PGDx elio tissue complete

K192063 · Personal Genome Diagnostics · PZM · Apr 24, 2020 · Pathology

Device Facts

• Record ID: K192063
• Device Name: PGDx elio tissue complete
• Applicant: Personal Genome Diagnostics
• Product Code: PZM · Pathology
• Decision Date: Apr 24, 2020
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.6080
• Device Class: Class 2
• Attributes: AI/ML, Software as a Medical Device, Real-World Evidence

Real-World Evidence

Submission	Device	Sponsor	RWD Sources	RWE Use Summary	Key Tags
K192063 · Apr 24, 2020	PGDx elio tissue complete	Personal Genome Diagnostics	Retrospective clinical FFPE tumor tissue samples	Retrospective clinical FFPE samples were used to evaluate the assay's invalid rates across >40 tumor types, establish analytical sensitivity (LoD), and assess accuracy through method comparison against orthogonal methods.	FFPE; Retrospective; Analytical Performance; Accuracy; Tumor Profiling

Clinical Evidence

Study Design	Population	Comparator	Key Endpoints
Acceptability Rates Study; Retrospective analysis of clinical cases	Clinical cases from >40 tumor types; Sample Size: 4173	Not applicable for this study	Acceptability/Pass rates
Method Comparison (Accuracy); Retrospective method comparison	582 clinical FFPE samples from 35 tumor types; Sample Size: 582	Validated NGS and PCR methods	PPA, NPA, PPV, NPV

AI Performance

Output	Algorithm	Acceptance	Observed	Dev DS	Dev Readers	Test DS	Test Readers
Somatic single nucleotide variants (SNVs)	Automated bioinformatics pipeline for variant calling	—	PPA 97.2% (SNVs with Evidence of Clinical Significance); PPA 86.4% (SNVs with Potential Clinical Significance); PPA 97.1% (Hotspot SNVs); PPA 85.1% (Non-hotspot SNVs)	—	—	Accuracy study: 582 clinical FFPE samples from 35 tumor types.	—
Small insertions and deletions (indels)	Automated bioinformatics pipeline for variant calling	—	PPA 100% (Hotspot indels); PPA 81.4% (Non-hotspot indels); PPA 80.8% (Insertions with Potential Clinical Significance); PPA 82.7% (Deletions with Potential Clinical Significance)	—	—	Accuracy study: 582 clinical FFPE samples from 35 tumor types.	—
ERBB2 amplification	Automated bioinformatics pipeline for fold change estimation	—	PPA 75.0% (All cases); PPA 87.0% (Excluding borderlines)	—	—	Accuracy study: 147 tumor tissues representing 20 tumor types.	—
ALK, RET, NTRK2, and NTRK3 translocations	Automated bioinformatics pipeline for fusion read detection	—	PPA 92.9% (ALK); PPA 1 (NTRK2); PPA 66% (NTRK3); PPA 55.6% (RET)	—	—	Accuracy study: clinical FFPE samples.	—
Microsatellite instability (MSI)	Linear classifier combining frequency of unstable tracts and mutation signatures	—	PPA 98.8% (18 tumor types); PPA 100% (CRC/endometrial); PPA 96.6% (Non-CRC/non-endometrial)	—	—	Accuracy study: 283 samples (CRC, endometrial, and other tumor types).	—
Tumor mutation burden (TMB)	Automated bioinformatics pipeline for mutation load calculation	—	Spearman correlation coefficient of 0.903 compared to matched tumor-normal whole exome sequencing.	—	—	Accuracy study: 118 cases across 8 tumor types.	—

Indications for Use

The PGDx elio™ tissue complete assay is a qualitative in vitro diagnostic device that uses targeted next generation sequencing of DNA isolated from formalin-fixed, paraffin-embedded tumor tissue from patients with solid malignant neoplasms to detect tumor gene alterations in a broad multi-gene panel. PGDx elio tissue complete is intended to provide tumor mutation profiling information on somatic alterations (SNVs, small insertions and deletions, one amplification and four translocations), microsatellite instability (MSI) and tumor mutation burden (TMB) for use by qualified healthcare professionals in accordance with professional guidelines in oncology for previously diagnosed cancer patients, and is not conclusive or prescriptive for labeled use of any specific therapeutic product.

Device Story

PGDx elio tissue complete is a targeted NGS assay for tumor mutation profiling; uses genomic DNA isolated from FFPE tumor tissue; workflow includes mechanical shearing, end-repair, adapter ligation, and hybrid capture enrichment; sequencing performed on Illumina NextSeq 550Dx; automated bioinformatics pipeline identifies SNVs, indels, ERBB2 amplification, ALK/RET/NTRK2/NTRK3 translocations, MSI, and TMB; results provided in case reports for qualified healthcare professionals; used in clinical oncology settings; aids in tumor profiling; does not provide prescriptive therapeutic guidance.

Clinical Evidence

Bench-only validation. Accuracy assessed against orthogonal methods (NGS panels, PCR, FISH, whole exome sequencing) across 582 samples. SNV/indel PPA/NPA >80% and >99% respectively. ERBB2 amplification concordance with FISH PPA 75-87%. ALK translocation PPA 92.9%. TMB Spearman correlation 0.903 vs. whole exome sequencing. MSI performance PPA 98.8% (excluding failed/indeterminate). Reproducibility assessed across 3 sites with 14 samples; APA/ANA >92% for most variants. Analytical sensitivity (LoD) established for SNVs, indels, amplifications, and translocations.

Technological Characteristics

Targeted NGS panel (505 genes); hybrid capture chemistry; FFPE-derived DNA input (50-100ng); Illumina NextSeq 550Dx platform; automated bioinformatics pipeline; proprietary PGDx elio server; software-based variant calling; no patient-matched normal sample required; database-based germline filtering.

Indications for Use

Indicated for previously diagnosed cancer patients with solid malignant neoplasms. Used for qualitative detection of somatic tumor gene alterations (SNVs, small indels, ERBB2 amplification, ALK/RET/NTRK2/NTRK3 translocations), MSI, and TMB from FFPE tumor tissue.

Regulatory Classification

Identification

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

Special Controls

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

Predicate Devices

• MSK-IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets) (DEN170058)

Submission Summary (Full Text)

{0}

Food and Drug Administration

10903 New Hampshire Avenue

Silver Spring, MD 20993-0002

www.fda.gov

# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION
DECISION SUMMARY

# I Background Information:

# A 510(k) Number

K192063

# B Applicant

Personal Genome Diagnostics

# C Proprietary and Established Names

PGDx elio™ tissue complete

# D Regulatory Information

| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| PZM | Class II | 21 CFR 866.6080 - Next Generation Sequencing Based Tumor Profiling Test | Pathology |

# II Submission/Device Overview:

# A Purpose for Submission:

New device

# B Measurand:

Somatic single nucleotide variants, insertions and deletions, select amplifications and translocations, microsatellite instability (MSI) and tumor mutation burden (TMB) in human genomic DNA obtained from formalin-fixed, paraffin-embedded tumor tissue. Refer to Appendix A for a list of the genes covered by the assay.

# C Type of Test:

Next-generation sequencing tumor profiling test

K192063 - Page 1 of 108

{1}

### III Intended Use/Indications for Use:

#### A Intended Use(s):

The PGDx elio™ tissue complete assay is a qualitative in vitro diagnostic device that uses targeted next generation sequencing of DNA isolated from formalin-fixed, paraffin-embedded tumor tissue from patients with solid malignant neoplasms to detect tumor gene alterations in a broad multi-gene panel.

PGDx elio tissue complete is intended to provide tumor mutation profiling information on somatic alterations (SNVs, small insertions and deletions, one amplification and four translocations), microsatellite instability (MSI) and tumor mutation burden (TMB) for use by qualified healthcare professionals in accordance with professional guidelines in oncology for previously diagnosed cancer patients, and is not conclusive or prescriptive for labeled use of any specific therapeutic product.

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

Same as above

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

Rx - For Prescription Use Only

For in vitro diagnostic use.

#### D Special Instrument Requirements:

Illumina NextSeq® 550Dx (qualified by PGDx)

### IV Device/System Characteristics:

#### A Device Description:

##### 1. Reagents

The PGDx elio tissue complete assay is for use as part of a test system with the NextSeq 550Dx and sequencing reagents. Components of the PGDx elio tissue complete assay are listed in Table 1. PGDx provided components include reagent kits, software for data analysis, and a server. The assay contains reagents for 2 full sequencing runs (i.e., 30 samples plus 2 external controls 2 NTC runs). Materials required but not provided are described in the text below Table 1. A detailed list of required instruments, software, reagents, consumables and storage conditions is described in the product labeling (PGDx elio tissue complete User Manual).

K192063 - Page 2 of 108

{2}

Table 1. Reagent Components of the PGDx elio tissue complete assay

| Storage Temp. (°C) | Component Name | Volume | Cap Label |
| --- | --- | --- | --- |
| Library Preparation Kit, Box 1 of 2 | | | |
| -25 to -15 | ER/AT Buffer | 302 μL | ER/AT Buffer |
| -25 to -15 | ER/AT Enzyme | 147 μL | ER/AT Enzyme |
| -25 to -15 | Ligation Buffer | 1.3 mL | Lig Buffer |
| -25 to -15 | DNA Ligase | 431 μL | DNA Ligase |
| -25 to -15 | Hot Start PCR Mix (2x) | 1.0 mL | PCR Mix |
| -25 to -15 | Primer Mix (10x) | 204 μL | Primer Mix |
| -25 to -15 | Nuclease-Free Water | 4.9 mL | None |
| -25 to -15 | MB_Reagents (Adapters; multiple) | 20 μL each | MB |
| Library Preparation Kit, Box 2 of 2 (A0220300) | | | |
| 2 to 8 | Pre-PCR Beads | 11.8 mL | None |
| Capture Kit, Box 1 of 4 (A0220400) | | | |
| -25 to -15 | Hyb Blocker 1 | 47 μL | Hyb Blocker 1 |
| -25 to -15 | 100 μM Primer 1 | 27 μL | 100 μM Primer 1 |
| -25 to -15 | 100 μM Primer 2 | 27 μL | 100 μM Primer 2 |
| -25 to -15 | Hyb Blocker 2 | 208 μL | Hyb Blocker 2 |
| -25 to -15 | RNase Block | 23 μL | RNase Block |
| -25 to -15 | Hybridization Buffer | 248 μL | Hyb Buffer |
| -25 to -15 | DNA Pol Buffer | 431 μL | PCR Buffer |
| -25 to -15 | DNA Pol Enzyme | 46 μL | PCR Enzyme |
| -25 to -15 | dNTP Mix | 23 μL | dNTP Mix |
| -25 to -15 | Nuclease-Free Water | 4.9 mL | None |
| Capture Kit, Box 2 of 4 (A0220500) | | | |
| 15 to 30 | Binding Buffer (0220501) | 37.3 mL | None |
| 15 to 30 | Wash Buffer 1 | 8.8 mL | None |
| 15 to 30 | Wash Buffer 2 | 56.4 mL | None |
| Capture Kit, Box 3 of 4 (A0220600) | | | |
| 2 to 8 | Post-PCR Beads | 6.8 mL | None |
| 2 to 8 | Capture Beads | 2.4 mL | Capture Beads |
| Capture Kit, Box 4 of 4 (A0220700) | | | |
| -85 to -65 | Capture Baits | 71 μL | Capture Baits |
| External Control (A0220900) | | | |
| 2 to 8 | External Control | 5 μL | Ext Control |

K192063 - Page 3 of 108

{3}

## 2. Materials Required but Not Provided

For a detailed list of required, but not provided reagents and consumables refer to the product labeling (PGDx elio tissue complete User Manual).

