWA-MEM alignment; PiVAT bioinformatics pipeline; standalone workstation (Ubuntu OS, no network connectivity); 2x150bp sequencing protocol.

## 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

- xT CDx ([P210011](/device/P210011.md))
- cobas® KRAS Mutation Test ([P140023](/device/P140023.md))
- Therascreen® KRAS RGQ PCR Kit ([P110030](/device/P110030.md); [P110027](/device/P110027.md))
- FoundationOne® CDx ([P170019](/device/P170019.md))
- Therascreen® EGFR RGQ PCR Kit ([P120022](/device/P120022.md)/S018)
- cobas® EGFR Mutation Test v2 ([P120019](/device/P120019.md)/S01)
- Oncomine™ Dx Target Test ([P160045](/device/P160045.md))

## Submission Summary (Full Text)

> This content was OCRed from public FDA records by [Innolitics](https://innolitics.com). If you use, quote, summarize, crawl, or train on this content, cite Innolitics at https://innolitics.com.
>
> Innolitics is a medical-device software consultancy. We help companies design, build, and clear FDA-regulated software and AI/ML devices, including [a PMA](https://innolitics.com/services/regulatory/), [a 510(k)](https://innolitics.com/services/510ks/), [a SaMD](https://innolitics.com/services/end-to-end-samd/), [an AI/ML medical device](https://innolitics.com/services/medical-imaging-ai-development/), or [an FDA regulatory strategy](https://innolitics.com/services/regulatory/).

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# SUMMARY OF SAFETY AND EFFECTIVENESS DATA (SSED)

## I. GENERAL INFORMATION

|  Device Generic Name: | Next generation sequencing oncology panel, somatic or germline variant detection system  |
| --- | --- |
|  Device Trade Name: | oncoReveal™ CDx  |
|  Device Procode: | PQP  |
|  Applicant's Name and Address: | Pillar Biosciences, Inc. 9 Strathmore Road Natick, MA 01760  |
|  Date(s) of Panel Recommendation: | None  |
|  Premarket Approval Application (PMA) Number: | P200011/S001  |
|  Date of FDA Notice of Approval: | April 18, 2024  |

The original PMA (P200011) for Pillar Biosciences was approved on July 30, 2021, for the detection of single nucleotide variants (SNVs) and deletions in two genes (*EGFR* and *KRAS*) in patients who may benefit from FDA-approved therapies for non-small cell lung cancer (NSCLC), and colorectal cancer (CRC).

The current supplement was submitted to expand the intended use and indication for use of oncoReveal CDx Assay to include tumor mutation profiling to be used by qualified health care professionals in accordance with professional guidelines in oncology for cancer patients with solid malignant neoplasms.

## II. INTENDED USE/INDICATIONS FOR USE

The oncoReveal™ CDx is a qualitative next generation sequencing based *in vitro* diagnostic test that uses amplicon-based target enrichment technology for detection of single nucleotide variants (SNVs), insertions and deletions in 22 genes using DNA isolated from formalin-fixed paraffin-embedded (FFPE) tumor tissue specimens and using the Illumina MiSeqDx®. The test is intended as a companion diagnostic to identify patients who may benefit from treatment with the targeted therapies listed in Table 1 in accordance with the approved therapeutic product labeling.

Additionally, oncoReveal™ CDx is intended to provide tumor mutation profiling to be used by qualified health care professionals in accordance with professional guidelines in oncology for previously diagnosed cancer patients with solid malignant neoplasms. Genomic findings other than those listed in Table 1 are not prescriptive or conclusive for labeled use of any specific therapeutic product.

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**Table 1. List of Somatic Variants for Therapeutic Use**

|  Indication | Gene | Variant | Targeted therapy  |
| --- | --- | --- | --- |
|  Colorectal Cancer (CRC) | *KRAS* | *KRAS* wild-type (absence of mutations in codons 12 and 13) | ERBITUX® (cetuximab), or VECTIBIX® (panitumumab)  |
|  Non-Small Cell Lung Cancer (NSCLC) | *EGFR* | Exon 19 Deletions and Exon 21 L858R Substitution Mutations | EGFR Tyrosine Kinase Inhibitors approved by FDA*  |

*For the most current information about the therapeutic products in this group, go to:
https://www.fda.gov/medicaldevices/productsandmedicalprocedures/invitrodiagnostics/ucm301431.htm

### III. CONTRAINDICATIONS

There are no known contraindications.

### IV. WARNINGS AND PRECAUTIONS

The warnings/precautions and limitations can be found in the oncoReveal™ CDx assay labeling.

### V. DEVICE DESCRIPTION

The oncoReveal CDx is an NGS *in vitro* diagnostic Test that uses amplicon-based target enrichment technology for detection of SNVs, insertions and deletions in 22 genes using DNA isolated from FFPE tumor tissue specimens and using the Illumina MiSeqDx instrument. In addition to the companion diagnostic (CDx) claims noted in Table 1 of the intended use/indications for use, the oncoReveal CDx also reports SNV, insertions and deletions in the 22 genes listed Table 2 to provide tumor mutation profiling to be used by qualified health care professionals in accordance with professional guidelines in oncology for previously diagnosed cancer patients with solid malignant neoplasms.

**Table 2. Genes Targeted by the oncoReveal™ CDx**

|  *AKT1* | *CTNNB1* | *ERBB2* | *FGFR1* | *KRAS* | *NOTCH1* | *PTEN* | *TP53*  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  *ALK* | *DDR2* | *ERBB4* | *FGFR2* | *MAP2K1* | *NRAS* | *SMAD4* |   |
|  *BRAF* | *EGFR* | *FBXW7* | *FGFR3* | *MET* | *PIK3CA* | *STK11* |   |

#### Test Output

The output of the test includes:

- Level 1: Companion Diagnostic (CDx) Claims noted in Table 1 of the Intended Use
- Level 2: Cancer Mutations with Evidence of Clinical Significance
- Level 3: Cancer Mutations with Potential Clinical Significance

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## Test Kit Contents

The Assay Kit is composed of 7 reagents to allow the processing of 48 reactions (46 patient samples and required controls), refer to Table 3. The reagents are liquids or suspensions, stored in individual vials, and segregated into four labeled sub-containers. The sub-containers allow kit components to be stored at the recommended temperature, which may be room temperature, 4°C, or -20°C, depending on the components. Safety Data Sheets are available from Pillar Biosciences. Outer packaging supports international frozen shipment.

**Table 3. Assay Kit Reagents**

|  Kit Box 1: GS-PCR Reagent | Quantity | Storage  |
| --- | --- | --- |
|  Gene Specific PCR Master Mix | 1 tube (red cap) | -25°C to -15°C  |
|  LC Oligo Pool | 1 tube (yellow cap) | -25°C to -15°C  |
|  Positive Control (PosCtrl) | 1 tube (clear cap) | -25°C to -15°C  |
|  Uracil-DNA glycosylase (UDG) | 1 tube (blue cap) | -25°C to -15°C  |
|  Kit Box 2: Indexing PCR Reagent | Quantity | Storage  |
|  Indexing PCR Master Mix | 1 tube (green cap) | -25°C to -15°C  |
|  Forward indexing primers (A501-A508) | 8 tubes (white caps) | -25°C to -15°C  |
|  Reverse indexing primers (A701-A706) | 6 tubes (orange caps) | -25°C to -15°C  |
|  Kit Box 3: PCR Product Purification Reagent | Quantity | Storage  |
|  Purification Beads | 1 bottle | 2°C to 8°C  |
|  Kit Box 4: Index Tube Caps | Quantity | Storage  |
|  White caps (for A501-A508 primers) | 24 caps | Ambient  |
|  Orange caps (for A701-A706 primers) | 18 caps | Ambient  |

Materials and equipment and software that are required for the test but are not provided with the assay kit are listed in Tables 4, 5 and 6.

**Table 4. Materials required but not provided in the oncoReveal™ CDx Kit**

|  Material | Source/Part Number  |
| --- | --- |
|  Reagent kit for extraction and purification of DNA from formalin-fixed paraffin-embedded (FFPE) tissues used in clinical diagnostic applications. | See DNA EXTRACTION equivalency section 5 below. Column- or bead-based kits for extraction and purification of DNA from FFPE tissues. Proteinase K treatment and final elution volume 25µL are recommended for optimal results with this assay.  |

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|  Material | Source/Part Number  |
| --- | --- |
|  Reagent kit for quantification of double-stranded DNA (dsDNA) in biological samples used in clinical diagnostic applications. | The assay should: - accurately measure dsDNA for initial sample concentrations from 0.2 ng/μL to 15 ng/μL. - be compatible with a variety of biological samples, including purified genomic DNA from FFPE tissues, and PCR products. - contain a fluorescent dsDNA-binding dye, appropriate buffer, and DNA standards for calibration. - be designed for use with a fluorometer instrument.  |
|  Reaction vessels intended for use with a fluorometer instrument for the quantification of dsDNA used in clinical diagnostic applications. | The reaction vessels should be compatible for use with dsDNA quantification assay and fluorometer instrument to provide consistent and accurate fluorescence measurements.  |
|  PhiX Library Control intended as a control in nucleic acid sequencing workflows used in clinical diagnostic applications. | Library of bacteriophage PhiX DNA fragment at or above 20 pM. The fragments should have an average size of 500 bp and consist of base composition at ~45% GC and ~55% AT. The control should be used as directed by the  |
|  MiSeqDx® Reagent Kit v3 (600 cycles) | Illumina/20037174  |
|  Ethanol, 200 proof for molecular biology | General lab supplier  |
|  Nuclease-free water | General lab supplier  |
|  10 mM Tris-HCl w/ 0.1% Tween-20, pH 8.5 | General lab supplier  |
|  10 N NaOH or 1 N NaOH | General lab supplier  |
|  1.5 mL microcentrifuge tubes | General lab supplier  |
|  96-well PCR plates, 0.2 mL | General lab supplier  |
|  Microplate sealing film | General lab supplier  |
|  Conical tubes, 15 mL | General lab supplier  |
|  Conical tubes, 50 mL | General lab supplier  |
|  Aerosol filter pipette tips | General lab supplier  |
|  Solution basin (trough or reservoir) | General lab supplier  |

Table 5. Equipment Required But Not Provided

|  Equipment | Source/Part Number  |
| --- | --- |
|  MiSeqDx® Instrument† | Illumina/DX-410-1001  |

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|  Fluorometer instrument^{††} | A fluorometer instrument compatible for use with DNA quantification kit.  |
| --- | --- |
|  Vortexer | General lab supplier  |
|  Magnetic stand intended for use with PCR product purification workflow. | General lab supplier  |
|  Microfuge | General lab supplier  |
|  Thermal cycler^{†} with heated lid capability | General lab supplier  |
|  Single- and multi-channel pipettes^{†}, 0.5 to 1000 μl | General lab supplier  |
|  Centrifuge adapted for PCR plates | General lab supplier  |

† Equipment should be maintained and/or calibrated according to the manufacturer's instructions

Table 6. Software Provided During System Set up.

|  Equipment | Source/Part Number  |
| --- | --- |
|  Pillar LC-HS module v2.1 or higher | Pillar Biosciences/SFW-2008  |
|  oncoReveal™ CDx PiVAT® Workstation with software version 2.1 or higher | Pillar Biosciences/SFW-2012  |
|  Pillar Sample Sheet Tool version 3.2 or higher | Pillar Biosciences/TL-0059  |

The PiVAT® software is for use with oncoReveal™ CDx. PiVAT® performs secondary analysis and report generation from sequencing runs that use the oncoReveal CDx.

PiVAT® is installed on a stand-alone computer system configured with an Ubuntu operating system and a Chromium browser. The system is configured with no network connectivity.

### Test Process

#### 1. Specimen Preparation/DNA extraction

Specimens must be deparaffinized and digested with protease to liberate the DNA target before purification.