- DNA extraction Kits for FFPE Tissue
- DNA Fragment analyzer reagents
- Sequencing Reagent Kit: The PGDx elio tissue complete is validated for use with the NextSeq 550Dx High Output Reagent Kits (300 Cycle). If using additional NextSeq 550 reagents, PGDx elio IVD assay requires that only PGDx qualified lots of NextSeq 550 reagents be used with the device. A list of NextSeq reagent lots that have been qualified by PGDx for use with PGDx elio IVD assays is available on the PGDx elio Portal. Reagents must only be used with the instructions for use contained in the package insert. The PGDx software is designed to prevent the use of unqualified lots with the software.

## 3. PGDx elio Server and Software

The proprietary PGDx elioTM server contains analysis and reporting software necessary for the PGDx elio tissue complete assay (software versions are displayed within the PGDx elio platform user interface and on reports). The software is compatible with NextSeq® 550Dx instruments. A list of compatible versions of NextSeq software is available through the PGDx elio Portal. The PGDx elio server saves reports only and does not provide storage or backup of raw sequencing data. PGDx

## 4. Instrument

The PGDx elio tissue complete is validated for use on the NextSeq 550Dx instrument as part of a test system. NextSeq 550Dx instruments must be qualified by a PGDx representative before use with the PGDx elio platform software. Qualification establishes the instrument as IVD for use with the PDx elio tissue complete assay only. Qualification is performed upon server installation and prior to use. The PGDx elioTM diversiPhi is used to qualify and maintain the instrument.

Other required equipment and the specifications for the specific equipment for use with the PGDx elio tissue complete assay are described in Table 2.

Table 1. Other Required Equipment, Not Provided

| Equipment | Notes |
| --- | --- |
| DNA shearing instrument | Mechanically shears DNA to the appropriate size. |
| DNA fragment analyzer | Automated sample processing determines size, quantity and purity for quick library QC. |
| Fluorometer | Uses detection of target-specific fluorescence to provide quantification of samples prior to library preparation and sequencing. Separate fluorometers are required in pre-PCR and post-PCR areas. |

K192063 - Page 4 of 108

{4}

| Magnetic stand | Designed for paramagnetic bead precipitation from standard and deep 96-well microplates. Separate magnetic stands are required in pre-PCR and post-PCR areas. |
| --- | --- |
| Mini-centrifuge or micro-centrifuge | Tabletop micro-centrifuge or mini-centrifuge capable of holding 0.5 mL to 2.0 mL tubes. Separate micro- or mini-centrifuges are required in pre-PCR and post-PCR areas. |
| Thermal cycler | One 96-well dual-block thermal cycler (or two 96-well single block thermal cyclers) is required in the post-PCR areas. |
| Tabletop 96-well plate centrifuge | Any plate centrifuge capable of maintaining 280 x g for at least 1 minute is sufficient. Separate plate centrifuges are required for pre-PCR and post-PCR areas. |
| Thermomixer | Thermomixer capable of temperatures ranging from 20 °C to 70 °C and shaking at 1700 rpm. Two thermomixers or two thermal cyclers (or one thermal cycler with multiple thermal blocks) are required in the pre-PCR area and one thermomixer is required in the post-PCR area. |
| Tabletop vortex mixer | Separate vortex mixers are required in pre-PCR and post-PCR areas. |
| Single-channel pipettors (P-2, P-10, P-20, P-200, P-1000) | Separate sets of pipettors are required in pre-PCR and post-PCR areas. Pipettors should be calibrated regularly and verified accurate within 5% of stated volume. |
| Multi-channel pipettor (P-20, P-200) | Separate sets of pipettors are required in pre-PCR and post-PCR areas. Pipettors should be calibrated regularly and verified accurate within 5% of stated volume. |

### 5. Sample Preparation:

The PGDx elio tissue complete assay requires genomic DNA isolated from FFPE tissue specimens. The tumor volume and minimum tumor content needed to obtain sufficient DNA for testing to achieve stated performance are shown in Table 3. If less than 100% of the tissue section contains ≥20% tumor purity, the tissue should be macro-dissected to select as much viable tumor as possible and minimize the amount of adjacent non-tumor tissue.

Table 3. Specimen Handling and Processing for Validated Specimen Types

| Tissue Type | Volume | Minimum Tumor Proportion | Macrodissection Requirements (based on tumor proportion) | Limitations | Storage |
| --- | --- | --- | --- | --- | --- |
| FFPE sections | The assay may require up to 10 slides at a minimum 5 microns thick | ≥ 20% of viable nuclei in the selected tumor area should consist of tumor cell nuclei | Samples less than 100% tumor nuclei should be macrodissected | Archival FFPE material >14.5 years post-resection is not suitable for analysis | Room temperature |

K192063 - Page 5 of 108

{5}

## 6. DNA Extraction:

PGDx elio tissue complete assay requires genomic DNA isolated from FFPE tissue using an appropriate commercially available DNA extraction method. DNA extraction kits should be able to yield 50 ng of DNA with a minimum concentration of 1 ng/μL. The recommended DNA input for PGDx elio tissue complete is 100 ng of total DNA recovered from tissue with a minimum 20% viable tumor nuclei. While recommended DNA input for the assay is 100ng, results can be obtained with DNA inputs down to 50 ng. The assay has been validated with extracted DNA stored at ≤ -20 °C for up to 9 months.

## 7. Library Preparation:

The PGDx elio tissue complete assay workflow begins with genomic DNA. Genomic DNA is quantified using a fluorometer. DNA molecules are mechanically sheared to a target size of 200 bp and subjected to a magnetic bead purification step to remove smaller fragments and perform an exchange of buffer. Fragmented DNA is end-repaired, phosphorylated, and adenylated. Indexed adapters are then ligated to the A-tailed DNA molecules.

Unincorporated adapters and reagents are removed by magnetic bead purification. Adapter-ligated DNA is enriched by PCR amplification. Primer dimers and residual reagents are removed by magnetic bead purification. Library quality is assessed using a DNA fragment analyzer prior to hybrid capture. Sample libraries must be ≥ 15 ng/μL within the 180-800 bp range with the average peak size ≥ 250 bp in length, and external control (EC) for the batch must be ≥ 15 ng/μL within the 180-800 bp range with the average peak size ≥ 250 bp in length, prior to proceeding to hybridization / target enrichment.

## 8. Hybrid Capture NGS:

The adapter-ligated library is hybridized with biotinylated RNA library baits and targeted regions are captured using magnetic streptavidin coated beads. Captured DNA libraries are purified to remove baits and incompletely hybridized DNA fragments. Captured libraries are enriched by PCR amplification. Primer dimers and residual reagents are removed by magnetic bead purification. Final library quality is assessed using a DNA fragment analyzer prior to sequencing. Samples and external control must be ≥ 10 nM within the 180-800 bp range with the average size ≥ 250 bp in length. If the level of primer/adapter dimers in sample library lanes (100-180 bp region) is > 5% of the library yield, the library has failed QC and steps starting from library preparation must be repeated.

## 9. Sequencing:

Sample libraries are quantified and normalized into a sequencing pool of up to 15 samples and an external control. Partial batches are supported using a filler of diverse material, such as PGDx elio diversiPhi, or previously captured libraries. Pooled sample libraries are fluorometrically quantified, loaded on a sequencing flow cell, and sequenced.

## 10. Data Analysis:

a) Data Management System (DMS): Sequence data is automatically processed using the PGDx elio platform software that tracks sample names, sample metadata and

K192063 - Page 6 of 108

{6}

processing status from sequencing through to analysis and reporting. Reports of identified alterations are available in a web-based user interface for download. Sequencing and sample metrics are available in run and case reports, including sample and sequencing quality.

b) Demultiplexing and FASTQ Generation: Demultiplexing software generates FASTQ files containing sequence reads and quality scores for each of the samples on a sequencing run. The FASTQ formatted data files are used for subsequent processing of samples.

c) Indexing QC check: Samples are checked for an expected yield of sequence reads identified to detect mistakes in pooling samples. Samples outside the expected range are marked as failed.

d) Read Alignment and BAM Generation: Genome alignment is performed to map sequence reads for each sample to the human reference genome (hg19/GRCh37). Alignments are saved as Binary Alignment Map (BAM) formatted files, which contain read placement information relative to the reference genome with quality scores. Aligned BAM files are further processed in a pipeline to identify genomic alterations.

e) Sample QC checks: Samples are checked for possible contamination through a bioinformatic analysis of genome haplotypes, based on an analysis of pre-defined SNP sites that are characteristic of populations and individuals. Samples containing more than one haplotype are considered potentially contaminated and are marked as failed. Sequence coverage is assessed across the panel requiring 90% of targeted regions with a minimum >100x coverage.

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

i. SNVs and Indels: Candidate mutations are evaluated and filtered for characteristics of high confidence somatic variants, including mutant allele frequency, sequence coverage and quality, genomic context, functional annotation, germline status, and prevalence in a database of normal controls. A minimum of 4 or 6 mutant observations and 0.4%, 2%, or 5% mutant allele fraction (MAF) are required depending on sequence coverage and status of the variant as a Variant with Evidence of Clinical Significance, somatic hotspot, or a Variant with Potential Clinical Significance. SNVs with lower bound 95% Confidence Interval <5% MAF based on sequence coverage are excluded from reporting. Common germline mutations present in dbSNP, ExAC, and gnomAD are identified and excluded from reporting. Additional germline mutations with ≥ 3 matches in ExAC and MAF ≥ 20% are also excluded from reporting.

ii. Amplifications: The assay is validated to detect ERBB2 amplification. The amplification is identified based on comparing normalized sequence coverage against a collection of normal controls run by PGDx elio tissue complete. A fold change from diploid is estimated from the observed change in coverage combined with an in-silico prediction of tumor purity. ERBB2 gene amplifications are

K192063 - Page 7 of 108

{7}

reported when predicted fold >2.5x are observed in >25% of evaluated regions of interest for the gene. The test is validated for reporting only amplifications in the ERBB2 gene.

iii. Translocations: The assay is validated to report 4 translocations only, ALK, RET and NTRK2, NTRK3. Translocations are identified based on observations of reads supporting gene fusions in genomic alignments of discordantly mapped or split read pairs.

iv. Microsatellite instability: Microsatellite instability is assessed from select mononucleotide tracts and signatures of genomic context from sequence mutations. A linear classifier determines an overall case status of microsatellite instability-high (MSI-H), microsatellite stable (MSS), or indeterminate by combining the frequency of unstable tracts and signatures of observed mutations.

v. Tumor Mutation Burden (TMB): TMB is calculated based on detected sequence mutations and indels. Filtering of sequence mutations is performed to exclude low mutant allele fraction mutations (<5% MAF), common somatic driver mutations, and common germline mutations. Both synonymous and non-synonymous alterations are considered for the mutation load. TMB is reported as the number of mutations per megabase (Muts/Mb)

## 11. Controls:

a) Negative Control: A no template control (NTC) can be processed to serve as a negative control to validate the acceptability of all the test samples processed through library preparation and capture steps by testing for sample or reagent contamination. The NTC is not included on the sequencing run.
b) Positive Control: An external control that is provided in the PGDx elio tissue complete assay reagent kit consists of cell line derived-DNA with multiple verified sequence mutations. The external control is processed from library preparation through sequencing to serve as an end to end control to demonstrate assay performance. The external control is checked for quality during library preparation and after sequencing. Failure of the external control to meet the pre-defined quality metrics will result in all test samples on the run being reported as “No result.”