All tissues must be formalin fixed and embedded in paraffin according to accepted histological methods. FFPE sections with ≥ 30% tumor content by area for processing without macro dissection. For FFPE sections that are less than 30% tumor content by area, tumor content is enriched by macro dissecting multiple sections to obtain ≥ 30% tumor content by area. Column-based DNA extraction kits with Proteinase K treatment with agitation and final elution with 25 μL volume per section are recommended for DNA extractions intended for use with this assay. Extracted DNA giving a dsDNA quantification of > 4.5 ng/μl can be used for the oncoReveal™ CDx assay.

#### 2. Library Preparation

Library preparation is performed using the oncoReveal CDx Kit. Briefly, purified DNA samples are treated with Uracil-DNA Glycosylase to render formalin damaged DNA non-amplifiable. The samples are then amplified using polymerase chain reaction (PCR) and a gene-specific primer pool to enrich the number of assay target

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sequences. The amplification products are purified from remaining primers and each sample is “barcoded” in a second PCR reaction using a unique pair of indexing primers to prepare the samples for pooling and multiplexed analysis. After purification of the amplification products from residual indexing primers, the indexed libraries are quantified, normalized, and pooled for sequencing.

### 3. Sample Sequencing

Sequence information is extracted from the sample library pool using Illumina’s MiSeqDx NGS analyzer and corresponding reagents according to the manufacturer’s protocols. The user selects the appropriate analysis panel type (oncoReveal™ CDx labeled as LC-HS within Local Run Manager) from the Pillar Module on the MiSeqDx to initiate the sequence analysis utilizing the preset parameters for that panel. The Pillar Module is an interface designed for the MiSeqDx by Illumina to interface with collaborator assays. Its main function is to automatically configure assay-specific sequencing parameters on the instrument, such as read length, when a particular assay is selected from the Module’s drop-down menu. As additional assays are developed, they can be added to extend the capabilities of the Module.

The user must also create a Sample Sheet in Illumina’s format that holds information about the samples such as name, whether the sample is a control sample (positive, negative, or no template), and what indices were used to tag that sample. Pillar Biosciences provides a tool that facilitates the aggregation of batches of libraries prepared across multiple days onto a single MiSeqDx v3 flow cell, provided that each batch has the required positive and No Template Control (NTC) controls. Up to 48 libraries may be multiplexed onto a single MiSeqDx v3 flow cell. A successful sequencing run will produce sequence data in Illumina’s proprietary Binary Base Call format that is converted to a more universal FASTQ sequence format by the Pillar Module.

The oncoReveal CDx requires the user to run positive control (PosCtrl) and NTC for each “Batch” of up to 46 samples (processed on the same plate). Up to 6 batches may be included in a single sequencing run and analyzed through the PiVAT software. PosCtrl is a cell line DNA containing the CDx variants with expected variant allele frequencies as shown in Table 7 below. The PosCtrl must generate expected mutations to be valid. If the PosCtrl is invalid, the PiVAT software will fail the entire batch and no results will be reported for all samples within the batch.

**Table 7. Positive Control (PosCtrl)**

|  Gene | Variant | Expected Allelic Frequency, %  |
| --- | --- | --- |
|  *EGFR* | ΔE746 - A750 | 2.0%  |
|  *EGFR* | L858R | 3.0%  |
|  *KRAS* | G13D | 15.0%  |
|  *KRAS* | G12D | 6.0%  |

The non template control (NTC) reaction is setup using DNA diluent or nuclease-free water with no template or DNA input. The NTC should not detect any mutations. If the

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NTC is invalid, the PiVAT software will fail the entire batch and no results will be reported for all samples within the batch.

#### 4. Bioinformatic Analysis

The PiVAT IVD bioinformatics pipeline is used to convert the raw FASTQ output from the MiSeqDx into genetic variation observed for each sample. The PiVAT IVD software is provided on a standalone workstation and raw sequence data are transferred from the MiSeqDx to the PiVAT IVD workstation for analysis using a USB drive. Once the required FASTQ files are uploaded to the PiVAT IVD workstation, the user can select sequence data to be analyzed from the browser-based PiVAT interface and begin the analysis workflow. The software will deconvolute the mixed sample sequence data using the unique sample index and consolidate matching forward and reverse sequence reads. Filters are used to minimize the result of random variation introduced during sample amplification steps and sequencing.

The resulting sequences are aligned to the hg19 human genome sequence framework using the BWA-MEM aligner. Local re-alignments are performed to identify longer insertions and deletions (indels). Filters are applied to isolate likely variation from sequencing noise (Table 8). oncoReveal CDx is designed to detect and report somatic variants in three levels:

Level 1: CDx variants listed in Table 1 of Indications for Use

Level 2: Cancer Mutations with Evidence of Clinical Significance

Level 3: Cancer Mutations with Potential Clinical Significance

Non-targeted variants including germline variants are not reported. After this step, the remaining variants are annotated using Human Genome Variation Society (HGVS) standards, and a PDF format summary report is generated in two parts.

- The Run Summary section of the PiVAT Customer PDF Report output file contains various statistics that reflect run quality and an overview of all variation to be reported across all samples analyzed within that run. Samples that fail to meet certain NGS quality criteria are reported as not valid and no genetic variants are reported for these samples.
- The Patient Report section of the PiVAT Patient Report output file aggregates any variation observed at the patient level along with previously established clinical validity. Samples that fail to meet certain NGS quality thresholds are reported as not valid and no genetic variants are reported for these samples. The user can interact with the PiVAT IVD pipeline utilizing a browser-based visual interface. At the end of analysis, all intermediate data files and reports may be downloaded to a USB drive and transferred to another location for permanent storage.

Table 8. NGS-QC in PiVAT®: Run, Sample and Variant Calling Passing Criteria

|  a. NGS run level quality control  |   |   |
| --- | --- | --- |
|  Category | QC Metrics | Passing Criteria  |
|  Run - Invalid if any QC metric(s) | PosCtrl | Expected mutations are detected  |
|   |   |  No unexpected mutation(s) detected  |

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|  fails | NTC | No mutation detected  |
| --- | --- | --- |
|   |   |  Maximum coverage <50x or <0.5% of median within-run sample coverage  |
|  b. NGS sample level quality control  |   |   |
|  Category | QC Metrics | Passing Criteria  |
|  Sample - NOT valid if any QC metric(s) fails | Sequencing base quality | Bases (with Q Score ≥Q30) ≥75%  |
|   |  Amplification specificity | Effective On-Target-Rate^{1}≥70%  |
|   |  Coverage^{2} | Minimum depths of the three amplicons covering CDx mutations ≥1000x  |
|  CDx mutations thresholds | non-C>T|G>A | 1. Variant coverage ≥10x AND Total coverage ≥1000x 2. Average variant base Q-score ≥ 30 AND VAF ≥ 1%  |
|   |  C>T|G>A | 1. Variant coverage ≥10x AND Total coverage ≥1000x 2. Average variant base Q-score ≥ 30 AND VAF ≥ 1.5%  |
|  ^{a}Group 1 non-CDx mutation thresholds** | non-C>T|G>A | 1. Variant coverage ≥10x AND Total coverage ≥1000x 2. Average variant base Q-score ≥ 30 AND VAF ≥ 1%  |
|   |  C>T|G>A | 1. Variant coverage ≥10x AND Total coverage ≥1000x 2. Average variant base Q-score ≥ 30 AND VAF ≥ 1.5%  |
|   |  No Call* | Coverage < 1000x  |
|  ^{a}Group 2 non-CDx mutation thresholds** | Variant | 1. Variant coverage ≥10x AND Total coverage ≥500x 2. Average variant base Q-score ≥ 30 AND VAF ≥ 3.2%  |
|   |  No Call* | Coverage < 500 x  |

$^{1}$ Effective On-Target Rate = Mapping rate * On-target rate

$^{2}$ Coverage: the coverage after paired-end assembly by PiVAT®. All markers in the assay are bi-directional sequenced with 2x150bp sequencing protocol due to the short amplicon sizes (144-162bp including primers). 1x coverage = 1x forward + 1x reverse of sequencing reads. Only uniquely mapped reads are analyzed.

a: Group 1 non-CDx mutations includes: EGFR G719X, T790M; KRAS A59X, Q61X, K117N, A146X; and BRAF V600E; all other non-CDx mutations in Group 2.

* No calls are only applicable to non-CDx variants. Variants on amplicons with coverage below the threshold are at risk of being false-negatives.

** Non-CDx variants may be detected and reported although variant coverage requirement are not met. Positive non-CDx variant calls for variants that did not meet the variant coverage requirement are at risk of being false positive calls.

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## 5. Processes for database and variant annotation

**Database:** For the oncoReveal CDx distributed kit, the PIVAT software includes database information regarding the variants and their assignment to either Level 1 CDx Mutations', 'Level 2 Mutations = (Variants with Evidence of Clinical Significance)' and 'Level 3 Mutations (Variants with Potential Clinical Significance)'. A description of the assignment and curation process was provided.

**Report Generation in oncoReveal CDx:** The PiVAT Run Summary Report provides a run summary of the applicable run. The PiVAT software generates reports for each batched run and sample processed. The report includes tumor type, detected variants reported in HGVS format as either Level 1, 2 or 3 mutations (CDRH's approach to tumor profiling), and pertinent no call regions. The PIVAT Sample Test Report is the primary report of identified alterations for a sample. The Test Report divides variants into 3 sections: 'Level 1 CDx Mutations', 'Level 1 Mutations = (Variants with Evidence of Clinical Significance)' and 'Level 3 Mutations (Variants with Potential Clinical Significance)'. The variants listed in the section 'Level 2 Mutations - Variants with Evidence of Clinical Significance' are determined based on the selected tumor type. Only variants clinically associated with the selected tumor type will appear on this Level 2 – Variants with Evidence of Clinical Significance. Any remaining variants, meeting the SOP requirements for Level 3, will appear in the 'Variants with Potential Clinical Significance' section. A qualified healthcare professional selects the appropriate tumor type to ensure the corresponding tumor profiling variants appear in the report.

## 6. Determination of PiVAT NGS Calling and QC Threshold

### a. Requirements on Amplicon and Base Coverage:

Depth of coverage (coverage): defined as the number of aligned reads that contain a given nucleotide position. In PiVAT, the sequencing reads are first aligned to human reference hg19, then go through local re-alignment to remove alignment errors. After local-realignments, the paired-reads (forward and reverse reads) for each pair are assembled into a single read for coverage assessment and variant calling.

Base quality score (Q-score): The quality score of each base within an assembled read is adjusted by the PiVAT software by considering the Q-scores from both sequencing directions. The reassigned base Q-scores are subject to a threshold of 30, corresponding to a 1/1000 chance of error.

### b. Statistical Determination of Depth Coverage Requirements

Assuming that alleles reported by reads at a given genomic position follow a Bernoulli random process, with each read representing an independent event, the total number of reads supporting the mutant allele is expected to follow a binomial distribution. The 95% confidence interval (CI) representing the range of observed variant allele frequencies (VAFs) from the true underlying VAFs greater than 2% was computed. Based on the power analysis, the observed VAFs for a true underlying VAF of 2% are estimated to fall between 1.2% to 3.1% with 1000x coverage. When the mutation is

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present at 5% with a coverage of 500x, the 95% CI ranges from 3.3% to 7.3%, refer to Table 9.

Based on a requirement of a minimum 10 mutation reads to support a positive call, sequence coverage of $\geq 1000x$ yields a $> 95\%$ probability of detecting a true mutation at 2% VAF (1.2% -3.1% at 95% CI). For a mutation at a 5% VAF, a sequencing coverage of 500x provides close to 100% statistical power for detection (3.3% to 7.3%, 95%CI).