## 12. Result Reporting:

PGDx elio tissue complete reports SNVs and indels in protein coding regions across all genes in the panel. In addition, amplifications are reported for ERBB2 as well as translocations for ALK, RET, NTRK2, and NTRK3. Germline mutations, including common polymorphisms in the population, present in dbSNP v150, ExAC v0.3.1, and gnomAD v2.0.2, are filtered and excluded from reports. SNVs and indels that are not Variants with Evidence of Clinical Significance or hotspots will also be removed from reporting if they have ≥ 3 ExAC hits and have a MAF ≥ 20%. The assay also reports on two genomic signatures, MSI and TMB.

K192063 - Page 8 of 108

{8}

Variants are reported in one of two levels of evidence¹: Variants with Evidence of Clinical Significance and Variants with Potential Clinical Significance. Variants reported as having evidence of clinical significance are defined by AMP/ASCO/CAP guidelines (Li et al., 2017), specifically, variants meeting Tier 1A evidence. The variants listed in the section Variants with Evidence of Clinical Significance are determined based on the selected tumor type. Only variants clinically associated with the tested tumor type will appear in the Variants with Evidence of Clinical Significance section. Any remaining detected variants will appear as the Variants with Potential Clinical Significance. Any variants clinically associated with tumor types other than the one selected will be reported in the section labeled 'Variants with Potential Clinical Significance. A list of all 505 genes is provided in Appendix A and a list of excluded exons in the genes or excluded regions due to challenging regions (e.g., low complexity/repeats) is provided in Appendix B and Appendix C, respectively.

Reporting software was designed to mask results that have low confidence allele frequencies levels near the calling threshold. The PGDx elio tissue complete analytical pipeline calculates a 95% CI around the estimated MAF for all sequence mutations. PGDx has applied a reporting filter to mask Level 3 non-hotspot SNV calls that have a lower bound 95% CI <5% MAF. By taking this approach, unreliable results at low MAF are filtered out of reporting, while high confidence calls in this range will still be reported.

Indeterminates: For select genes and regions, quality metrics are assessed to check for low coverage or incomplete data needed to identify an alteration. Indeterminate status is reported when 1) no evidence of the alteration was found, but minimum coverage was not met to support the verified limit of detection, or 2) insufficient evidence of the alteration was observed, but minimum coverage thresholds were not met to report the variant. Supporting evidence of detected alterations and coverage in read data is available in the Complete Case Record (CCR). Indeterminate status is reported when evidence of a sequence mutation is observed in regions of low coverage below < 80x. Indeterminate status is also reported for select genes and codons when low coverage is observed and there is no evidence of an alteration. The minimum coverage threshold range is 116x - 248x in cases where select genes and codons are called negative.

### 13. Quality Metrics

Reporting takes in account the quality metrics outlined in Table 4. Quality metrics are assessed across the following categories:

- Batch-level: Metrics that are quantified per sequencing run; failing batch-level metrics generates “No result” reports samples failing these criteria. If the external control fails these criteria, “No result” is reported for the entire batch of samples.
- Sample-level: Metrics that are quantified per sample; generates “No result” report for a sample failing QC.
- Analyte-level: Metrics that are quantified for individual alteration types and positions, such as sequence coverage. Variants passing analyte-level QC are reported.

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

K192063 - Page 9 of 108

{9}

Table 4. Summary of PGDx elio tissue complete Post-Sequencing Quality Control Metrics

| Quality Metric | Level of Qualification | Passing Criteria |
| --- | --- | --- |
| Cluster Density | Batch-level | Sequencer Cluster Density ≥ 130 |
| Q30 Reads | Batch-level | %Q30 (Read1 and Read4) ≥ 80% %Q30 (Read2 and Read3) ≥ 85% |
| External Control | Batch-level | All expected sequence mutations are detected and passes all other quality criteria |
| Percent Regions Covered | Sample-level | ≥ 90% exons with > 100x Median Distinct Coverage |
| Percent Reads Identified | Sample-level | Percent Reads Identified 15%-35% |
| Contamination QC | Sample-level | Estimated contamination levels < 2% |
| Select SNVs and Indels with Evidence of Clinical Significance | Analyte-level | Mutant reads ≥ 4 MAF ≥ 0.4% |
| Hotspot SNVs and Indels | Analyte-level | Mutant reads ≥ 4 MAF ≥ 2% |
| Non-hotspot SNVs | Analyte-level | Mutant reads ≥ 6 MAF with lower bound 95% CI ≥ 5% |
| Non-hotspot Indels | Analyte-level | Mutant reads ≥ 6 MAF ≥ 5% |
| Homopolymer Indels | Analyte-level | Homopolymer regions < 5 bp or Homopolymer regions ≥ 5 bp with MAF ≥ 12% |
| ERBB2 Amplification | Analyte-level | Fold change ≥ 2.5 in ≥ 25% regions covered |
| Translocations (ALK, NTRK2, NTRK3 and RET) | Analyte-level | Fusion reads ≥ 3 |

### B Principle of Operation:

PGDx elio™ tissue complete is an in vitro diagnostic assay that uses targeted next generation sequencing to detect tumor gene alterations in genomic DNA isolated from formalin- fixed paraffin-embedded (FFPE) tumor tissue in a 505 gene panel. PGDx elio

K192063 - Page 10 of 108

{10}

tissue complete targets cancer-associated genes that are enriched from genomic libraries using a hybrid capture-based chemistry. Genomic libraries are prepared and captured. Samples are pooled for sequencing. After sequencing, automated software executes a bioinformatics analysis pipeline to identify genomic alterations in sequence data. The PGDx elio tissue complete assay workflow does not use a patient-matched normal sample but filters polymorphisms using databases. A summary of the alterations found, including a PDF case report, are reported in output files and provided in a user interface as part of the PGDx elio platform software.

# C Determination of assay thresholds:

# 1. Requirements on exon coverage:

A power analysis was conducted to determine the sequence coverage necessary to detect mutations with true underlying MAFs as low as 2%. Statistical power was estimated based on a requirement of 4 mutant observations to make a positive call. Sequence coverage of >400x provides 95% statistical power for detection of true mutations at 2% MAF (95% CI, 0.8% - 3.5% MAF). For mutations with 5% underlying MAF, sequence coverage of >150x provides 95% statistical power for detection (95% CI, 2.0%-8.6% MAF).

Summary statistics were calculated for individual exons across a cohort of samples to identify exons with consistent below-target coverage. These regions were removed from PGDx elio tissue complete and are not included in variant analysis or reporting. Additional repeat and low complexity regions are also excluded from reporting. The excluded regions are listed in Appendix B and Appendix C. No Variants with Evidence of Clinical Significance or somatic hotspot mutations are masked from reports.

Sequence coverage was evaluated in the remaining regions across a cohort of 175 FFPE samples, and 99.5% of targeted regions (6,991 of 7,026 regions) were sequenced to a depth of 100x or greater with >98% of all regions sequenced to a depth of 250x or greater. Regions with somatic hotspot mutations exhibited sequence coverage >250x. Prediction of tumor mutation burden was maintained in low coverage samples, as demonstrated by low variability across replicates in simulations with coverage loss of up to 10% of exons. Overall, coefficient of variation (CV) estimates are below 25.6%, with an average CV of 11.7%. Excluding TMB scores below limit of blank (LoB), CV estimates are below 20%.

# 2. Requirements on sample coverage:

Sequence coverage was evaluated across a range of FFPE samples (n=175 across 8 different tissue types) to obtain sample summary statistics. Overall sample coverage was high in targeted regions of interest (Figure 1; Figure 1 shows a bar graph demonstrating the mutant frequency relative to the mean read depth) with high percentage of targeted exons covered (Figure 2). The mean coverage across all targeted regions for the FFPE samples was 915x (SD=375).

Sequence coverage was further evaluated to establish minimum criteria for the analysis and reporting of variants. Based on a power analysis, a minimum sequence coverage of 100x is necessary to call mutations with true underlying mutation frequency of 8% or greater. The number of exons for an individual sample meeting this coverage threshold was evaluated to established a per sample threshold. The samples evaluated included a range of DNA quality

K192063 - Page 11 of 108

{11}

estimates based on DNA fragment analysis. Of 175 samples evaluated, >97% of samples (171 of 175 samples) demonstrated ≥ 100x coverage across at least 90% of targeted regions of interest (Figure 3 and Figure 4). The consistently high coverage supports tolerance of occasional low coverage regions that may be seen with varying sample quality. A threshold of 90% of Regions of Interest (ROIs) with at least 100x coverage was selected and is used to determine if a sample is sequenced to sufficient depth for analysis and reporting.


Figure 1. Distribution of mean and median coverage values for targeted regions of PGDx elio tissue complete. Dashed line indicates coverage at 100x.


Figure 2. Evaluation of ROI loss and impact on TMB estimation. For each sample with at least 90% ROIs with ≥ 100x coverage (n=175), loss of 10% of ROIs was simulated (n=10,000 simulations per sample) to evaluate the effect on TMB estimates.

K192063 - Page 12 of 108

{12}


Figure 3. Distribution of mean distinct coverage per sample in PGDx elio tissue complete across 175 FFPE samples.


Figure 4. Distribution of mean coverage values per sample (x-axis) and percentage of ROIs in PGDx elio tissue complete with ≥ 100x coverage per sample (y-axis). Dashed line indicates 90% ROIs with ≥ 100x coverage.

### 3. Requirements on mutation coverage, allele depth and frequency for positive calls:

Variant calling parameters such as sequence coverage, mutation coverage, and mutation frequency were assessed as filters for specificity while maintaining the ability to detect

K192063 - Page 13 of 108

{13}

true positive calls. Thresholds were established to ensure specificity is maintained at targeted MAF levels for reporting. Mutation frequency thresholds were established at 0.4%, 2%, and 5% for sequence mutations based on a categorization of Variants with Evidence of Clinical Significance, somatic hotspots, and non-hotspots positions.

Additional filtering of Variants with Potential Clinical Significance excludes reporting of insertions and deletions in homopolymer regions (5 bp or greater) below 12% MAF as well as sequence mutations with lower bound 95% Confidence Interval <5% MAF based on sequence coverage. Additional quality metrics, such as base quality and strand bias were also incorporated in assessments of confidence for pipeline filters. A cohort of normal FFPE tissue (n=36) was used to provide empirical evidence that specificity was maintained using the pipeline thresholds and filters.

### D Substantial Equivalence Information:

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

MSK-IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets): a Hybridization-Capture Based Next Generation Sequencing Assay

#### 2. Predicate 510(k) Number(s):

DEN170058

#### 3. Comparison with Predicate(s):

| Characteristics | Predicate Device: MSK-IMPACT (DEN170058) | Subject Device: elio Tissue Complete |
| --- | --- | --- |
| Similarities | | |
| Indications for Use | The MSK-IMPACT assay is a qualitative in vitro diagnostic test that uses targeted next generation sequencing of formalin-fixed paraffin-embedded tumor tissue matched with normal specimens from patients with solid malignant neoplasms to detect tumor gene alterations in a broad multi gene panel. The test is intended to provide information on somatic mutations (point mutations and small insertions and deletions) and microsatellite instability for use by qualified health care professionals in accordance with professional guidelines and is not conclusive or prescriptive for labeled use of any specific therapeutic product. MSK-IMPACT is a single-site assay performed at Memorial Sloan Kettering Cancer Center. | The PGDx elioTM tissue complete assay is a qualitative in vitro diagnostic device that uses targeted next generation sequencing of DNA isolated from formalin-fixed, paraffin-embedded tumor tissue from patients with solid malignant neoplasms to detect tumor gene alterations in a broad multi-gene panel.PGDx elio tissue complete is intended to provide tumor mutation profiling information on somatic alterations (SNVs, small insertions and deletions, one amplification and four translocations), microsatellite instability (MSI) and tumor mutation burden (TMB) for use by qualified healthcare professionals in accordance with professional guidelines in oncology for previously diagnosed cancer patients, and is not conclusive or prescriptive for labeled use of any specific therapeutic product. |

K192063 - Page 14 of 108

{14}

| Technology | Hybrid Capture | Same |
| --- | --- | --- |
| Specimen Types | Formalin-fixed, paraffin-embedded (FFPE) tumor tissue matched with normal specimens from patients with solid malignant neoplasms | Formalin-fixed, paraffin-embedded (FFPE) tumor tissue from patients with solid malignant neoplasms |
| Target Population | Patients with solid malignant neoplasms | Same |
| Characteristics | Predicate Device: MSK-IMPACT (DEN170058) | Subject Device: elio Tissue Complete |
| Differences | | |
| Test Environment | Single-site assay (performed at Memorial Sloan Kettering Cancer Center) | Kit |
| Genes on Panel | 468 | 505 |
| Black List | 73 exons | 58 genes/exons excluded from reporting due to consistently low coverage and low complexity and repeat genomic regions in 254 genes |
| Variant types | Intended to provide information on somatic mutations (point mutations and small insertions and deletions), and microsatellite instability | Same except elio Tissue complete includes 1 amplification and 4 fusions and provides information on tumor mutational burden (TMB) |
| Instrument | Illumina HiSeq® 2500 Sequencing System (qualified by MSK) | Illumina NextSeq 550Dx (qualified by PGDx) |
| Determination of Pipeline Thresholds | • Based on >200X target coverage, • 100X for ≥ 98% target exons, • hotspot mutation calling threshold (mutation coverage (DP) ≥ 20, mutant reads (AD) ≥ 8, mutation frequency (VF) ≥ 2%, and non-hotspot mutation threshold (DP ≥ 20, AD ≥ 10, VF ≥ 5%) | Sequence coverage of >400x provides 95% statistical power for detection of true mutations at 2% MAF (95% CI, 0.8% - 3.5% MAF). For mutations with 5% underlying MAF, sequence coverage of >150x provides 95% statistical power for detection (95% CI, 2.0%-8.6% MAF). |
| Assay cut-off | MSK-IMPACT does not report mutations below 2% for known hotspot mutations and 5% for non-hotspot mutations. | A minimum of 4 or 6 mutant observations and 0.4%, 2%, or 5% mutant allele fraction (MAF) are required depending on sequence coverage and status of the variant as a Variant with Evidence of Clinical Significance, somatic hotspot, or a Variant with Potential Clinical Significance. SNVs with lower bound 95% Confidence Interval <5% MAF based on sequence coverage are excluded from reporting. Common germline mutations present in dbSNP, ExAC, and gnomAD are identified and excluded from reporting. Additional germline mutations with ≥ 3 matches in ExAC and MAF ≥ 20% are also excluded from reporting. |

K192063 - Page 15 of 108

{15}

| **Controls** | - Matched normal - Positive control - Negative control No template control (NTC) | - Positive control - No template control (NTC) - Normalized to database of common germline SNPs |
| --- | --- | --- |
| **Clinical Evidence Curation** Oncopanel results are reported under one of these two categories: - “Cancer Mutations with Evidence of Clinical Significance” or - “Cancer Mutations with Potential Clinical Significance.” | Uses OncoKB, knowledge base that includes biologic, clinical and therapeutic information curated from professional guidelines and recommendations, therapeutic labeling, disease specific expert and advocacy group recommendations, and medical literature. Classification criteria were developed by MSK to communicate the level of clinical evidence available for individual mutations in the test report. OncoKB undergoes periodic updates through the review of new information by a panel of experts | Variant calls are organized into Variants with Evidence of Clinical Significance or Variants with Potential Clinical Significance; with Variants with Evidence of Clinical Significance aligning with Tier 1A of the AMP/ASCO/CAP guidelines, based on the selected tumor type for use in tumor profiling. Tumor type selection should align with the clinical diagnosis and all available information. In the case of metastasis of unknown origin, unknown primary site, or uncertainty of the tumor type, ‘Other’ should be selected. |

## E Standards/Guidance Documents Referenced:

The following FDA guidance documents were consulted:

1. 1. Guidance on Informed Consent for In Vitro Diagnostic Device Studies Using Leftover Human Specimens that are Not Individually Identifiable; Guidance for Sponsors, Institutional Review Boards, Clinical Investigators, and Food and Drug Administration Staff (April 25, 2006);
2. 2. eCopy Program for Medical Device Submissions; Guidance for Industry and Food and Drug Administration Staff (December 3, 2015);
3. 3. Refuse to Accept Policy for 510(k)s; Guidance for Industry and Food and Drug Administration Staff (February 21, 2019);
4. 4. Guidance for the Content of Premarket Submissions for Software Contained in Medical Devices; Guidance for Industry and FDA Staff (May 11, 2005);
5. 5. Content of Premarket Submissions for Management of Cybersecurity in Medical Devices; Guidance for Industry and Food and Drug Administration Staff (Draft, October 18, 2018);
6. 6. Medical Device Accessories – Describing Accessories and Classification Pathways; Guidance for Industry and FDA Staff (December 20, 2017);
7. 7. Format for Traditional and Abbreviated 510(k)s – Guidance for Industry and FDA Staff (August 12, 2005);
8. 8. Off-The-Shelf Software Use in Medical Devices; Guidance for Industry, FDA Reviewers, and Compliance (September 9, 1999);
9. 9. Information to Support a Claim of Electromagnetic Compatibility (EMC) of Electrically-Powered Medical Devices; Guidance for Industry and Food and Drug Administration Staff (July 11, 2016); and

K192063 - Page 16 of 108

{16}

10. Benefit-Risk Factors to Consider When Determining Substantial Equivalence in Premarket Notifications (510(k)) with Different Technological Characteristics; Guidance for Industry and Food and Drug Administration Staff (September 25, 2018).

# F Performance Characteristics:

# 1. Analytical Performance- General:

The PGDx elio tissue complete is a targeted NGS panel with 505 genes. The targeted regions of interest in PGDx elio tissue complete are designed to detect single nucleotide variants (SNVs) as well as small insertions and deletions (indels) < 30bp in length in the coding exons of the targeted genes, as well as ERBB2 amplifications, ALK, RET, NTRK2, and NTRK3 translocations, MSI, and TMB. For SNVs and indels, A representative approach to validation of the targeted genes in the panel was submitted with data representing variant types for SNVs and indels, and at the gene level for amplification and translocations indicated with this assay. In addition, the assay was evaluated for performance regarding the panel wide quality metrics.

# a) Invalid Rates

Multiple factors can influence overall robustness and performance of complex molecular tests, including pre-analytical factors and overall sample quality. If key in-process or automated data quality metrics are not met, PGDx elio tissue complete supports repeating samples through the workflow. Performance throughout verification and validation of the device was tracked and a summary of the rates for first pass (no repeat) and overall pass (allowing for a single repeat) are presented below. Data were aggregated for clinical cases from >40 tumor types. Resulting pass rates for all samples (clinical samples and cell lines) are presented in Table 5, while Table 6 shows invalid rate by tumor type across the workflow. The data shows that there the performance across tumor types is supportive of a pan tumor profiling.

Table 5. Acceptability Rates of PGDx elio tissue complete

| All Samples | Acceptability Rate (n/N) (2-sided 95% CI) |
| --- | --- |
| First Pass | 83.4% (3481/4173) (82.3%, 84.5) |
| After Repeat Test | 94.2% (3931/4173) (93.5%, 94.9) |
| Clinical FFPE Samples | Acceptability Rate (n/N) (2-sided 95% CI) |
| First Pass | 81.8% (2352/2874) (80.4%, 83.2) |
| After Repeat Test | 92.9% (2671/2874) (91.9%, 93.8) |

K192063 - Page 17 of 108

{17}

Table 6. Comparability of Tumor Pass Rates for the PGDx elio tissue complete

| Tumor Type | Totals Samples | Total Failures | Total Passes | Failed Tumor Purity | Failed Pre-Library Prep | Failed Post-Library Prep | Pass Rate |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Adenocarcinoma, NOS | 160 | 37 | 123 | 14 | 4 | 19 | 0.77 |
| Bladder Cancer | 154 | 28 | 126 | 13 | 5 | 10 | 0.82 |
| Brain Cancer | 54 | 3 | 51 | - | 2 | 1 | 0.94 |
| Breast Cancer | 77 | 15 | 62 | 2 | 5 | 8 | 0.81 |
| Cholangiocarcinoma | 41 | 5 | 36 | 1 | 3 | 1 | 0.88 |
| Colorectal Cancer (CRC) | 744 | 122 | 622 | 34 | 19 | 69 | 0.84 |
| Esophageal Cancer | 105 | 14 | 91 | 9 | - | 5 | 0.87 |
| Gastric Cancer | 64 | 12 | 52 | 4 | 3 | 5 | 0.81 |
| Gastrointestinal Stromal Tumor (GIST) | 17 | 1 | 16 | - | 1 | - | 0.94 |
| Head And Neck Cancer | 72 | 9 | 63 | 6 | - | 3 | 0.88 |
| Kidney Cancer | 70 | 8 | 62 | 4 | 2 | 2 | 0.89 |
| Liver Cancer | 69 | 11 | 58 | 4 | 2 | 5 | 0.84 |
| Lung Cancer, Non-Small Cell (NSCLC) | 99 | 13 | 86 | 6 | 1 | 6 | 0.87 |
| Lung Cancer, NOS | 1025 | 197 | 828 | 83 | 46 | 68 | 0.81 |
| Lung Cancer, Squamous Cell Carcinoma (SCC) | 94 | 16 | 78 | 2 | 3 | 11 | 0.83 |
| Melanoma | 131 | 20 | 111 | 2 | 4 | 14 | 0.85 |
| Mesothelioma | 11 | - | 11 | - | - | - | 1 |
| Pancreatic Cancer | 107 | 21 | 86 | 10 | 5 | 6 | 0.8 |
| Prostate Cancer | 613 | 169 | 444 | 33 | 37 | 99 | 0.72 |
| Sarcoma, NOS | 26 | 4 | 22 | - | 1 | 3 | 0.85 |
| Small Cell Lung Cancer | 16 | 3 | 13 | 2 | - | 1 | 0.81 |
| Thyroid Cancer | 40 | 13 | 27 | 3 | 3 | 7 | 0.68 |

## 2. Precision/Reproducibility:

### a) Interlaboratory Reproducibility

Interlaboratory reproducibility of the PGDx elio tissue complete assay was assessed across 3 different sites, using DNA extracted from 13 FFPE tissue specimens and 1 cell line. Together these 14 samples represented a range of SNVs, indels, ERBB2 amplifications, ALK, RET, and NTRK3 translocations, MSI, and TMB. Each of the

K192063 - Page 18 of 108

{18}

14 samples were tested in duplicate by 2 different operators on 12 sequencing runs across 3 non- consecutive days at each of the 3 independent laboratory sites using a single kit lot (36 total sequencing runs and 504 total replicates). Allele frequencies for the variants in the specimens spanned all ranges. Each replicate began with the workflow post-DNA extraction. The samples used in the multi-site reproducibility study, along with their expected variants, are presented in Table 7 below.