**Table 9. Power Analysis Results by Exact Binomial Distribution Model**

|  True_VAF | 95% confidence interval for computed observed-VAF, as function of coverage  |   |   |   |
| --- | --- | --- | --- | --- |
|   |  200X | 500x | 800x | 1000x  |
|  1% | (0.1%, 3.6%) | (0.3%, 2.3%) | (0.4%, 2.0%) | (0.5%, 1.8%)  |
|  2% | (0.6%, 5.0%) | (1.0%, 3.7%) | (1.2%, 3.2%) | **1.2%, 3.1%**  |
|  3% | (1.1%, 6.4%) | (1.7%, 4.9%) | (1.9%, 4.4%) | (2.0%, 4.3%)  |
|  3.7% | (1.4%, 7.1%) | (2.3%, 5.9%) | (2.5%, 5.3%) | **(2.6%, 5.1%)**  |
|  4% | (1.7%, 7.7%) | (2.5%, 6.1%) | (2.8%, 5.6%) | (2.9%, 5.4%)  |
|  **5%** | (2.4%, 9.0%) | **3.3%, 7.3%** | (3.6%, 6.8%) | (3.7%, 6.5%)  |
|  6% | (3.1%, 10.3%) | (4.1%, 8.5%) | (4.5%, 7.9%) | (4.6%, 7.7%)  |
|  7% | (3.9%, 11.5%) | (4.9%, 9.6%) | (5.3%, 9.0%) | (5.5%, 8.8%)  |
|  8% | (4.6%, 12.7%) | (5.8%, 10.7%) | (6.2%, 10.1%) | (6.4%, 9.9%)  |
|  9% | (5.4%, 13.9%) | (6.6%, 11.9%) | (7.1%, 11.2%) | (7.3%, 11.0%)  |
|  10% | (6.2%, 15.0%) | (7.5%, 13.0%) | (8.0%, 12.3%) | (8.2%, 12.0%)  |

### c. Confirmation of Theoretical VAF Estimates and Coverage Requirements

To evaluate the actual NGS VAFs compared to those estimated from the power analysis, NGS data was obtained from two sequencing runs. These runs yielded a total of 536 VAF measurements across 15 unique hotspot mutations at 10 expected VAF levels, derived from a total of 55 libraries encompassing 8 different DNA or FFPE samples. These samples consisted of mixed cell lines covering multiple endogenous SNPs and deletions, with mutation frequencies confirmed by ddPCR. The coverages for the 15 hotspot mutations typically varied within ranges that greatly exceeded 500x and 1000x in the samples. To evaluate the observed VAF distribution as function of coverage, the data was down-sampled in silico to approximate mean coverages of 500x and 1000x for hotspot mutations with $\geq 5\%$ VAF and $\geq 2\%$ VAF, respectively.

#### i. Direct Comparison of the NGS-Derived VAF with Expected ddPCR VAFs

The detailed variant and sample information, along with the high-level results, are presented in Tables 10a and 10b. The VAFs detected by NGS were highly correlated with and similar to those measured by droplet digital PCR (ddPCR), yielding an R-squared value of 0.99 (see Figures 1 and 2 below).

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**Table 10a. Sample, Variant, and General Run Information**

|  Sample | Horizon Cat# | Note | Library-prep # | Gene covered | Targeted Somatic mutations per sample | Total VAF measurements | ddPCR VAFs | Mean Base coverage (Range)  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  1 | HD850 | FFPE | 1 | *EGFR* | 5 | 5 | 1% | Run1: mean = **4302** (3382 – 4992)  |
|  2 | HD300 | FFPE | 1 | *EGFR* |   | 5 | 5%  |   |
|  3 | HD301 | FFPE | 1 | *KRAS, NRAS* | 6 | 6 | 5%  |   |
|  4 | HD701 | gDNA | 8 | *BRAF, EGFR, KRAS, NRAS, PIK3CA* | 10 | 80 | 1%, 2%, 3%*, 6%, 9%, 10.5%  |   |
|  5 | HD-C749 | Formalin-Compromised DNA I (Mild) | 8 |   |   | 80 | 12.5%, 15%, 17.5% | Run2: mean = **6475** (5523 – 7254)  |
|  6 | HD803 | Formalin-Compromised DNA I (Severe) | 6 |   |   | 60 | 24.5%  |   |
|  7 | HD799 | Formalin-Compromised DNA I (moderate) | 24 |   |   | 240 |   |   |
|  7 | HD701 | gDNA | 6 |   |   | 60 |   |   |
|  Total |  |  | 55 |  |  | 536 |  |   |

\*Intended to be 3% VAF, but the actual lot is 3.5%

**Table 10b. Summary of Results for 15 Unique Mutations Across 536 Observations at Each Tested VAF Level**

|  Expected ddPCR VAF | NGS observations | Mean NGS VAF (%) | %CV of NGS VAFs | Mutations  |
| --- | --- | --- | --- | --- |
|  1%-VAF | 57 | 1.1 | 31% | *EGFR*: T790M; *EGFR*: G719S, L858R, E746_A750del, T790M, L861Q  |
|  2%-VAF | 52 | 2.0 | 24% | *EGFR*: E746_A750del  |
|  3%-VAF | 52 | 3.5 | 22% | *EGFR*: L858R  |
|  5%-VAF | 11 | 5.4 | 16% | *KRAS*: G13D, G12D, A146T, Q61H *NRAS*: Q61K, G12V *EGFR*: G719S, L858R, E746_A750del, T790M, L861Q  |
|  6%-VAF | 52 | 6.3 | 15% | *KRAS*: G12D  |
|  9%-VAF | 52 | 8.5 | 12% | *PIK3CA*: E545K  |
|  10.5%-VAF | 52 | 12.6 | 14% | *BRAF*: V600E  |
|  12.5%-VAF | 52 | 12.2 | 12% | *NRAS*: Q61K  |

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|  Expected ddPCR VAF | NGS observations | Mean NGS VAF (%) | %CV of NGS VAFs | Mutations  |
| --- | --- | --- | --- | --- |
|  15%-VAF | 52 | 15.4 | 11% | *KRAS*: G13D  |
|  17.5%-VAF | 52 | 18.4 | 10% | *PIK3CA*: H1047R  |
|  24.5%-VAF | 52 | 24.7 | 6% | *EGFR*: G719S  |
|  Total | 536 |  |  |   |

![img-0.jpeg](img-0.jpeg)

**Figure 1: Correlation between NGS and ddPCR VAFs.** Comparison between variant allele frequencies (VAF) measured by NGS or ddPCR. Samples used in analysis were eight different FFPE or mixed cell line-derived DNA reference materials. A total of 55 libraries were prepared and sequenced from two independent sequencing runs. 536 observations across 15 unique hotspot mutations estimated to be at 10 different VAF levels were compared. (n=11-57 NGS observations per ddPCR determined VAF level) A. Correlation between VAFs measured by oncoReveal NGS and ddPCR method. Error bars represent SD in NGS VAF measurement.

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![img-1.jpeg](img-1.jpeg)

Figure 2: Boxplot of NGS VAF distribution across a total of 536 VAF measurements. Box and whisker plot of NGS determined VAF across all 536 observations (Figure 1), binned by ddPCR VAF. Box and whisker plot with outliers: the boxes represent the interquartile ranges (IQR) from the first quartiles to the third quartiles. The vertical lines go through the boxes at the median with mean value marked. The whiskers extend down or up from each quartile to the minimum data value or maximum data value within 1.5 times the IQR. Values outside of this range are considered to be outliers and represented by a small filled-in circle.

## ii. Variant Total Coverage Requirements

In silico down sampling analysis was conducted on the 55 libraries listed in Table 10a to normalize hotspot coverage to approximately 500x or 1000x. For the expected underlying VAFs of 2% and 3.5%, at a coverage of ~1000x, the observed VAFs were detected at 1.94% (ranging from 0.7% - 3.1%) and 3.68% (ranging from 1.6% to 6.4%) respectively. At a coverage of ~500x, the observed VAFs for an expected 5% VAF ranged from 3.9% to 8.4% with a mean VAF of 5.48%; for an expected 6% VAF, the observed range was 2.7% to 9.1% with a mean VAF at 6.31%. These ranges are roughly in agreement with theoretical statistical estimates (see Figure 3 below). The data support the use of a 1 – 1.5% VAF threshold to detect CDx and Group1 variants with true underlying VAFs of 2-4% at a coverage of ≥ 1000x; a threshold of 3.2% is recommended to report Group 2 variants with true underlying VAFs of 5-6% at a coverage of ≥ 500x.

For Group-2 variants with a VAF cut-off of 3.2%, a “No call” is reported if the position coverage is sequenced at less than 500x and no positive variant is detected at this position. Similarly, for Group-1 variants with a VAF cut-off of 1-1.5%, a “No call” is reported if the position coverage is sequenced at less than 1000x. A “No call” designation indicates that there is a risk of false-negative calls, especially for true variants at VAFs levels around the Limit of detection (LoD).

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A.

![img-2.jpeg](img-2.jpeg)

B.

![img-3.jpeg](img-3.jpeg)

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C.

![img-4.jpeg](img-4.jpeg)

D.

![img-5.jpeg](img-5.jpeg)

**Figure 3. Comparison of observed and theoretical statistically estimated VAFs.** *In silico* down sampling analysis of 55 sequenced libraries was used to determine cutoffs for variants with true VAF values of (A) 2%, (B) 3.5%, (C) 6%, and (D) 5%. Observed VAFs after down-sampling libraries to 1000x read coverage with variants at 2% or 3.5% are shown in A and B, respectively. Observed VAFs after down-sampling libraries to 500x read coverage with variants at 6% or 5% are shown in C and D, respectively. The computed lower bound (dark orange) and upper bound (light orange) 95% CI of VAF measurements as a function of coverage as well as VAF cutoffs (green dashed lines) are shown.

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### iii. Requirements on Sample Quality and Sequencing Metrics for Confirmed Coverage Requirements:

Fifty-four (54) normal (diploid) FFPE samples from 9 different tissue types were tested with normalized 10ng of DNA input to provide the assessment for the pre-NGS library yield, effective on-target rate and amplicon/base coverage (Table 11).

**Table 11. Normal FFPE from 9 Tissue Types**

|  FFPE Tissue type | Count  |
| --- | --- |
|  Normal Bladder | 5  |
|  Normal Breast | 8  |
|  Normal Cervix | 2  |
|  Normal Kidney | 6  |
|  Normal Liver | 7  |
|  Normal Pancreas | 7  |
|  Normal Skin | 6  |
|  Normal Thyroid | 9  |
|  Normal Uterus | 4  |

The results shown in Figure 4 demonstrate that a lower mean coverage depth and a higher percentage of bases with less than 500x coverage are associated with low library yield and low effective on-target rate (calculated as Mapping rate x on-target rate). The minimal requirement of 3.5nM for library yield and 70% for the effective on-target rate effectively removed low-quality libraries characterized by insufficient base coverage. The 36 normal samples that passed the filter yielded a mean base coverage of 6359x, ranging from 4619x to 10487x, with 100% of bases covered at least 200x. On average, 99.5% of bases achieved coverage exceeding 500x, with a range from 96.9 % to 100%. Statistical analysis indicates that a minimum of 10 mutation reads at a 95% power level, and 200x coverage, can detect a true underlying variant frequency of 8.5% with a 95% CI ranging from 5% to 13.3%.

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![img-6.jpeg](img-6.jpeg)

**Figure 4. Establishing QC Thresholds Using 54 Normal FFPE Samples**

Library yield and effective on-target rate are associated with mean coverage depth and percentage of bases below 500x coverage. Library yield cut off value of 3.5 nM (dash grey line) and effective on-target rate cut off value of 70% (solid grey line) can filter out poor quality libraries.

With the established threshold of 3.5 nM for pre-NGS library yield, sequence coverage was then evaluated across a range of FFPE samples (n= 373), with DNA input varying from 6.5 ng to 513 ng with median input of approximately 30ng. Among these, three samples had effective on-target rates below the 70% threshold and were thus deemed failures. The remaining 370 samples achieved a median average-base-coverage of 5971x with an effective on-target rate of 96.1+-2.59 (mean ± SD). Notably, only two samples did not attain 100% base coverage at a depth of 200x (Table 12).