Table 7. Samples used in the multi-site reproducibility study

| Tissue Type | Expected SNVs with Evidence of Clinical Significance | Number of Variants with Potential Clinical Significance | Translocation (trans) or Amplification (amp) | Mean TMB score (Muts/Mb) | MSI Status |
| --- | --- | --- | --- | --- | --- |
| Cell Line | 0 | 9 | NTRK3 trans | 7.1 | MSS |
| Mediastinum | 0 | 0 | ALK trans | 2.3 | MSS |
| Colorectal | 0 | 9 | RET trans | 8.7 | MSS |
| Sarcoma | 0 | 7 | RET trans | 9.0 | MSS |
| Colorectal | 0 | 43 | ALK trans | 50.1 | MSI-H |
| Lung – NOS^{1} | KRAS G12A | 19 | 0 | 18.8 | MSS |
| Lung NSCLC^{1} | 0 | 26 | ERBB2 amp | 22.6 | MSS |
| Colorectal | BRAF V600E | 77 | 0 | 64.5 | MSI-H |
| Colorectal | BRAF V600E | 87 | 0 | 97.9 | MSI-H |
| Endometrial | KRAS G12C | 28 | 0 | 28.0 | MSI-H |
| Colorectal | BRAF V600E | 93 | 0 | 81.3 | MSI-H |
| Melanoma | BRAF V600K | 31 | 0 | 41.8 | MSS |
| Appendix | NRAS G13D | 8 | 0 | 6.2 | MSS |
| Endometrial | KRAS G12C & BRCA2 W2574* | 22 | 0 | 23.9 | MSI-H |

$^{1}$NOS: not otherwise specified; NSCLC: non-small cell lung cancer.

### b) Panel-wide Reproducibility

Reproducibility was assessed for each positive variant detected across all 36 replicates (Positive call rate) – The positive call rate was calculated based on the total number of mutations along with the two-sided 95% confidence interval.

Table 8 summarizes the positive call rates stratified by mutation type (SNV, insertions, and deletions) and mutant allele frequency (MAF). Overall call rate 86.2% across all samples and replicates (14493/16813, 85.7%-86.7% CI) with increased positive call rate at higher mutant allele frequency (MAFs). In terms of invalid rate, the first pass rate was 90.3% (455/504) and the overall pass rate of the study after repeat testing was 98.2% (495/504) allowing a maximum of 1 round of repeat testing.

The positive call rates for individual sequence mutations assessed in the Interlaboratory Reproducibility study, along with the MAF range, mean, SD, and CV are presented in Appendix D. A total of 337 SNVs and 137 indels (22 insertions, 115 deletions) are

K192063 - Page 19 of 108

{19}

provided. Variants are listed by specimen; each specimen is separated by a dark gray line. Discordant cases are denoted in light grey.

Table 8. Interlaboratory Reproducibility Positive Call Rates

| Mutation Type | MAF Threshold | Positive Call Rate Among All Observed Mutations | Total Unique Variants | Mean MAF Ranges | Mean AD Range | Mean DP Range |
| --- | --- | --- | --- | --- | --- | --- |
| All | MAF ≥ 0 | 86.2% (14493/16813) | 474 | 0.8-99.6 | 4-4881 | 74.3-6569.5 |
| | MAF ≥ 5 | 88.0% (14483/16458) | 464 | 5.9-99.6 | 9-4881 | 74.3-6569.5 |
| | MAF ≥ 8 | 91.9% (13921/15146) | 427 | 8.1-99.6 | 9-4881 | 74.3-6569.5 |
| | MAF ≥ 10 | 93.1% (13404/14400) | 406 | 10.1-99.6 | 9-4881 | 74.3-6569.5 |
| | MAF ≥ 15 | 96.4% (12387/12846) | 362 | 15.1-99.6 | 25.8-4881 | 74.3-6569.5 |
| All SNVs | MAF ≥ 0 | 88.4% (10549/11937) | 337 | 0.8-99.6 | 4-4881 | 109-6569.5 |
| | MAF ≥ 5 | 91.0% (10539/11582) | 327 | 6.1-99.6 | 13.5-4881 | 109-6569.5 |
| | MAF ≥ 8 | 95.7% (10070/10519) | 297 | 8.1-99.6 | 13.5-4881 | 109-6569.5 |
| | MAF ≥ 10 | 97.7% (9618/9845) | 278 | 10.1-99.6 | 13.5-4881 | 109-6569.5 |
| | MAF ≥ 15 | 97.8% (8773/8966) | 253 | 15.1-99.6 | 39.2-4881 | 172.4-6569.5 |
| All Insertions | MAF ≥ 0 | 82.8% (649/784) | 22 | 6.9-39 | 20.7-1094.4 | 153.5-2976.5 |
| | MAF ≥ 5 | 82.8% (649/784) | 22 | 6.9-39 | 20.7-1094.4 | 153.5-2976.5 |
| | MAF ≥ 8 | 86.9% (619/712) | 20 | 10.4-39 | 34-1094.4 | 153.5-2976.5 |
| | MAF ≥ 10 | 86.9% (619/712) | 20 | 10.4-39 | 34-1094.4 | 153.5-2976.5 |
| | MAF ≥ 15 | 95.9% (614/640) | 18 | 15.7-39 | 37.6-1094.4 | 153.5-2976.5 |
| All Deletions | MAF ≥ 0 | 80.5% (3295/4092) | 115 | 5.9-82 | 9-2093.4 | 74.3-4615.3 |
| | MAF ≥ 5 | 80.5% (3295/4092) | 115 | 5.9-82 | 9-2093.4 | 74.3-4615.3 |
| | MAF ≥ 8 | 82.6% (3232/3915) | 110 | 8.1-82 | 9-2093.4 | 74.3-4615.3 |
| | MAF ≥ 10 | 82.4% (3167/3843) | 108 | 11.8-82 | 9-2093.4 | 74.3-4615.3 |
| | MAF ≥ 15 | 92.6% (3000/3240) | 91 | 15.1-82 | 25.8-2093.4 | 74.3-4615.3 |
| ALK | N/A | 100% (70/70) | 2 | N/A | 6-155 | 337-5162.5 |
| NTRK2 | N/A | 100% (8/8) | 1 | N/A | 24-44 | 200-1707.5 |
| NTRK3 | N/A | 100% (36/36) | 1 | N/A | 321-911 | 1855-6721.5 |
| RET | N/A | 100% (71/71) | 2 | N/A | 55-396 | 963-6208 |

*≥ refers to all variants greater than the designated MAF; Mean AD: Average allele depth across replicates per variant; Mean DP: Average distinct coverage across replicates per variant

K192063 - Page 20 of 108

{20}

# c) Per Specimen:

The modal positive and negative call rates for sequence mutations (SNVs and indels) in each specimen are summarized in Table 9. A modal analysis yielded a 97.8% positive call rate among all positives (410 SNVs and indels).

Table 9. Interlaboratory Reproducibility Modal Call Rates per Specimen

| Specimen | Total Unique Mutations Detected Across All Replicates | Modal Positive Call Rate^{1} (n/N) (two-sided 95% CI) | Modal Negative Call Rate^{2} (n/N) (two-sided 95% CI) |
| --- | --- | --- | --- |
| 1 | 10 | 99.6% (251/252) (97.8%, 99.9%) | 97.2% (105/108) (92.2%, 99.1%) |
| 2^{3} | 0 | - | - |
| 3 | 9 | 100% (315/315) (98.8%, 100%) | - |
| 4 | 7 | 100% (216/216) (98.3%, 100%) | 97.2% (35/36) (85.8%, 99.5%) |
| 5 | 43 | 99.5% (1462/1470) (98.9%, 99.7%) | 91.4% (32/35) (77.6%, 97.0%) |
| 6 | 20 | 98.9% (639/646) (97.8%, 99.5%) | 88.2% (30/34) (73.4%, 95.3%) |
| 7 | 26 | 97.8% (678/693) (96.5%, 98.7%) | 95.2% (157/165) (90.7%, 97.5%) |
| 8 | 81 | 96.4% (1991/2065) (95.5%, 97.1%) | 88.7% (683/770) (86.3%, 90.8%) |
| 9 | 88 | 97.8% (2710/2772) (97.1%, 98.3%) | 80.3% (318/396) (76.1%, 83.9%) |
| 10 | 30 | 99.0% (998/1008) (98.2%, 99.5%) | 97.2% (70/72) (90.4%, 99.2%) |
| 11 | 94 | 96.1% (2907/3024) (95.4%, 96.8%) | 83.1% (299/360) (78.8%, 86.6%) |
| 12 | 33 | 99.4% (1109/1116) (98.7%, 99.7%) | 97.2% (70/72) (90.4%, 99.2%) |
| 13 | 9 | 100% (216/216) (98.3%, 100%) | 93.5% (101/108) (87.2%, 96.8%) |
| 14 | 24 | 96.8% (732/756) (95.3%, 97.9%) | 88.0% (95/108) (80.5%, 92.8%) |

$^{1}$ Positive call rate was calculated based on variants with majority call detected as positive.

$^{2}$ Negative call rate was calculated based on variants detected at least once, but with majority or equal call as negative. For all other locations, the negative call rates are 100%.

$^{3}$ Specimen 2 was selected for presence of ALK translocation and had no detected SNVs or indels.

K192063 - Page 21 of 108

{21}

### d) Analysis of Source of Variance

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

TMB was assessed using %CV of the TMB score across test sample replicates for samples with a reference TMB above LoB (7.2 Muts/Mb). The results are shown in (Table 10).