**Table 12. Base Coverage Distribution for 370 Valid FFPE samples**

|  Base coverage stats | Sample | % sample  |
| --- | --- | --- |
|  100% base coverage>200x | 368 | 99.5%  |
|  100% base coverage>300x | 356 | 96.2%  |
|  100% base coverage>500x | 303 | 81.9%  |

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#### iv. Requirements on Variant Coverage, Allele Depth and Frequency for Positive Calls:

Variant filtering parameters, as detailed in Table 8, which include coverage, variant allele depth, and variant allele frequency, were established to maximize the probability of true positive calls and minimize false positive calls.

The variant calling thresholds were empirical supported by the 57 libraries, which contained various known level of variants, and the cohort of 36 normal FFPE samples that passed the sample level QC as described in the previous sections.

### VI. ALTERNATIVE PRACTICES AND PROCEDURES

There are several FDA-approved CDx alternatives for the detection of genetic alterations using FFPE tumor specimens, as listed in the oncoReveal™ CDx intended use statement. The approved CDx tests are listed in Table 13 below; for additional details see FDA List of Cleared or Approved CDx Devices at https://www.fda.gov/medical-devices/in-vitro-diagnostics/list-cleared-or-approved-companion-diagnostic-devices-in-vitro-and-imaging-tools. Each alternative has its own advantages and disadvantages. A patient should fully discuss these alternatives with his/her physician to select the method that best meets expectations and lifestyle.

**Table 13. FDA-approved companion diagnostic (CDx) Alternatives to oncoReveal CDx**

|  Indicatio | Gene | Device | Company | Technolog | Therapy  |
| --- | --- | --- | --- | --- | --- |
|  CRC | *KRAS*, *NRAS* | xT CDx (P210011) | Tempus Labs, Inc. | NGS | ERBITUX (cetuximab) or VECTIBIX (panitumumab).  |
|  CRC | *KRAS* | **cobas**® KRAS Mutation Test (P140023) | Roche Molecular Systems, Inc. | PCR | ERBITUX® (cetuximab) VECTIBIX (panitumumab)  |
|  CRC | *KRAS* | *Therascreen*® KRAS RGQ PCR Kit (P110030; P110027) | QIAGEN Manchester Ltd. | PCR | ERBITUX® (cetuximab) VECTIBIX (panitumumab)  |
|  CRC | *KRAS* | FoundationOne® CDx (P170019) | Foundation Medicine, Inc. | NGS | ERBITUX® (cetuximab) VECTIBIX® (panitumumab)  |

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|  NSCLC | *EGFR* | *Therascreen*® EGFR RGQ PCR Kit (P120022/S018) | QIAGEN Manchester Ltd. | PCR | IRESSA® (gefitinib) GILOTRIF® (afatinib) VIZIMPRO® (dacomitinib)  |
| --- | --- | --- | --- | --- | --- |
|  NSCLC | *EGFR* | **cobas**® EGFR Mutation Test v2 (P120019/S01) | Roche Molecular Systems, Inc. | PCR | EGFR Tyrosine Kinase Inhibitors approved by FDA  |
|  NSCLC | *EGFR* | FoundationOne® CDx (P170019) | Foundation Medicine, Inc. | NGS | GILOTRIF® (afatinib) IRESSA® (gefitinib) TARCEVA® (erlotinib) TAGRISSO® (osimertinib)  |
|  NSCLC | *EGFR* | Oncomine™ Dx Target Test (P160045; | Life Technologie s Corp. | NGS | IRESSA® (gefitinib)  |

**Abbreviations:** NSCLC = Non-Small Cell Lung Cancer, CRC=Colorectal Cancer

## **VII. MARKETING HISTORY**

The oncoReveal CDx Premarket Approval (P200011) was originally approved on July 30, 2021, by FDA and has been commercially available in the U.S. since August 21, 2021.

## **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 positive results may undergo treatment with one of the therapies listed in the above intended use statement without clinical benefit and may experience adverse reactions associated with the 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 the indicated therapy.

For the specific adverse events related to the approved therapeutics, please see approved drug product labels.

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# IX. SUMMARY OF NONCLINICAL STUDIES

# A. Laboratory Studies

Performance characteristics of the oncoReveal™ CDx were established using DNA derived from a wide range of FFPE tumor tissue specimens. Studies included reportable CDx variants indicated in Table 1 of the intended use statement and a wide range of representative variant types (SNV, deletion and insertion) across 22 genes.

# 1. Analytical Accuracy

Analytical accuracy was performed to demonstrate the concordance between the oncoReveal CDx and two externally validated NGS (evNGS) comparator methods (A & B) to support the accuracy of the oncoReveal CDx to detect reportable SNVs, deletions and insertions for tumor profiling in 22 genes. The concordance analysis was done for overall agreement, by variant types, and per gene.

# a. Comparator Method A

A total of 271 samples represented by 10 tumor types (colorectal cancer, pancreatic adenocarcinoma, non-small cell lung cancer, breast cancer, melanoma, bladder cancer, uterine corpus endometrial cancer, hepatocellular carcinoma, clear cell renal cell carcinoma, and papillary thyroid cancer) were tested across 15 genes using comparator method A. Of these samples, 257 samples yielded valid results (181 positive and 65 negative) by both assays and were included in the agreement analysis. There was a total of 11 discordant samples including 7 oncoReveal™ CDx positive (+) evNGS A negative (-), and 4 samples positive concordant call in both assays for one mutation but contained an additional variant(s) called by only one assay. In 3 samples an additional low VAF variant (<3%) was reported by oncoReveal™ CDx and not comparator A and in 1 sample, a low VAF variant (<5%) was reported in comparator A but not oncoReveal™ CDx. The three discordant variants with high VAFs (33%- 75%), two 15bp deletions and one 6-bp insertion, were confirmed positive in a third orthogonal method. All other discordant mutations are expected due to the low allelic fractions and the differences in VAF cut-offs applied by oncoReveal CDx and comparator method A. The overall sample level concordance was 95.7% (246/257).

The aggregated results at the variant-type level are shown in Table 14 and gene level is shown in Table 15 below. As the accuracy study samples were enrolled by the oncoReveal™ CDx, the positive predictive value (PPV) and negative predictive value (NPV) were direct calculations; however, the positive percent agreement (PPA) and negative percent agreement (NPA) values were adjusted using the proportion of positive variants detected by oncoReveal CDx assay.

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**Table 14. Comparator Method A: Overall Variant-level Agreement and Binned by Variant Type**

|  Variant Type | # Samples | # Variants | PPA (n/N) (95% CI) | NPA (n/N) (95% CI) | PPV (n/N) (95% CI) | NPV (n/N) (95% CI) | Adjusted PPA (95% CI) | Adjusted NPA (95% CI)  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  **All** | 257 | 243636 | 99.6% (245/246) (97.7%, 99.9%) | 99.9% (243380/243390) (99.9%, 99.9%) | 96.1% (245/255) (92.9%, 97.9%) | 99.9% (243380/243381) (99.9%, 99.9%) | 99.0% (94.4%, 99.8%) | 99.9% (99.9%, 99.9%)  |
|  **SNV** | 257 | 141864 | 99.6% (228/229) (97.6%, 99.9%) | 99.9% (141629/141635) (99.9%, 99.9%) | 97.4% (228/234) (94.5%, 98.8%) | 99.9% (141629/141630) (99.9%, 99.9%) | 99.3% (95.9%, 99.9%) | 99.9% (99.9%, 99.9%)  |
|  **MNV** | 257 | 31354 | 100.0% (4/4) (51.0%, 100.0%) | 100.0% (31350/31350) (99.9%, 100.0%) | 100.0% (4/4) (51.0%, 100.0%) | 100.0% (31350/31350) (99.9%, 100.0%) | 100.0% (16.5%, 100.0%) | 100.0% (99.9%, 100.0%)  |
|  **Deletion** | 257 | 42148 | 100.0% (11/11) (74.1%, 100.0%) | 99.9% (42134/42137) (99.9%, 99.9%) | 78.6% (11/14) (52.4%, 92.4%) | 100.0% (42134/42134) (99.9%, 100.0%) | 100.0% (31.0%, 100.0%) | 99.9% (99.9%, 99.9%)  |
|  **Insertion** | 257 | 28270 | 100.0% (2/2) (34.2%, 100.0%) | 99.9% (28267/28268) (99.9%, 99.9%) | 66.7% (2/3) (20.8%, 93.9%) | 100.0% (28267/28267) (99.9%, 100.0%) | 100.0% (6.4%, 100.0%) | 99.9% (99.9%, 99.9%)  |

SNV=single nucleotide variants, MNV=multi-nucleotide variant

**Table 15. Comparator Method A: Variant-level Agreement by Gene**

|  Gene | PPA (n/N) (95% CI) | NPA (n/N) (95% CI) | PPV (n/N) (95% CI) | NPV (n/N) (95% CI)  |
| --- | --- | --- | --- | --- |
|  *AKT1* | 100.0% (4/4) (51.0%, 100.0%) | 100.0% (767/767) (99.5%, 100.0%) | 100.0% (4/4) (51.0%, 100.0%) | 100.0% (767/767) (99.5%, 100.0%)  |
|  *ALK* | Not Evaluable | 100.0% (8995/8995) (99.9%, 100.0%) | Not Evaluable | 100.0% (8995/8995) (99.9%, 100.0%)  |
|  *BRAF* | 100.0% (34/34) (89.8%, 100.0%) | 99.9% (21296/21297) (99.9%, 99.9%) | 97.1% (34/35) (85.5%, 99.5%) | 100.0% (21296/21296) (99.9%, 100.0%)  |
|  *CTNNB1* | 100.0% (11/11) (74.1%, 100.0%) | 100.0% (44964/44964) (99.9%, 100.0%) | 100.0% (11/11) (74.1%, 100.0%) | 100.0% (44964/44964) (99.9%, 100.0%)  |
|  *DDR2* | Not Evaluable | 100.0% (514/514) (99.3%, 100.0%) | Not Evaluable | 100.0% (514/514) (99.3%, 100.0%)  |

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|  Gene | PPA (n/N) (95% CI) | NPA (n/N) (95% CI) | PPV (n/N) (95% CI) | NPV (n/N) (95% CI)  |
| --- | --- | --- | --- | --- |
|  EGFR | 100.0% (30/30) (88.6%, 100.0%) | 99.9% (68586/68589) (99.9%, 99.9%) | 90.9% (30/33) (76.4%, 96.9%) | 100.0% (68586/68586) (99.9%, 100.0%)  |
|  ERBB2 | 80.0% (4/5) (37.6%, 96.4%) | 99.9% (12330/12331) (99.9%, 99.9%) | 80.0% (4/5) (37.6%, 96.4%) | 99.9% (12330/12331) (99.9%, 99.9%)  |
|  ERBB4 | Not Evaluable | Not Evaluable | Not Evaluable | Not Evaluable  |
|  FBXW7 | Not Evaluable | Not Evaluable | Not Evaluable | Not Evaluable  |
|  FGFR1 | Not Evaluable | 100.0% (771/771) (99.5%, 100.0%) | Not Evaluable | 100.0% (771/771) (99.5%, 100.0%)  |
|  FGFR2 | 100.0% (4/4) (51.0%, 100.0%) | 100.0% (4365/4365) (99.9%, 100.0%) | 100.0% (4/4) (51.0%, 100.0%) | 100.0% (4365/4365) (99.9%, 100.0%)  |
|  FGFR3 | 100.0% (5/5) (56.6%, 100.0%) | 100.0% (4621/4621) (99.9%, 100.0%) | 100.0% (5/5) (56.6%, 100.0%) | 100.0% (4621/4621) (99.9%, 100.0%)  |
|  KRAS | 100.0% (93/93) (96.0%, 100.0%) | 99.9% (25603/25607) (99.9%, 99.9%) | 95.9% (93/97) (89.9%, 98.4%) | 100.0% (25603/25603) (99.9%, 100.0%)  |
|  MAP2K1 | 100.0% (1/1) (20.7%, 100.0%) | 99.9% (7194/7195) (99.9%, 99.9%) | 50.0% (1/2) (9.5%, 90.5%) | 100.0% (7194/7194) (99.9%, 100.0%)  |
|  MET | Not Evaluable | 100.0% (2827/2827) (99.9%, 100.0%) | Not Evaluable | 100.0% (2827/2827) (99.9%, 100.0%)  |
|  NOTCH1 | Not Evaluable | Not Evaluable | Not Evaluable | Not Evaluable  |
|  NRAS | 100.0% (10/10) (72.2%, 100.0%) | 100.0% (12069/12069) (99.9%, 100.0%) | 100.0% (10/10) (72.2%, 100.0%) | 100.0% (12069/12069) (99.9%, 100.0%)  |
|  PIK3CA | 100.0% (49/49) (92.7%, 100.0%) | 100.0% (28478/28478) (99.9%, 100.0%) | 100.0% (49/49) (92.7%, 100.0%) | 100.0% (28478/28478) (99.9%, 100.0%)  |
|  PTEN | Not Evaluable | Not Evaluable | Not Evaluable | Not Evaluable  |
|  SMAD4 | Not Evaluable | Not Evaluable | Not Evaluable | Not Evaluable  |
|  STK11 | Not Evaluable | Not Evaluable | Not Evaluable | Not Evaluable  |
|  TP53 | Not Evaluable | Not Evaluable | Not Evaluable | Not Evaluable  |