Table 10. Interlaboratory Reproducibility of PGDx elio tissue complete

| Alteration Type | Metric | Overall (95% CI) | Inter-Site (95% CI) | Inter-Operator (95% CI) | Inter-Day (95% CI) | Repeatability (Within-Run) (95% CI) |
| --- | --- | --- | --- | --- | --- | --- |
| SNVs | APA | 97.8% (97.7%, 97.9%) | 97.8% (97.7%, 97.9%) | 97.9% (97.7%, 98.0%) | 97.9% (97.7%, 98.1%) | 97.8% (97.5%, 98.1%) |
| | ANA | 99.9% (99.9%, 100%) | 99.9% (99.9%, 100%) | 99.9% (99.9%, 100%) | 99.9% (99.9%, 100%) | 99.9% (99.9%, 100%) |
| Insertions | APA | 95.6% (95.2%, 96.0%) | 95.7% (95.2%, 96.2%) | 95.4% (94.3%, 96.2%) | 95.5% (94.2%, 96.5%) | 96.4% (94.6%, 97.6%) |
| | ANA | 99.9% (99.9%, 100%) | 99.9% (99.9%, 100%) | 99.9% (99.9%, 100%) | 99.9% (99.9%, 100%) | 99.9% (99.9%, 100%) |
| Deletions | APA | 94.4% (94.2%, 94.6%) | 94.2% (93.9%, 94.4%) | 94.9% (94.4%, 95.3%) | 95.0% (94.4%, 95.5%) | 95.5% (94.7%, 96.2%) |
| | ANA | 99.9% (99.9%, 100%) | 99.9% (99.9%, 100%) | 99.9% (99.9%, 100%) | 99.9% (99.9%, 100%) | 99.9% (99.9%, 100%) |
| MSI | APA | 99.1% (98.7%, 99.4%) | 99.1% (98.7%, 99.4%) | 99.0% (97.9%, 99.6%) | 99.0% (97.6%, 99.6%) | 99.0% (96.6%, 99.7%) |
| | ANA | 99.3% (99.0%, 99.5%) | 99.3% (99.0%, 99.6%) | 99.3% (98.4%, 99.7%) | 99.3% (98.1%, 99.7%) | 99.3% (97.4%, 99.8%) |
| ERBB2 Amplification | APA | 100% (99.3%, 100%) | 100% (98.9%, 100%) | 100% (95.9%, 100%) | 100% (94.0%, 100%) | 100% (88.6%, 100%) |
| | ANA | 100% (100%, 100%) | 100% (99.9%, 100%) | 100% (99.7%, 100%) | 100% (99.6%, 100%) | 100% (99.2%, 100%) |
| ALK translocation | APA | 98.6% (97.7%, 99.1%) | 98.6% (97.5%, 99.2%) | 98.6% (95.8%, 99.5%) | 98.6% (94.8%, 99.6%) | 98.6% (92.2%, 99.7%) |
| | ANA | 99.8% (99.6%, 99.9%) | 99.8% (99.6%, 99.9%) | 99.8% (99.3%, 99.9%) | 99.8% (99.1%, 99.9%) | 99.8% (98.7%, 100%) |
| NTRK3 translocation | APA | 92.7% (90.4%, 94.5%) | 92.5% (89.6%, 94.6%) | 93.1% (86.7%, 96.5%) | 93.5% (85.4%, 97.3%) | 92.3% (79.1%, 97.4%) |
| | ANA | 99.4% (99.2%, 99.5%) | 99.3% (99.1%, 99.5%) | 99.4% (98.8%, 99.7%) | 99.4% (98.7%, 99.8%) | 99.3% (98.0%, 99.8%) |
| RET translocation | APA | 98.7% (97.8%, 99.2%) | 98.7% (97.7%, 99.3%) | 98.6% (95.9%, 99.5%) | 98.6% (95.0%, 99.6%) | 98.6% (92.4%, 99.8%) |
| | ANA | 99.8% (99.6%, 99.9%) | 99.8% (99.6%, 99.9%) | 99.8% (99.3%, 99.9%) | 99.8% (99.1%, 99.9%) | 99.8% (98.7%, 100%) |
| TMB | CV | 3.5% | 0.9% | 0.4% | 0.8% | 3.0% |

K192063 - Page 22 of 108

{22}

Independently ERBB2 amplification and ALK, NTRK2 and NTRK3, RET translocations were evaluated (one specimen each) and analyzed by ANA and APA. The overall APA for these variants was 97.7% and ANA was 99.9%. For BRCA1 deleterious variants, the APA was 11.8% because 3 replicates from a single case showed detection of a mutation not present in the other replicates. The MAF values for these 3 observations were 1.2%, 0.9%, and 0.8%, respectively (data not shown).

# e) Precision for MSI:

Precision of MSI was evaluated across 8 MSS and 6 MSI-H samples with a range of MSI scores. The mean MSI score, MSI range, SD and % CV for the score along with positive call rates are provided for results with 36 replicates obtained in the 3-site reproducibility study. The results demonstrate that precision of MSI values is supported by the PGDx elio tissue complete. Data is shown in Table 11.

Table 11. MSI Performance in the Interlaboratory Reproducibility Study

| Case No. | Modal Status | Total Replicates | Mean MSI Score | MSI Score Range | SD | %CV | Positive Call Rate (95% CI) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | MSS | 36 | 10.5 | (4.4, 19.1) | 3.7 | 35.1 | 100% (90.4%,100%) |
| 2 | MSS | 35 | 13.6 | (5.8, 20.4) | 3.6 | 26.8 | 100% (90.4%,100%) |
| 3 | MSS | 35 | 13.8 | (7.2, 20.2) | 3.7 | 26.8 | 100% (90.4%,100%) |
| 4 | MSS | 36 | 10.5 | (3.9, 19.8) | 3.2 | 30.4 | 100% (90.4%,100%) |
| 5 | MSI-H | 35 | 209.7 | (203.7, 216.5) | 3.6 | 1.7 | 100% (90.4%,100%) |
| 6 | MSS | 34 | 9.6 | (4.7, 16.0) | 3.0 | 31.3 | 100% (90.4%,100%) |
| 7 | MSS | 33 | -21.9 | (-29.9, -13.2) | 5.0 | -22.8 | 100% (90.4%,100%) |
| 8 | MSI-H | 35 | 223.5 | (213.1, 236.3) | 6.0 | 2.7 | 100% (90.4%,100%) |
| 9 | MSI-H | 36 | 271.6 | (261.6, 287.2) | 5.9 | 2.2 | 100% (90.4%,100%) |
| 10 | MSI-H | 36 | 77.5 | (62.6, 102.5) | 6.6 | 8.5 | 100% (90.4%,100%) |
| 11 | MSI-H | 36 | 219.0 | (212.6, 224.0) | 2.9 | 1.3 | 100% (90.4%,100%) |
| 12 | MSS | 36 | -56.1 | (-61.5, -41.5) | 4.1 | -7.3 | 100% (90.4%,100%) |
| 13 | MSS | 36 | 16.4 | (10.4, 25.1) | 3.9 | 23.7 | 100% (90.4%,100%) |
| 14 | MSI-H | 36 | 49.3 | (36.7, 61.7) | 6.3 | 12.8 | 94.4% (81.9%, 98.5%) |

K192063 - Page 23 of 108

{23}

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

Precision of TMB was evaluated across 11 samples (with TMB scores near the analytical borderline value of TMB LoB of 7.2 Muts/Mb) in the 3-site reproducibility site. The distribution of replicates by site, operator, and day is across site, operator, and day per samples and score are provided in Figure 5 (Figure 5 shows the distribution of replicates per site and operator for each of representative specimens by TMB score and test day). The data demonstrates high precision for TMB scores.


Figure 5: TMB Performance in the Interlaboratory Reproducibility Study by Site, Operator, and Day.

# g) Precision – cell lines:

Prior to performing the 3-site reproducibility study with clinical specimens, a study with DNA extracted from 6 blended cell line samples and a single colorectal FFPE sample that was MSI-H were evaluated. Together these samples represented a variety of DNA alterations, including over 600 unique alterations across a range of mutant allele fractions (MAFs). Each of the 7 samples was tested in duplicate by 2 different operators on 6 distinct sequencing runs at each of the 3 independent laboratory sites using a single kit lot. The positive call rate observed for the distinct variant types was consistent with the data observed with the clinical specimens (Table 12) though the cell line data had a larger number of variants with low MAFs

K192063 - Page 24 of 108

{24}

due to a dilution effect. Cell lines were blended at low concentrations reducing the MAF of insertions below thresholds and left only 20 total insertions (and only 3 at ≥15% MAF) for assessment.

Table 12. Multi-Site Reproducibility Study with Cell Lines:

| Mutation Type | Positive Call Rate | Variants |
| --- | --- | --- |
| All | 83.8% (24497/29232) | 812 |
| SNVs | 85.6% (21543/25164) | 699 |
| Insertions | 81.6% (248/304) | 19 |
| Deletions | 76.8% (2571/3348) | 93 |

### h) Lot-to-lot Precision

Performance of PGDx elio tissue complete was assessed across 3 unique kit lots by determining concordance of variant calls in FFPE tissue samples. The 3 unique kit lots were utilized to process 5 test cases in triplicate for a total of 45 observations. All batches were sequenced on the same instrument. Table 13 lists the Average Positive Agreement (APA) and Average Negative Agreement (ANA) used to assess lot to lot performance. APA for all variants is > 86%, and %CV for TMB analyses is < 10%. The performance is consistent with that of the reproducibility study.

Table 13. Lot-to-Lot Precision of PGDx elio tissue complete

| Variant Type | Performance | Between Lot 1 & Lot 2 | Between Lot 1 & Lot 3 | Between Lot 2 & Lot 3 |
| --- | --- | --- | --- | --- |
| Variants with Evidence of Clinical Significance | APA | 98.7% (93.0%, 99.8%) | 96.1% (89.2%, 98.7%) | 97.4% (91.1%, 99.3%) |
| | ANA | 99.9% (99.6%, 100%) | 99.8% (99.4%, 99.9%) | 99.9% (99.5%, 100%) |
| MSI | APA | 100% (75.8%, 100%) | 100% (75.8%, 100%) | 100% (75.8%, 100%) |
| | ANA | 100% (82.4%, 100%) | 100% (82.4%, 100%) | 100% (82.4%, 100%) |
| SNVs | APA | 92.1% (90.8%, 93.2%) | 91.9% (90.6%, 93.0%) | 91.9% (90.7%, 93.0%) |
| | ANA | 99.9% (99.9%, 100%) | 99.9% (99.9%, 100%) | 99.9% (99.9%, 100%) |
| Insertions | APA | 88.9% (80.2%, 94.0%) | 88.9% (80.2%, 94.0%) | 87.2% (78.0%, 92.9%) |
| | ANA | 99.9% (99.9%, 100%) | 99.9% (99.9%, 100%) | 99.9% (99.9%, 100%) |
| Deletions | APA | 86.2% (82.6%, 89.1%) | 89.8% (86.7%, 92.2%) | 87.3% (83.9%, 90.0%) |
| | ANA | 99.9% (99.9%, 100%) | 99.9% (99.9%, 100%) | 99.9% (99.9%, 100%) |

K192063 - Page 25 of 108

{25}

| ERBB2 Amplification | APA | 100% (61.0%, 100%) | 100% (61.0%, 100%) | 100% (61.0%, 100%) |
| --- | --- | --- | --- | --- |
| | ANA | 100% (86.2%, 100%) | 100% (86.2%, 100%) | 100% (86.2%, 100%) |
| ALK Translocation | APA | 100% (61.0%, 100%) | 100% (61.0%, 100%) | 100% (61.0%, 100%) |
| | ANA | 100% (96.7%, 100%) | 100% (96.7%, 100%) | 100% (96.7%, 100%) |
| TMB | CV | 9.5% | 7.9% | 7.1% |

### 3. Analytical Sensitivity - Limit of Detection (LoD):

The recommended DNA input for PGDx elio tissue complete is 100 ng of total DNA with a minimum 20% tumor purity. The LoD of the PGDx elio tissue complete assay is defined as the mutant allele fraction (MAF) at which 95% of replicates for a variant type are reliably detected. The LoD study was comprised of two steps: LoD establishment using cell lines and LoD confirmation with 10 FFPE clinical tumor samples from clinical cases across a diverse set of cancers (4 SNVs, 4 insertions and 4 deletions). Select specimens were used to evaluate specific mutations with evidence of clinical significance. Specimens were selected for allele frequencies near the claimed cut-offs. Details of the data are discussed and shown below.