### b. Comparator Method B

A second validated Comparator Method B (evNGS B) was used to include 6 additional genes not targeted by Method A by testing samples from 10 cancer types (colorectal cancer, pancreatic adenocarcinoma, non-small cell lung cancer, breast cancer, melanoma, bladder cancer, uterine corpus endometrial cancer, hepatocellular carcinoma, clear cell renal cell carcinoma, and papillary thyroid cancer). From the total enrolled 212 samples, 187 samples yielded valid results (158 positive and 10 negative) for both assays and included in the agreement study. There was a total of 19 discordant samples consisting of 2 oncoReveal™

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CDx positive (+) evNGS B negative (-) and 17 samples that showed positive concordance in the targeted variant(s) but contained additional positive variant calls in either oncoReveal CDx or comparator B. The overall sample level concordance was 90% (168/187).

The aggregated results at the variant-level is shown in Table 16 and gene level is shown in Table 17 below. As the accuracy study samples were enrolled by the oncoReveal$^{TM}$ CDx, the PPV and NPV were direct calculations; however, the PPA and NPA values were adjusted using the proportion of positive variants detected by oncoReveal CDx assay.

**Table 16. Comparator B: Overall Variant-level Agreement and Binned by Variant Type**

|  Variant Type | # Samples | # Variants | PPA (n/N) (95% CI) | NPA (n/N) (95% CI) | PPV (n/N) (95% CI) | NPV (n/N) (95% CI) | Adjusted PPA (95% CI) | Adjusted NPA (95% CI)  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  **All** | 187 | 661045 | 98.6% (345/350) (96.7%, 99.4%) | 99.9% (660677/66 0695) (99.9%, 99.9%) | 95.0% (345/363) (92.3%, 96.8%) | 99.9% (660677/ 660682) (99.9%, 99.9%) | 94.3% (91.1%, 96.3%) | 99.9% (99.9%, 99.9%)  |
|  **SNV** | 187 | 250954 | 98.7% (308/312) (96.8%, 99.5%) | 99.9% (250627/25 0642) (99.9%, 99.9%) | 95.4% (308/323) (92.5%, 97.2%) | 99.9% (250627/ 250631) (99.9%, 99.9%) | 94.4% (90.9%, 96.5%) | 99.9% (99.9%, 99.9%)  |
|  **MNV** | 187 | 37587 | 100.0% (6/6) (61.0%, 100.0%) | 100.0% (37581/375 81) (99.9%, 100.0%) | 100.0% (6/6) (61.0%, 100.0%) | 100.0% (37581/3 7581) (99.9%, 100.0%) | 100.0% (27.5%, 100.0%) | 100.0% (99.9%, 100.0%)  |
|  **Deletion** | 187 | 238051 | 100.0% (21/21) (84.5%, 100.0%) | 99.9% (238028/23 8030) (99.9%, 99.9%) | 91.3% (21/23) (73.2%, 97.6%) | 100.0% (238028/ 238028) (99.9%, 100.0%) | 89.3% (66.0%, 96.8%) | 100.0% (99.9%, 100.0%)  |
|  **Insertion** | 187 | 134453 | 90.9% (10/11) (62.3%, 98.4%) | 99.9% (134441/13 4442) (99.9%, 99.9%) | 90.9% (10/11) (62.3%, 98.4%) | 99.9% (134441/ 134442) (99.9%, 99.9%) | 86.7% (44.1%, 97.6%) | 99.9% (99.9%, 99.9%)  |

SNV=single nucleotide variants, MNV=multi-nucleotide variant

**Table 17. Comparator Method B: Variant-level Agreement by Gene**

|  Gene | PPA (n/N) (95% CI) | NPA (n/N) (95% CI) | PPV (n/N) (95% CI) | NPV (n/N) (95% CI)  |
| --- | --- | --- | --- | --- |
|  *AKT1* | 100.0% (4/4) (51.0%, 100.0%) | 100.0% (370/370) (99.0%, 100.0%) | 100.0% (4/4) (51.0%, 100.0%) | 100.0% (370/370) (99.0%, 100.0%)  |

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|  Gene | PPA (n/N) (95% CI) | NPA (n/N) (95% CI) | PPV (n/N) (95% CI) | NPV (n/N) (95% CI)  |
| --- | --- | --- | --- | --- |
|  *ALK* | 100.0% (1/1) (20.7%, 100.0%) | 100.0% (6357/6357) (99.9%, 100.0%) | 100.0% (1/1) (20.7%, 100.0%) | 100.0% (6357/6357) (99.9%, 100.0%)  |
|  *BRAF* | 100.0% (37/37) (90.6%, 100.0%) | 99.9% (15296/15297) (99.9%, 99.9%) | 97.4% (37/38) (86.5%, 99.5%) | 100.0% (15296/15296) (99.9%, 100.0%)  |
|  *CTNNB1* | 94.7% (18/19) (75.4%, 99.1%) | 100.0% (32706/32706) (99.9%, 100.0%) | 100.0% (18/18) (82.4%, 100.0%) | 99.9% (32706/32707) (99.9%, 99.9%)  |
|  *DDR2* | Not Evaluable | 100.0% (561/561) (99.3%, 100.0%) | Not Evaluable | 100.0% (561/561) (99.3%, 100.0%)  |
|  *EGFR* | 100.0% (22/22) (85.1%, 100.0%) | 100.0% (49533/49533) (99.9%, 100.0%) | 100.0% (22/22) (85.1%, 100.0%) | 100.0% (49533/49533) (99.9%, 100.0%)  |
|  *ERBB2* | 100.0% (7/7) (64.6%, 100.0%) | 100.0% (8782/8782) (99.9%, 100.0%) | 100.0% (7/7) (64.6%, 100.0%) | 100.0% (8782/8782) (99.9%, 100.0%)  |
|  *ERBB4* | 100.0% (6/6) (61.0%, 100.0%) | 100.0% (3734/3734) (99.9%, 100.0%) | 100.0% (6/6) (61.0%, 100.0%) | 100.0% (3734/3734) (99.9%, 100.0%)  |
|  *FBXW7* | 100.0% (13/13) (77.2%, 100.0%) | 100.0% (16443/16443) (99.9%, 100.0%) | 100.0% (13/13) (77.2%, 100.0%) | 100.0% (16443/16443) (99.9%, 100.0%)  |
|  *FGFR1* | 100.0% (1/1) (20.7%, 100.0%) | 100.0% (934/934) (99.6%, 100.0%) | 100.0% (1/1) (20.7%, 100.0%) | 100.0% (934/934) (99.6%, 100.0%)  |
|  *FGFR2* | 100.0% (7/7) (64.6%, 100.0%) | 100.0% (2985/2985) (99.9%, 100.0%) | 100.0% (7/7) (64.6%, 100.0%) | 100.0% (2985/2985) (99.9%, 100.0%)  |
|  *FGFR3* | 100.0% (3/3) (43.9%, 100.0%) | 99.9% (3362/3363) (99.8%, 99.9%) | 75.0% (3/4) (30.1%, 95.4%) | 100.0% (3362/3362) (99.9%, 100.0%)  |
|  *KRAS* | 96.8% (30/31) (83.8%, 99.4%) | 99.9% (17919/17921) (99.9%, 99.9%) | 93.8% (30/32) (79.9%, 98.3%) | 99.9% (17919/17920) (99.9%, 99.9%)  |
|  *MAP2K1* | 100.0% (2/2) (34.2%, 100.0%) | 100.0% (5608/5608) (99.9%, 100.0%) | 100.0% (2/2) (34.2%, 100.0%) | 100.0% (5608/5608) (99.9%, 100.0%)  |
|  *MET* | Not Evaluable | 100.0% (2431/2431) (99.8%, 100.0%) | Not Evaluable | 100.0% (2431/2431) (99.8%, 100.0%)  |
|  *NOTCH1* | Not Evaluable | Not Evaluable | Not Evaluable | Not Evaluable  |
|  *NRAS* | 100.0% (21/21) (84.5%, 100.0%) | 100.0% (8394/8394) (99.9%, 100.0%) | 100.0% (21/21) (84.5%, 100.0%) | 100.0% (8394/8394) (99.9%, 100.0%)  |

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|  Gene | PPA (n/N) (95% CI) | NPA (n/N) (95% CI) | PPV (n/N) (95% CI) | NPV (n/N) (95% CI)  |
| --- | --- | --- | --- | --- |
|  *PIK3CA* | 100.0% (57/57) (93.7%, 100.0%) | 99.9% (20699/20700) (99.9%, 99.9%) | 98.3% (57/58) (90.9%, 99.7%) | 100.0% (20699/20699) (99.9%, 100.0%)  |
|  *PTEN* | 100.0% (40/40) (91.2%, 100.0%) | 99.9% (131789/131795) (99.9%, 99.9%) | 87.0% (40/46) (74.3%, 93.9%) | 100.0% (131789/131789) (99.9%, 100.0%)  |
|  *SMAD4* | 100.0% (10/10) (72.2%, 100.0%) | 99.9% (32713/32715) (99.9%, 99.9%) | 83.3% (10/12) (55.2%, 95.3%) | 100.0% (32713/32713) (99.9%, 100.0%)  |
|  *STK11* | 100.0% (6/6) (61.0%, 100.0%) | 100.0% (15702/15702) (99.9%, 100.0%) | 100.0% (6/6) (61.0%, 100.0%) | 100.0% (15702/15702) (99.9%, 100.0%)  |
|  *TP53* | 95.2% (60/63) (86.9%, 98.4%) | 99.9% (284359/284364) (99.9%, 99.9%) | 92.3% (60/65) (83.2%, 96.7%) | 99.9% (284359/284362) (99.9%, 99.9%)  |

The results of the accuracy study support the accuracy of variant (SNVs, insertions and deletions) calling by the oncoReveal CDx assay.

## 2. Analytical Sensitivity

### a. Limit of Blank (LoB)

An LoB study was conducted by evaluating DNA samples extracted from 16 FFPE specimens from normal tissues for ten cancer types. The following normal tissue were evaluated: lung, colon, bladder, breast, uterus, kidney, liver, pancreas, skin, and thyroid. Each sample was tested with 4 to 18 replicates at the maximum specified DNA input for oncoReveal™ CDx which is 80 ng, with two reagent lots, two to three replicates over two to three sequencing runs. All 105 replicate measurements yielded valid results. As shown in Table 18, no false positive calls were observed confirming the false positive rate at 80 ng DNA input as zero.

**Table 18. LoB Study Results**

|  Tissue Type | Number of Valid Samples | CDx Positive | Level 2 Positive | Level 3 Positive  |
| --- | --- | --- | --- | --- |
|  Normal Lung | 18 | 0/18 | 0/18 | 0/18  |
|  Normal Colon | 18 | 0/18 | 0/18 | 0/18  |
|  Normal Bladder | 10 | 0/10 | 0/10 | 0/10  |
|  Normal Breast | 8 | 0/8 | 0/8 | 0/8  |
|  Normal Uterus | 10 | 0/10 | 0/10 | 0/10  |
|  Normal Kidney | 9 | 0/9 | 0/9 | 0/9  |

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|  Tissue Type | Number of Valid Samples | CDx Positive | Level 2 Positive | Level 3 Positive  |
| --- | --- | --- | --- | --- |
|  Normal Liver | 10 | 0/10 | 0/10 | 0/10  |
|  Normal Pancreas | 9 | 0/9 | 0/9 | 0/9  |
|  Normal Skin | 4 | 0/4 | 0/4 | 0/4  |
|  Normal Thyroid | 9 | 0/9 | 0/9 | 0/9  |
|  Positive calls/Valid Results | 105 | 0/105 | 0/105 | 0/105  |
|  Percent False Positive Rate |   | 0% | 0% | 0%  |

### b. Limit of Detection (LoD)

Eleven (11) NSCLC and CRC specimens containing 14 tumor profiling variants (13 SNVs, and 1 insertion) were evaluated in this study. The LoD for tumor profiling variants were estimated using the hit rate approach where LoD is defined as the VAF detected at ≥95% hit rate. A minimum of 5 titration levels were tested with 20 replicates per level with two reagent lots (10 replicates per lot) using the minimum specified DNA input for the oncoReveal CDx, which is 30 ng.

The estimated LoD of each variant is summarized in Table 19 below. LoD of CDx variants were previously reported in P200011, which included a deletion (refer to Section IX.A.2b for the P200011 SSED) for which an LoD of 1.7% VAF was established. Based on the established LoD results presented in Table 20, the LoD ranges from 1.4% to 4.8% VAF.

Table 19. Summary of oncoReveal CDx assay non-CDx Variant Limit of Detection

|  Gene | Nucleotide Change | Amino Acid Change | Variant Type | LoD (%VAF)  |
| --- | --- | --- | --- | --- |
|  EGFR | c.2155G>T | p.Gly719Cys | SNV | 1.6  |
|  EGFR | c.2369C>T | p.Thr790Met | SNV | 3.0  |
|  KRAS | c.182A>T | p.Gln61Leu | SNV | 2.2  |
|  KRAS | c.436G>A | p.Ala146Thr | SNV | 2.8  |
|  PIK3CA | c.1624G>A | p.Glu542Lys | SNV | 4.4  |
|  SMAD4 | c.533C>G | p.Ser178Ter | SNV | 3.7  |
|  TP53 | c.880G>T | p.Glu294Ter | SNV | 4.5  |
|  TP53 | c.818G>A | p.Arg273His | SNV | 4.7  |
|  PIK3CA | c.3140A>G | p.His1047Arg | SNV | 4.1  |
|  EGFR | c.2303G>T | p.Ser768Ile | SNV | 4.8  |
|  TP53 | c.817C>T | p.Arg273Cys | SNV | 4.1  |
|  TP53 | c.892G>T | p.Glu298Ter | SNV | 4.7  |
|  BRAF | c.1799T>A | p.Val600Glu | SNV | 1.4  |
|  EGFR | c.2314_2319dup | p.Pro772_His773dup | Insertion | 2.2  |

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## LoD confirmation

LoD established using NSCLC and CRC specimens for the 3 variant types above (SNVs, insertion and deletion) were subsequently confirmed using six replicates across 20 samples which included 10 tumor types (specimens from bladder, breast, renal, colon, liver, skin, lung, pancreatic, thyroid and uterine/endometrial cancer). Each specimen had one or more variants between 1x-1.5x LoD and was confirmed with six replicates. Some variants were at 1.5x -2x LoD in the LoD confirmation study. The LoD of 10 SNVs, seven deletions and seven insertions were confirmed as shown in Table 20 below.

Table 20. Observed Hit Rate of LoD Confirmation Stratified by Variant Type.

|  Variant Type | Positive Detected | Total Positive | Negative Detected | Total Negative | Positive call Rate (n/N) 95% CI | Negative call Rate (n/N) 95% CI  |
| --- | --- | --- | --- | --- | --- | --- |
|  SNV | 60 | 60 | 8340 | 8340 | 100.0% (60/60) (94.0%, 100.0%) | 100.0% (8340/8340) (100.0%, 100.0%)  |
|  Insertion | 41 | 42^{1} | 4344 | 4344 | 97.6% (41/42) (87.7%, 99.6%) | 100.0% (4344/4344) (99.9%, 100.0%)  |
|  Deletion | 41 | 42^{1} | 7830 | 7830 | 97.6% (41/42) (87.7%, 99.6%) | 100.0% (7830/7830) (100.0%, 100.0%)  |
|  ALL | 142 | 144 | 20514 | 20514 | 98.6% (142/144) (95.1%, 99.6%) | 100.0% (20514/20514) (100.0%, 100.0%)  |

$^{1}$ One replicate from a sample that contained an insertion, and a deletion was reported as invalid by PiVAT software. When invalid samples are excluded from the concordance estimation, total insertions and deletions are 41 each.

### c. DNA Input

The recommended DNA input range of the oncoReveal™ CDx is between 30 ng to 80 ng. A DNA input study testing DNA inputs ranging from 5 ng to 160 ng was previously conducted with CRC and NSCLC tumor samples positive for CDx variants which included SNVs and insertions to assess the robustness of the oncoReveal™ CDx to variations in DNA input, refer to Section IX.A.2.d. of the P200011 SSED.

To assess the robustness of detection insertion variants in the recommend DNA input, five clinical samples representing 3 tumor types, NSCLC, CRC and melanoma were tested at DNA input outside the recommended DNA input range (13 to 318 ng). Concordance at each DNA input level evaluated was determined

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against an evNGS comparator. Insertions 1 – 9 bases in length in four genes, EGFR, ERBB2, TP53 and PTEN at 2.5 – 77% VAFs were evaluated. All insertion were detected at the DNA inputs evaluated by both oncoReveal CDx and the evNGS assay, except for a PTEN insertion evaluated at 5.1% VAF and 21 ng DNA input level, which was not detected by the evNGS comparator. The discordance may be explained by the variant being below the LoD of the evNGS comparator.

In addition, a supplemental evaluation was performed to confirm performance at 30 ng DNA input extracted from FFPE of eight additional tumor types (other than CRC and NSCLC). The tissues include melanoma, bladder, breast, endometrial, liver, melanoma, pancreatic, cancer, and thyroid cancers. Eighty-four (84) libraries were prepared with 100% valid results that included a total of 106 positive mutation calls confirmed using evNGS comparator methods for evaluation. These results confirm that the assay produced accurate results across eight additional tumor types using minimum DNA input at 30 ng with PPA 100% and NPA of 99.97%.

### 3. Analytical Specificity

#### a. Interfering Substances

Potential impact of interfering substances on the performance of the oncoReveal™ CDx were evaluated in three tumor types (from melanoma, breast cancer and thyroid cancer). One hundred eighty-one (181) libraries were analyzed with seven exogenous substances and 142 libraries were analyzed with three endogenous substances (refer to Table 21 for the potential interfering substances evaluated and levels tested).

Table 21. Potential Interfering Substances Tested

|  Exogenous/Endogenous | Int Substance | Min/Max | Value  |
| --- | --- | --- | --- |
|  Exogenous | Xylene | Min | 0.000002% (v/v)  |
|  Exogenous | Xylene | Max | 0.000267% (v/v)  |
|  Exogenous | Qiagen Proteinase K | Min | 0.000004 mg/mL  |
|  Exogenous | Qiagen Proteinase K | Max | 0.000043 mg/mL  |
|  Exogenous | Buffer ATL | Min | 0.0002% (v/v)  |
|  Exogenous | Buffer ATL | Max | 0.0019% (v/v)  |
|  Exogenous | Buffer AL | Min | 0.0002% (v/v)  |
|  Exogenous | Buffer AL | Max | 0.0021% (v/v)  |
|  Exogenous | Qiagen AW1 | Min | 0.06% (v/v)  |
|  Exogenous | Qiagen AW1 | Max | 0.33% (v/v)  |
|  Exogenous | Qiagen AW2 | Min | 5.7% (v/v)  |
|  Exogenous | Qiagen AW2 | Max | 16.7% (v/v)  |
|  Exogenous | Ethanol | Min | 4.0% (v/v)  |
|  Exogenous | Ethanol | Max | 11.9% (v/v)  |
|  Exogenous | Control | Control | N/A  |
|  Endogenous | Hemoglobin | Min | 2% (g/g)  |

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|  Exogenous/Endogenous | Int Substance | Min/Max | Value  |
| --- | --- | --- | --- |
|  Endogenous | Hemoglobin | Max | 4% (g/g)  |
|  Endogenous | Melanin | Min | 0.009% (g/g)  |
|  Endogenous | Melanin | Max | 4% (g/g)  |
|  Endogenous | Triglycerides | Min | 2% (g/g)  |
|  Endogenous | Triglycerides | Max | 12% (g/g)  |
|  Endogenous | Control | Control | N/A  |

Ten (10) clinical samples representing 11 variants (4 CDx, 7 tumor profiling variants) were evaluated in the study. Agreement analysis of valid samples against no substance controls showed PPA and NPA at 100% for all substances and levels tested. No impact on the performance of the oncoReveal CDx was observed for each substance and at each level tested.

The impact of necrosis on the performance of oncoReveal CDx was also evaluated by assessing the valid rate of the samples processed in the accuracy study. Of the 312 samples with necrotic tissue content (0 - 60%) available, 284 samples (with 9 insertion variants) passed oncoReveal and comparator QC metrics and were included in the concordance analysis. For samples with 0-10% necrosis, the concordance was 92%, For samples with 10-20% necrosis, the concordance was 83%. For this group, there were 4 discordant. One sample had low VAF levels near LoD and one had low quality by the comparator, not the oncoReveal CDx. The reason for discordance of two out of the 4 samples are likely not due to necrosis but the reason is unknown. For samples with 20-30%, 30-40%, 40-50% and 50-60% necrosis, the concordances were 100%, respectively. Overall concordance was about 93%.

### 4. Precision and Reproducibility

#### a. Three (3)-site reproducibility study

A multi-site reproducibility study was performed to support oncoReveal™ CDx performance to detect tumor profiling mutations from different cancer indications. The reproducibility of the oncoReveal™ CDx was evaluated using 10 clinical samples with target tumor profiling variants adjusted to %VAF in the range of 1-1.5x LoD. The sample panel included FFPE tissues from six tumor types, including: bladder, colorectal, melanoma, NSCLC, pancreatic, and uterine/endometrial cancers and is summarized in Table 22. The study was conducted at three sites performing 3 runs on non-consecutive days. One sequencing instrument and one reagent lot were used at each site. Each sample was tested with up to 4 replicates in each run for a total of up to 36 possible results (3 sites by 3 runs by 4 replicates). The study produced a total of 348 test results.