#### a) LoD - SNVs, Insertions, and Deletions:

b)

Target levels for detection were first established in a dilution series from cell lines with up to 5 target MAF levels. The analytical sensitivity and LOD95 was then confirmed in clinical FFPE specimens. Data was aggregated across 2 reagent kit lots when possible, otherwise the lot with the higher MAF was used. Cell lines were used to establish the LoD MAF range for 451 SNVs and 31 indels across the panel. A total of 150 observations were generated (3 samples with 10 replicates at 5 dilution levels). Positive call status and MAF was evaluated for select variants identified in 10 FFPE clinical specimens diluted with normal DNA derived from FFPE tissue. Each specimen was processed with 2 kit lots of PGDx elio tissue complete across 10 replicates for a total of 200 observations (Table 14). The established analytical sensitivity ranges were confirmed at ≥ 95% call rate with FFPE clinical cases on a per variant level (Table 14) and using all somatic variants identified in FFPE clinical cases representing a range of MAFs for hotspot and non-hotspot positions (Table 15). A summary of the LoD mean mutant allele frequency and range as well as the positive call rate are displayed for each variant type by variant type for the entire panel across all replicates is shown in the Table 25 below. A range of 5.9-12.6% MAF was observed using the lowest average MAF where the positive call rates was ≥ 95%. The observed sequencing depth (DP), allele depth (AD), mutation allele frequency (MAF) range and average MAF are included.

K192063 - Page 26 of 108

{26}

Table 14. Analytical Sensitivity (LoD MAF) for SNVs and Indels in FFPE Tumor Tissue

| Mut Type | Gene | AA Change | DP Range | AD Range | MAF Range | Mean MAF | Positive Call Rate |
| --- | --- | --- | --- | --- | --- | --- | --- |
| SNV | BRAF | V600E | 491-804 | 6-31 | 1.0-5.3% | 3.1 | 100% |
| SNV | EGFR | L858R | 1257-2460 | 19-80 | 1.5-4.7% | 3.3 | 100% |
| SNV | BRCA2 | Splice Site Acceptor | 934-1600 | 27-61 | 2.3-5.2% | 3.4 | 100% |
| SNV | TP53 | Q331* | 537-696 | 15-43 | 2.4-6.2% | 4.3 | 100% |
| SNV | KRAS | G12V | 280-396 | 6-24 | 2.1-8.4% | 5.0 | 100% |
| SNV | NRAS | G13D | 530-1635 | 25-85 | 2.7-9.3% | 6.6 | 100% |
| SNV | TERT | Promoter | 255-431 | 13-33 | 3.9-8.5% | 5.9 | 100% |
| INS | TSC1 | Q654Tfs*34 | 547-1443 | 28-164 | 5.1-11.4% | 8.1 | 100% |
| INS | BRCA2 | S3366Nfs*5 | 731-973 | 58-113 | 6.9-12.7% | 8.9 | 100% |
| INS | TSC2 | D1690Gfs*27 | 463-877 | 51-114 | 8.7-16.9% | 12.0 | 95.0% |
| INS | BBC3 | R243Qfs*7 | 781-1530 | 95-254 | 12.2-19.2% | 14.3 | 95.0% |
| DEL | EGFR | L747_E749del | 479-969 | 22-48 | 3.5-6.7% | 4.6 | 100% |
| DEL | SMARCA 4 | E525Afs*8 | 1312-2150 | 99-195 | 6.4-9.7% | 8.2 | 95.0% |
| DEL | SOX9 | S484Wfs*? | 2133-3350 | 209-360 | 9.2-12.8% | 10.7 | 95.0% |
| DEL | KDM6A | S700Lfs*29 | 2062-2697 | 209-315 | 10.1-12.2% | 11.3 | 100% |

Table 15. Analytical Sensitivity (LoD MAF) for Representative SNVs and Indels

| Variant | Established MAF Range | Cell Line Variants | Number of Variants in Clinical Cases in the Established Range |
| --- | --- | --- | --- |
| Hotspot SNVs | 3.1% to 5.4% | 8 | 2 |
| Non-hotspot SNVs | 6.3% to 17.8% | 443 | 176 |
| Indels at homopolymer context\( ^{1} \) | 13.7% to 17.5% | 10 | 9 |
| Indels at non-homopolymer context | 6.1% to 10.9% | 19 | 4 |
| Insertions | 6.1% to 15.8% | 4 | 6 |
| Deletions | 6.5% to 17.5% | 25 | 13 |

\( ^{1} \) Greater than or equal to 5 bp repeat

K192063 - Page 27 of 108

{27}

Additional evaluations of analytical sensitivity performance used dilution series of FFPE clinical specimens. The positive call rates, sequence coverage, and mutant allele fraction are provided for a total of 11 SNVs, 3 insertions, and 5 deletions from 5 clinical FFPE specimens with 5 replicates per dilution level. A range of 5.9-12.6% MAF was observed using the lowest average MAF where the positive call rates was ≥ 95%. (Table 16 - Table 34).

An in-depth variant analysis of cell-line samples from LoD establishment studies were assessed to further demonstrate analytical sensitivity. The positive call rates, sequence coverage (DP), allele depth (AD), and mutant allele fraction (MAF) are provided for a total of 13 SNVs, 4 insertions, and 4 deletions from a cell-line based dilution series of 3 samples with 10 replicates at 5 dilution levels (Table 35-Table 55).

Table 16: KRAS G12D SNV

| KRAS SNV (Clinical Dilution Series) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Dilution | AA Change | DP Range | AD Range | Average MAF | Positive Call Rate |
| 40% | G12D | 487-565 | 87-133 | 20.4 | 100% (5/5) |
| 30% | | 519-580 | 79-92 | 15.6 | 100% (5/5) |
| 20% | | 554-593 | 35-73 | 10.2 | 100% (5/5) |
| 15% | | 583-624 | 33-39 | 5.9 | 100% (3/3) |

Table 17: APC R213* SNV

| APC SNV (Clinical Dilution Series) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Dilution | AA Change | DP Range | AD Range | Average MAF | Positive Call Rate |
| 40% | R213* | 264-343 | 59-85 | 23.1 | 100% (5/5) |
| 30% | | 309-374 | 56-98 | 21.7 | 100% (5/5) |
| 20% | | 302-327 | 33-42 | 12.0 | 100% (5/5) |
| 15% | | 305-326 | 16-23 | 6.1 | 100% (3/3) |

Table 18: PIK3CA Y1021C SNV

| PIK3CA SNV (Clinical Dilution Series) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Dilution | AA Change | DP Range | AD Range | Average MAF | Positive Call Rate |
| 40% | Y1021C | 609-790 | 137-192 | 25.0 | 100% (5/5) |
| 30% | | 620-791 | 91-115 | 15.1 | 100% (5/5) |
| 20% | | 684-835 | 74-87 | 10.8 | 100% (5/5) |
| 15% | | 727-817 | 43-61 | 6.5 | 100% (3/3) |

Table 19: MEN1 R206H SNV

| MEN1 SNV (Clinical Dilution Series) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Dilution | AA Change | DP Range | AD Range | Average MAF | Positive Call Rate |
| 40% | R206H | 1476-1802 | 332-431 | 22.8 | 100% (5/5) |
| 30% | | 1585-1746 | 230-286 | 15.6 | 100% (5/5) |
| 20% | | 1586-1911 | 170-207 | 11.0 | 100% (5/5) |
| 15% | | 1939-2230 | 130-148 | 6.6 | 100% (3/3) |

K192063 - Page 28 of 108

{28}

Table 20: ACVR1 R160*SNV

| ACVR1 SNV (Clinical Dilution Series) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Dilution | AA Change | DP Range | AD Range | Average MAF | Positive Call Rate |
| 40% | R160* | 1192-1501 | 274-351 | 22.8 | 100% (5/5) |
| 30% | | 1266-1691 | 251-358 | 19.9 | 100% (5/5) |
| 20% | | 1329-1561 | 163-207 | 12.4 | 100% (5/5) |
| 15% | | 1432-1572 | 103-107 | 6.9 | 100% (3/3) |

Table 21: PDCD1 R272Q SNV

| PDCD1 SNV (Clinical Dilution Series) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Dilution | AA Change | DP Range | AD Range | Average MAF | Positive Call Rate |
| 40% | R272Q | 1116-1360 | 211-276 | 20.0 | 100% (5/5) |
| 30% | | 1271-1523 | 193-310 | 17.6 | 100% (5/5) |
| 20% | | 1147-1447 | 88-173 | 10.5 | 100% (5/5) |
| 15% | | 1360-1507 | 95-103 | 7.0 | 100% (3/3) |

Table 22: SMAD3 V294M SNV

| SMAD3 SNV (Clinical Dilution Series) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Dilution | AA Change | DP Range | AD Range | Average MAF | Positive Call Rate |
| 40% | V294M | 1091-1487 | 280-321 | 23.6 | 100% (5/5) |
| 30% | | 1384-1618 | 240-301 | 18.4 | 100% (5/5) |
| 20% | | 1425-1523 | 147-169 | 10.6 | 100% (5/5) |
| 15% | | 1552-1673 | 104-129 | 7.0 | 100% (3/3) |

Table 23: CREBBP P885H SNV

| CREBBP SNV (Clinical Dilution Series 5) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Dilution | AA Change | DP Range | AD Range | Average MAF | Positive Call Rate |
| 40% | P885H | 1853-2160 | 455-542 | 24.6 | 100% (5/5) |
| 30% | | 1888-2670 | 366-502 | 19.6 | 100% (5/5) |
| 20% | | 2009-2420 | 202-289 | 11.2 | 100% (5/5) |
| 15% | | 2156-2564 | 160-181 | 7.1 | 100% (3/3) |

Table 24: PIK3CG T128M SNV

| PIK3CG SNV (Clinical Dilution Series) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Dilution | AA Change | DP Range | AD Range | Average MAF | Positive Call Rate |
| 40% | T128M | 2194-2894 | 431-618 | 21.2 | 100% (5/5) |
| 30% | | 2545-2866 | 376-458 | 15.4 | 100% (5/5) |
| 20% | | 2707-3333 | 224-356 | 9.5 | 100% (5/5) |
| 15% | | 3083-3423 | 215-255 | 7.1 | 100% (3/3) |

K192063 - Page 29 of 108

{29}

Table 25: NOTCH1 R4904* SNV

| NOTCH1 SNV (Clinical Dilution Series) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Dilution | AA Change | DP Range | AD Range | Average MAF | Positive Call Rate |
| 40% | R4904* | 1451-1657 | 307-393 | 23.0 | 100% (5/5) |
| 30% | | 1547-1924 | 274-359 | 18.4 | 100% (5/5) |
| 20% | | 1633-1791 | 179-213 | 11.3 | 100% (5/5) |
| 15% | | 1591-1826 | 109-147 | 7.3 | 100% (3/3) |