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Table 22. Multi-site Reproducibility Study 31 Variants (12 genes) 10 Clinical Samples.

|  Sample | Gene | Exon | Nucleotide Change | Amino Acid Change | Variant type | Variant level*** | Ratio Mean VAF/LoD | Mean VAF | Median | SD | CV %  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  1 | BRAF | 15 | 1799T>A | V600E | SNV | T-2 | 6.76 | 12.84 | 12.88 | 0.53 | 4.1  |
|  1 | FBXW7 | 10 | 1436G>A | R479Q | SNV | T-3 | 1.97 | 8.85 | 8.76 | 0.67 | 7.6  |
|  1 | PIK3CA | 10 | 1634A>G | E545G | SNV | T-3 | 2.82 | 12.71 | 12.72 | 0.49 | 3.8  |
|  1 | PTEN | 1 | 17_18del | K6RfsTer4 | Del | T-3 | 2.11 | 9.50 | 9.44 | 0.63 | 6.6  |
|  1** | PTEN | 7 | 710dup | F238VfsTer5 | Ins | T-3 | **0.93** | 4.18 | 4.12 | 0.35 | 8.4  |
|  1 | PTEN | 7 | 800del | K267RfsTer9 | Del | T-3 | 1.15 | 5.16 | 5.11 | 0.58 | 11.2  |
|  1 | PTEN | 8 | 968del | N323MfsTer21 | Del | T-3 | 1.54 | 6.95 | 6.91 | 0.38 | 5.5  |
|  1 | TP53 | 7 | 714dup | N239Ter | Ins | T-3 | 1.39 | 6.26 | 6.25 | 0.54 | 8.7  |
|  2 | FBXW7 | 9 | 1417dup | R473KfsTer4 | Ins | T-3 | 1.52 | 6.84 | 6.78 | 0.68 | 9.9  |
|  2 | NRAS | 3 | 182A>G | Q61R | SNV | T-3 | 1.08 | 4.88 | 4.83 | 0.60 | 12.3  |
|  2 | TP53 | 5 | 455del | P152RfsTer18 | Del | T-3 | 2.23 | 10.02 | 10.19 | 1.42 | 14.1  |
|  3 | EGFR | 20 | 2300_2308dup | A767_V769dup | Ins | T-2 | 1.57 | 2.98 | 3.03 | 0.69 | 23.0  |
|  4** | ERBB2 | 20 | 2321_2326dup | A775_G776insVA | Ins | T-3 | **0.94** | 4.23 | 4.24 | 0.52 | 12.4  |
|  5 | BRAF | 15 | 1798_1799delinsAG | V600R | MN V | T-2 | 1.76 | 7.93 | 7.93 | 0.50 | 6.3  |
|  6 | FGFR3 | 9 | 1118A>G | Y373C | SNV | T-2 | 1.08 | 4.86 | 4.87 | 0.82 | 16.8  |
|  7 | FGFR3 | 9 | 1118A>G | Y373C | SNV | T-2 | 1.44 | 6.49 | 6.50 | 0.88 | 13.5  |
|  8 | EGFR | 20 | 2303_2311dup | S768_D770dup | Ins | T-3 | 2.80 | 5.32 | 5.39 | 0.53 | 10.  |
|  8 | FGFR2 | 12 | 1647T>G | N549K | SNV | T-3 | 1.17 | 5.27 | 5.26 | 0.58 | 11.  |
|  8 | PIK3CA | 10 | 1637A>G | N546R | SNV | T-3 | 1.02 | 4.60 | 4.61 | 0.32 | 6.9  |
|  8 | PTEN | 5 | 313del | C105VfsTer8 | Del | T-3 | 1.03 | 4.64 | 4.58 | 0.41 | 8.8  |
|  8 | PTEN | 8 | 968del | N323MfsTer21 | Del | T-3 | 1.08 | 4.85 | 4.87 | 0.33 | 6.8  |
|  9 | EGFR | 20 | 2303_2311dup | S768_D770dup | Ins | T-3 | 1.83 | 3.47 | 3.58 | 0.49 | 14.2  |
|  9* | PIK3CA | 10 | 1637A>G | Q546R | SNV | T-3 | **0.76** | 3.19 | 3.17 | 0.27 | 8.0  |
|  9* | PTEN | 5 | 313del | C105VfsTer8 | Del | T-3 | **0.72** | 3.24 | 3.18 | 0.29 | 9.0  |
|  9* | PTEN | 8 | 968del | N323MfsTer21 | Del | T-3 | **0.81** | 3.63 | 3.60 | 0.36 | 10.0  |
|  9* | FGFR2 | 12 | 1647T>G | N549Lys | SNV | T-3 | **0.82** | 3.64 | 3.60 | 0.31 | 8.5  |
|  10 | KRAS | 2 | 34G>T | G12C | SNV | T-1 | 1.96 | 3.72 | 3.68 | 0.42 | 11.3  |
|  10 | KRAS | 2 | 35G>A | G12D | SNV | T-1 | 6.57 | 17.07 | 16.91 | 1.01 | 5.9  |
|  10 | ERBB2 | 20 | 2321_2326dup | A775_G776insVA | Ins | T-3 | 7.46 | 33.59 | 34.48 | 3.27 | 9.7  |
|  10 | SMAD4 | 6 | 778dup | Y260LfsTer4 | Ins | T-3 | 2.54 | 11.42 | 11.38 | 0.74 | 6.5  |
|  10 | TP53 | 5 | 378dup | S127LfsTer22 | Ins | T-3 | 1.34 | 6.03 | 5.99 | 0.72 | 11.9  |

*Mean observed VAF falls in 0.7 – 0.8x LoD were analyzed with inclusion and exclusion.

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** Mean observed VAF is > 0.9x LoD and is included for agreement analysis.

*** Variant Level refer to tumor profiling levels 1 through 3

Ins=Insertion, Del=Deletion

C= colorectal, P= Pancreas, L= non-small cell lung cancer, M= Melanoma, BL= Bladder, U= Uterine/Endometrial

### Site to Site Reproducibility:

Site to site reproducibility was assessed via positive and negative call rate for each test site (Table 23). The concordance analysis was performed with and without variants with allele frequencies 0.7-0.9x below the LoD of the device. The overall positive agreement across all sites was 96.0% (1044/1088; 94.6-97.0% CI) when assessed using all 31 variants detected in the sample panel and 100% (944/944; 99.6-100.0% CI) when assessed excluding four variants below the LoD (0.7 – 0.9x LoD) of the device. Negative call rate agreement was 100% when assessed both with (1263916/1263936; 99.998-100.0% CI) and without (1263916/1263936; 99.998-100.0% CI) variants below LoD.

**Table 23. Multi-site Agreement by Site.**

|  SITE | # samples | # libraries | # variants | Positive Call Rate (n/N) (2-sided 95% CI) | Negative Call Rate (n/N) (2-sided 95% CI)  |
| --- | --- | --- | --- | --- | --- |
|  ALL | 10 | 348 | 31 | 96.0% (1044/1088) (94.6%, 97.0%) | 100.0% (1263916/1263936) (99.998%, 100.0%)  |
|  Site 1 | 10 | 120 | 31 | 96.0% (357/372) (93.5%, 97.5%) | 100.000% (435840/435840) (99.999%, 100.0%)  |
|  Site 2 | 10 | 108 | 31 | 95.6% (329/344) (92.9%, 97.3%) | 99.995% (392236/392256) (99.992%, 100.0%)  |
|  Site 3 | 10 | 120 | 31 | 96.2% (358/372) (93.8%, 97.7%) | 100.0% (435840/435840) (100.0%, 100.0%)  |
|  **Excluded 4 variants with allele frequencies between 0.7-0.9x LoD**  |   |   |   |   |   |
|  ALL | 10 | 348 | 27 | 100.0% (944/944) (99.6%, 100.0%) | 100.0% (1263916/1263936) (99.998%, 100.0%)  |
|  Site 1 | 10 | 120 | 27 | 100.0% (324/324) (98.8%, 100.0%) | 100.000% (435840/435840) (99.999%, 100.0%)  |
|  Site 2 | 10 | 108 | 27 | 100.0% (296/296) (98.7%, 100.0%) | 99.995% (392236/392256) (99.992%, 100.0%)  |
|  Site 3 | 10 | 120 | 27 | 100.0% (324/324) (98.8%, 100.0%) | 100.0% (435840/435840) (100.0%, 100.0%)  |

### Agreements Per Variant Type:

The positive call rates for SNV, MNV, insertions, and deletions, stratified by variant allele frequency (VAF) relative to the LoD of the device, are summarized in Table 24. Overall positive call rate for variants above LoD (1x - >5x) was 100% for all variant types, including variants 1x-2x above the LoD (592/592; 99.4-100.0% CI). Variants detected by the oncoReveal CDx below the LoD of the device had an overall positive call rate of 79.2

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(168/212; 73.3-84.2% CI). Number of variants and VAF range for each stratum is reported.

**Table 24. Multi-site Agreement Analysis by Variant Type.**

|  Mutation type | Mean VAF range | # variants | Positive Call Rate (n/N) (2-sided 95% CI) | Mean VAF range  |
| --- | --- | --- | --- | --- |
|  All | 0.7 – 1x LoD | 6 | 79.2% (168/212) (73.3%, 84.2%) | 3.2 – 4.2  |
|   |  1 – 2x LoD | 17 | 100.0% (592/592) (99.4%, 100.0%) | 3.0 – 8.9  |
|   |  2 – 5x LoD | 5 | 100.0% (176/176) (97.9%, 100.0%) | 5.3 – 12.7  |
|   |  >5x LoD | 3 | 100.0% (108/108) (96.6%, 100.0%) | 12.8 – 33.6  |
|  SNV | 0.7 – 1x LoD | 2 | 70.8% (51/72) (59.5%, 80.1%) | 3.2 – 3.6  |
|   |  1 – 2x LoD | 7 | 100.0% (240/240) (98.4%, 100.0%) | 3.7 – 8.9  |
|   |  2 – 5x LoD | 1 | 100.0% (36/36) (90.4%, 100.0%) | 12.7 – 12.7  |
|   |  >5x LoD | 2 | 100.0% (72/72) (94.9%, 100.0%) | 12.8 – 17.1  |
|  Insertion | ~1x LoD | 2 | 100.0% (68/68) (94.7%, 100.0%) | 4.2 – 4.2  |
|   |  1 – 2x LoD | 5 | 100.0% (180/180) (97.9%, 100.0%) | 3.0 – 6.8  |
|   |  2 – 5x LoD | 2 | 100.0% (68/68) (94.7%, 100.0%) | 5.3 – 11.4  |
|   |  >5x LoD | 1 | 100.0% (36/36) (90.4%, 100.0%) | 33.6 – 33.6  |
|  Deletion | 0.7 – 1x LoD | 2 | 68.1% (49/72) (56.6%, 77.7%) | 3.2 – 3.6  |
|   |  1 – 2x LoD | 4 | 100.0% (136/136) (97.3%, 100.0%) | 4.6 – 6.9  |
|   |  2 – 5x LoD | 2 | 100.0% (72/72) (94.9%, 100.0%) | 9.5 – 10.0  |
|  MNV | 1 – 2x LoD | 1 | 100.0% (36/36) (90.4%, 100.0%) | 7.9 – 7.9  |

### Analysis of Source of Variance:

Variance due to site, operator, or day of run was assessed using Average Positive Agreement (APA) and Average Negative Agreement (ANA). Results are summarized in Table 25.

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**Table 25. Inter-site, Inter-operator, and Inter-day/Run Analysis of the Multi-site Reproducibility Study.**

|  Pair Name | INTER-SITE |   | INTER-OPERATOR |   | INTER-DAY/RUN  |   |
| --- | --- | --- | --- | --- | --- | --- |
|   |  APA (2-sided 95%CI) | ANA (2-sided 95%CI) | APA (2-sided 95%CI) | ANA (2-sided 95%CI) | APA (2-sided 95%CI) | ANA (2-sided 95%CI)  |
|  ALL | 99.0% (98.5%, 99.4%) | 100.0% (100.0%, 100.0%) | 99.5% (99.1%, 99.7%) | 100.0% (100.0%, 100.0%) | 98.3% (98.0%, 98.6%) | 100.0% (100.0%, 100.0%)  |
|  SNV | 100.0% (98.5%, 100.0%) | 100.0% (100.0%, 100.0%) | 99.1% (98.3%, 99.5%) | 100.0% (100.0%, 100.0%) | 97.8% (97.2%, 98.2%) | 100.0% (100.0%, 100.0%)  |
|  Insertion | 100.0% (98.3%, 100.0%) | 100.0% (100.0%, 100.0%) | 100.0% (99.6%, 100.0%) | 100.0% (100.0%, 100.0%) | 100.0% (99.9%, 100.0%) | 100.0% (100.0%, 100.0%)  |
|  Deletion | 100.0% (97.7%, 100.0%) | 100.0% (100.0%, 100.0%) | 99.3% (98.3%, 99.7%) | 100.0% (100.0%, 100.0%) | 96.7% (95.8%, 97.4%) | 100.0% (100.0%, 100.0%)  |
|  MNV | 100.0% (86.2%, 100.0%) | 100.0% (100.0%, 100.0%) | 100.0% (96.2%, 100.0%) | 100.0% (100.0%, 100.0%) | 100.0% (98.7%, 100.0%) | 100.0% (100.0%, 100.0%)  |

### Agreement Per Sample

Positive and negative call rates were assessed for each of the 10 samples within the sample panel used in the multi-site reproducibility study. Samples contained between 1-8 variants per sample. One sample contained five total variants, four of which were below the LoD of the device. Positive call rate and negative call rate were assessed with and without including the four variants below LoD. Results are summarized in Table 26.

**Table 26. Positive and Negative Call Rates per Sample used in Multi-Site Reproducibility.**

|  Sample | # libraries | # variants | Positive Call Rate (n/N) (2-sided 95% CI) | Negative Call Rate (n/N) (2-sided 95% CI)  |
| --- | --- | --- | --- | --- |
|  1 | 36 | 8 | 100.0% (288/288) (98.7%, 100.0%) | 100.0% (131580/131580) (99.997%, 100.0%)  |
|  2 | 36 | 3 | 100.0% (108/108) (96.6%, 100.0%) | 99.998% (131758/131760) (99.994%, 100.0%)  |
|  3 | 36 | 1 | 100.0% (36/36) (90.4%, 100.0%) | 99.999% (131831/131832) (99.996%, 100.0%)  |
|  4 | 32 | 1 | 100.0% (32/32) (89.3%, 100.0%) | 100.0% (117184/117184) (100.0%, 100.0%)  |
|  5 | 36 | 1 | 100.0% (36/36) (90.4%, 100.0%) | 99.998% (131830/131832) (99.994%, 100.0%)  |

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|  Sample | # libraries | # variants | Positive Call Rate (n/N) (2-sided 95% CI) | Negative Call Rate (n/N) (2-sided 95% CI)  |
| --- | --- | --- | --- | --- |
|  6 | 36 | 1 | 100.0% (36/36) (90.4%, 100.0%) | 99.998% (131829/131832) (99.993%, 100.0%)  |
|  7 | 32 | 1 | 100.0% (32/32) (89.3%, 100.0%) | 99.995% (117178/117184) (99.989%, 100.0%)  |
|  8 | 32 | 5 | 100.0% (160/160) (97.7%, 100.0%) | 99.998% (117054/117056) (99.994%, 100.0%)  |
|  9 | 36 | 5 | 75.6% (136/180) (68.8%, 81.3%) | 99.998% (131685/131688) (99.993%, 100.0%)  |
|   |  36 | 1^{1} | 100.0% (36/36) (90.4%, 100.0%) | 99.998% (131685/131688) (99.993%, 100.0%)  |
|  10 | 36 | 5 | 100.0% (180/180) (97.9%, 100.0%) | 99.999% (131687/131688) (99.996%, 100.0%)  |

$^{1}$ 4 variants at 0.7 – 0.9x LoD excluded.

A summary of the panel-wide precision results was presented in Table 27 below. The precision analysis was performed for the 31 variants (as listed in Table 22). A total 13 SNVs, 8 deletions and 10 insertions were evaluated. The results showed that all mutations have 100% concordance in all replicates except for 4 mutations. These 4 mutations were believed to be discordant because they have below the LoD (0.7 – 0.9x LoD) of the device.

The coefficient of variation (%CV) for the mutation allele frequency was also calculated for all 36 replicates. 20 out 31 samples had %CV ≤10%, 10/31 had between 10 and 14% and one sample had 23%. All runs passed the quality metrics criteria.

**Table 27. Panel-Wide Precision Summary Results for All Replicates Tested by the 3-sites.**

|  Gene Exon | Mutation (cDNA/Protein Changes) | NC* range | VAF range | VAF mean | VAF median | VAF (SD) | VAF (%CV) | Positive /Total Calls | Positive Call Rate (two-sided 95% CI)  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  *ERBB2 exon20* | 2321_2326dup A775_G776ins VA | 1.03 - 1.37 | 24.16 - 36.8 | 33.59 | 34.48 | 3.27 | 10% | 36/36 | 100.0% (90.4%, 100.0%)  |
|  *BRAF exon15* | 1799T>A V600E | 1.22 - 1.58 | 12.01 - 14.41 | 12.84 | 12.88 | 0.53 | 4% | 36/36 | 100.0% (90.4%, 100.0%)  |
|  *KRAS exon2* | 35G>A G12D | 1.03 - 1.19 | 15.2 - 18.97 | 17.07 | 16.91 | 1.01 | 6% | 36/36 | 100.0% (90.4%, 100.0%)  |
|  *PIK3CA exon10* | 1634A>G E545G | 1.5 - 1.8 | 11.85 - 13.82 | 12.71 | 12.72 | 0.49 | 4% | 36/36 | 100.0% (90.4%, 100.0%)  |
|  *EGFR exon20* | 2303_2311dup S768_D770dup | 0.34 - 0.46 | 4.03 - 6.33 | 5.32 | 5.39 | 0.53 | 10% | 32/32 | 100.0% (89.3%, 100.0%)  |
|  *SMAD4* | 778dup | 1.06 - | 10.01 | 11.42 | 11.38 | 0.74 | 6% | 36/36 | 100.0%  |

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|  Gene Exon | Mutation (cDNA/Protein Changes) | NC* range | VAF range | VAF mean | VAF median | VAF (SD) | VAF (%CV) | Positive /Total Calls | Positive Call Rate (two-sided 95% CI)  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  exon6 | Y260Lfs*4 | 1.34 | - 13.11 |  |  |  |  |  | (90.4%, 100.0%)  |
|  TP53 exon5 | 455del P152Rfs*18 | 0.4 - 0.61 | 7.39 - 12.55 | 10.02 | 10.19 | 1.42 | 14% | 36/36 | 100.0% (90.4%, 100.0%)  |
|  PTEN exon1 | 17_18del K6Rfs*4 | 0.94 - 1.19 | 8.18 - 10.86 | 9.50 | 9.44 | 0.63 | 7% | 36/36 | 100.0% (90.4%, 100.0%)  |
|  FBXW7 exon10 | 1436G>A R479Q | 1.19 - 1.59 | 7.88 - 10.44 | 8.85 | 8.76 | 0.67 | 8% | 36/36 | 100.0% (90.4%, 100.0%)  |
|  KRAS exon2 | 34G>T G12C | 1.03 - 1.19 | 2.54 - 4.5 | 3.72 | 3.68 | 0.42 | 11% | 36/36 | 100.0% (90.4%, 100.0%)  |
|  EGFR exon20 | 2303_2311dup S768_D770dup | 0.36 - 0.45 | 2.5 - 4.45 | 3.47 | 3.58 | 0.49 | 14% | 36/36 | 100.0% (90.4%, 100.0%)  |
|  BRAF exon15 | 1798_1799delinsAG V600R | 1.39 - 1.8 | 6.88 - 8.88 | 7.93 | 7.93 | 0.50 | 6% | 36/36 | 100.0% (90.4%, 100.0%)  |
|  EGFR exon20 | 2300_2308dup A767_V769dup | 0.51 - 0.66 | 1.72 - 4.45 | 2.98 | 3.03 | 0.69 | 23% | 36/36 | 100.0% (90.4%, 100.0%)  |
|  PTEN exon8 | 968del N323Mfs*21 | 0.84 - 1.25 | 6.37 - 7.73 | 6.95 | 6.91 | 0.38 | 5% | 36/36 | 100.0% (90.4%, 100.0%)  |
|  FBXW7 exon9 | 1417dup R473Kfs*4 | 0.89 - 1.02 | 5.17 - 8.11 | 6.84 | 6.78 | 0.68 | 10% | 36/36 | 100.0% (90.4%, 100.0%)  |
|  FGFR3 exon9 | 1118A>G Y373C | 0.19 - 0.28 | 5.03 - 8.72 | 6.49 | 6.50 | 0.88 | 13% | 32/32 | 100.0% (89.3%, 100.0%)  |
|  TP53 exon7 | 714dup N239* | 0.67 - 0.83 | 5.06 - 7.48 | 6.26 | 6.25 | 0.54 | 9% | 36/36 | 100.0% (90.4%, 100.0%)  |
|  TP53 exon5 | 378dup S127Lfs*22 | 0.53 - 0.79 | 4.66 - 7.65 | 6.03 | 5.99 | 0.72 | 12% | 36/36 | 100.0% (90.4%, 100.0%)  |
|  FGFR2 exon12 | 1647T>G N549K | 0.61 - 0.78 | 3.96 - 6.56 | 5.27 | 5.26 | 0.58 | 11% | 32/32 | 100.0% (89.3%, 100.0%)  |
|  PTEN exon7 | 800del K267Rfs*9 | 1.01 - 1.3 | 4.21 - 6.86 | 5.16 | 5.11 | 0.58 | 11% | 36/36 | 100.0% (90.4%, 100.0%)  |
|  NRAS exon3 | 182A>G Q61R | 1.05 - 1.58 | 3.6 - 6.18 | 4.88 | 4.83 | 0.60 | 12% | 36/36 | 100.0% (90.4%, 100.0%)  |

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|  Gene Exon | Mutation (cDNA/Protein Changes) | NC* range | VAF range | VAF mean | VAF median | VAF (SD) | VAF (%CV) | Positive /Total Calls | Positive Call Rate (two-sided 95% CI)  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  FGFR3 exon9 | 1118A>G Y373C | 0.13 - 0.19 | 3.55 - 7.16 | 4.82 | 4.82 | 0.82 | 17% | 36/36 | 100.0% (90.4%, 100.0%)  |
|  PTEN exon8 | 968del N323Mfs*21 | 0.83 - 1.25 | 4.21 - 5.41 | 4.85 | 4.87 | 0.33 | 7% | 32/32 | 100.0% (89.3%, 100.0%)  |
|  PTEN exon5 | 313del C105Vfs*8 | 1.21 - 1.45 | 3.79 - 5.77 | 4.64 | 4.58 | 0.41 | 9% | 32/32 | 100.0% (89.3%, 100.0%)  |
|  PIK3CA exon10 | 1637A>G Q546R | 1.5 - 1.73 | 4.09 - 5.19 | 4.60 | 4.61 | 0.32 | 7% | 32/32 | 100.0% (89.3%, 100.0%)  |
|  ERBB2 exon20 | 2321_2326dup A775_G776ins VA | 0.55 - 0.72 | 3.38 - 5.26 | 4.23 | 4.24 | 0.52 | 12% | 32/32 | 100.0% (89.3%, 100.0%)  |
|  PTEN exon7 | 710dup F238Vfs*5 | 1.01 - 1.3 | 3.64 - 5.34 | 4.18 | 4.12 | 0.35 | 8% | 36/36 | 100.0% (90.4%, 100.0%)  |
|  FGFR2 exon12 | 1647T>G N549K | 0.6 - 0.87 | 3.2 - 4.31 | 3.70 | 3.63 | 0.26 | 7% | 33/36 | 91.7% (78.2%, 97.1%)  |
|  PTEN exon8 | 968del N323Mfs*21 | 0.82 - 1.18 | 3…

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**Source:** [https://fda-staging.innolitics.com/device/P200011S001](https://fda-staging.innolitics.com/device/P200011S001)

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