Table 26: KMT2D R4904* SNV

| KMT2D SNV (Clinical Dilution Series) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Dilution | AA Change | DP Range | AD Range | Average MAF | Positive Call Rate |
| 40% | R4904* | 1451-1657 | 307-393 | 23.0 | 100% (5/5) |
| 30% | | 1547-1924 | 274-359 | 18.4 | 100% (5/5) |
| 20% | | 1633-1791 | 179-213 | 11.3 | 100% (5/5) |
| 15% | | 1591-1826 | 109-147 | 7.3 | 100% (3/3) |

Table 27: APC Insertion

| APC INS (Clinical Dilution Series) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Dilution | AA Change | DP Range | AD Range | Average MAF | Positive Call Rate |
| 40% | T1556Nfs*3 | 331-383 | 69-90 | 21.8 | 100% (5/5) |
| 30% | | 305-479 | 52-93 | 15.8 | 100% (5/5) |
| 20% | | 424-461 | 30-55 | 10.1 | 100% (5/5) |
| 15% | | 385-457 | 21-32 | 6.4 | 100% (3/3) |

Table 28: TLR9 Insertion

| TLR9 INS (Clinical Dilution Series) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Dilution | AA Change | DP Range | AD Range | Average MAF | Positive Call Rate |
| 40% | R389Pfs*76 | 2007-2500 | 470-606 | 23.9 | 100% (5/5) |
| 30% | | 2044-2341 | 342-371 | 15.9 | 100% (5/5) |
| 20% | | 2376-2652 | 249-303 | 11.0 | 100% (5/5) |
| 15% | | 2597-2815 | 179-187 | 6.8 | 100% (3/3) |

Table 29: ARID1A Insertion

| ARID1A Insertion (Clinical Dilution Series) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Dilution | AA Change | DP Range | AD Range | Average MAF | Positive Call Rate |
| 40% | H688Sfs*129 | 1228-1571 | 304-348 | 23.3 | 100% (5/5) |
| 30% | | 1395-1634 | 256-402 | 21.2 | 100% (5/5) |
| 20% | | 1374-1484 | 164-187 | 12.6 | 100% (5/5) |
| 15% | | 1513-1661 | 98-126 | 7.1 | 100% (3/3) |

K192063 - Page 30 of 108

{30}

Table 30: ARID1A Deletion

| ARID1A DEL (Clinical Dilution Series) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Dilution | AA Change | DP Range | AD Range | Average MAF | Positive Call Rate |
| 40% | Q1493Hfs*6 | 1362-1631 | 219-303 | 18.0 | 100% (5/5) |
| 30% | | 1284-1692 | 135-229 | 12.6 | 100% (5/5) |
| 20% | | 1658-1658 | 149-149 | 9.0 | 20.0% (1/5) |
| 15% | | 1678-1811 | 128-131 | 7.4 | 66.7% (2/3) |

Table 31: RAD51C Deletion

| RAD51C Deletion (Clinical Dilution Series) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Dilution | AA Change | DP Range | AD Range | Average MAF | Positive Call Rate |
| 40% | D202Ifs*37 | 213-272 | 22-44 | 13.0 | 100% (5/5) |
| 30% | | 237-319 | 20-36 | 9.8 | 100% (5/5) |
| 20% | | 220-270 | 16-20 | 7.5 | 80.0% (4/5) |
| 15% | | 265-327 | 17-25 | 7.4 | 100% (3/3) |

Table 32: TET2 Deletion

| TET2 DEL (Clinical Dilution Series) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Dilution | AA Change | DP Range | AD Range | Average MAF | Positive Call Rate |
| 40% | K117Rfs*11 | 515-593 | 108-137 | 21.9 | 100% (5/5) |
| 30% | | 521-657 | 91-109 | 16.7 | 100% (5/5) |
| 20% | | 609-741 | 57-85 | 11.1 | 100% (5/5) |
| 15% | | 656-726 | 51-62 | 8.3 | 100% (3/3) |

Table 33: NKX3-1 Deletion

| NKX3-1 DEL (Clinical Dilution Series) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Dilution | AA Change | DP Range | AD Range | Average MAF | Positive Call Rate |
| 40% | P58_E59del | 939-1190 | 161-202 | 17.4 | 100% (5/5) |
| 30% | | 953-1281 | 92-185 | 11.5 | 100% (5/5) |
| 20% | | 1093-1112 | 75-109 | 8.4 | 40.0% (2/5) |
| 15% | | 963-963 | 82-82 | 8.5 | 33.3% (1/3) |

Table 34: B2M Deletion

| B2M DEL (Clinical Dilution Series) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Dilution | AA Change | DP Range | AD Range | Average MAF | Positive Call Rate |
| 40% | L15Ffs*41 | 2175-2731 | 475-592 | 22.0 | 100% (5/5) |
| 30% | | 2322-2866 | 432-626 | 19.3 | 100% (5/5) |
| 20% | | 2120-2439 | 239-348 | 12.6 | 100% (5/5) |
| 15% | | 2252-2479 | 196-220 | 8.7 | 100% (3/3) |

K192063 - Page 31 of 108

{31}

Table 35: BRAF V600E SNV

| BRAF SNV (Cell Line Dilution Series) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Dilution | AA Change | DP Range | AD Range | Average MAF | Positive Call Rate |
| Level 1 | V600E | 879-1167 | 71-110 | 9.0 | 100% (10/10) |
| Level 2 | | 948-1126 | 51-107 | 6.6 | 100% (10/10) |
| Level 3 | | 860-1114 | 17-38 | 2.8 | 100% (10/10) |
| Level 4 | | 878-1147 | 20-36 | 2.5 | 100% (10/10) |
| Level 5 | | 1017-1257 | 10-29 | 1.6 | 90.0% (9/10) |

Table 36: EGFR L858R SNV

| EGFR SNV (Cell Line Dilution Series) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Dilution | AA Change | DP Range | AD Range | Average MAF | Positive Call Rate |
| Level 1 | L858R | 1819-2593 | 210-288 | 11.4 | 100% (10/10) |
| Level 2 | | 1942-2383 | 149-204 | 8.1 | 100% (10/10) |
| Level 3 | | 1344-1763 | 35-77 | 3.5 | 100% (10/10) |
| Level 4 | | 1601-2131 | 46-70 | 2.9 | 100% (10/10) |
| Level 5 | | 1844-2333 | 27-42 | 1.7 | 100% (10/10) |

Table 37: KRAS SNV

| KRAS SNV (Cell Line Dilution Series) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Dilution | AA Change | DP Range | AD Range | Average MAF | Positive Call Rate |
| Level 1 | G13D | 897-1046 | 71-107 | 9.5 | 100% (10/10) |
| Level 2 | | 1059-1320 | 56-115 | 7.1 | 100% (10/10) |
| Level 3 | | 951-1349 | 32-58 | 4.2 | 100% (10/10) |
| Level 4 | | 930-1123 | 24-43 | 3.2 | 100% (10/10) |
| Level 5 | | 982-1147 | 18-30 | 2.1 | 100% (10/10) |

Table 38: EGFR SNV

| EGFR SNV (Cell Line Dilution Series) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Dilution | AA Change | DP Range | AD Range | Average MAF | Positive Call Rate |
| Level 1 | T790M | 2600-3602 | 294-497 | 12.5 | 100% (10/10) |
| Level 2 | | 2551-3501 | 230-308 | 8.6 | 100% (10/10) |
| Level 3 | | 1808-2231 | 79-110 | 4.4 | 100% (10/10) |
| Level 4 | | 1991-2980 | 45-93 | 2.8 | 100% (10/10) |
| Level 5 | | 2443-3117 | 41-69 | 2.1 | 100% (10/10) |

K192063 - Page 32 of 108

{32}

Table 39: NRAS SNV

| NRAS SNV (Cell Line Dilution Series) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Dilution | AA Change | DP Range | AD Range | Average MAF | Positive Call Rate |
| Level 1 | Q61K | 776-996 | 64-105 | 10.0 | 100% (10/10) |
| Level 2 | | 953-1112 | 50-88 | 7.1 | 100% (10/10) |
| Level 3 | | 743-1136 | 34-67 | 5.0 | 100% (10/10) |
| Level 4 | | 679-1006 | 19-44 | 3.4 | 100% (10/10) |
| Level 5 | | 860-963 | 16-33 | 2.4 | 100% (10/10) |

Table 40: NRAS SNV

| NRAS SNV (Cell Line Dilution Series) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Dilution | AA Change | DP Range | AD Range | Average MAF | Positive Call Rate |
| Level 1 | A146T | 1018-1208 | 102-154 | 12.0 | 100% (10/10) |
| Level 2 | | 1089-1453 | 81-117 | 7.7 | 100% (10/10) |
| Level 3 | | 1363-1564 | 66-102 | 5.5 | 100% (10/10) |
| Level 4 | | 1025-1572 | 30-53 | 3.3 | 100% (10/10) |
| Level 5 | | 1040-1307 | 21-40 | 2.4 | 100% (10/10) |

Table 41: PIK3CA SNV

| PIK3CA SNV (Cell Line Dilution Series) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Dilution | AA Change | DP Range | AD Range | Average MAF | Positive Call Rate |
| Level 1 | G118D | 152-252 | 7-23 | 8.1 | 100% (10/10) |
| Level 2 | | 199-268 | 10-16 | 5.4 | 100% (10/10) |
| Level 3 | | 200-313 | 5-11 | 2.7 | 60.0% (6/10) |
| Level 4 | | 251-296 | 6-7 | 2.4 | 30.0% (3/10) |
| Level 5 | | 259-329 | 7-7 | 2.4 | 20.0% (2/10) |

Table 42: TP53 SNV

| TP53 SNV (Cell Line Dilution Series) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Dilution | AA Change | DP Range | AD Range | Average MAF | Positive Call Rate |
| Level 1 | R273H | 902-1326 | 126-202 | 14.6 | 100% (10/10) |
| Level 2 | | 971-1296 | 89-126 | 10.1 | 100% (10/10) |
| Level 3 | | 832-1242 | 33-63 | 4.5 | 100% (10/10) |
| Level 4 | | 1008-1240 | 27-38 | 3.0 | 90.0% (9/10) |
| Level 5 | | 1123-1242 | 27-29 | 2.3 | 30.0% (3/10) |

K192063 - Page 33 of 108

{33}

Table 43: CTNNB1 SNV

| CTNNB1 SNV (Cell Line Dilution Series) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Dilution | AA Change | DP Range | AD Range | Average MAF | Positive Call Rate |
| Level 1 | S33Y | 1075-1269 | 115-189 | 13.7 | 100% (10/10) |
| Level 2 | | 1276-1599 | 95-145 | 8.2 | 100% (10/10) |
| Level 3 | | 1493-1794 | 68-122 | 5.2 | 100% (10/10) |
| Level 4 | | 1250-1705 | 37-64 | 3.1 | 100% (10/10) |
| Level 5 | | 1101-1534 | 28-48 | 2.5 | 70.0% (7/10) |

Table 44: EGFR SNV

| EGFR SNV (Cell Line Dilution Series) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Dilution | AA Change | DP Range | AD Range | Average MAF | Positive Call Rate |
| Level 1 | G719S | 941-1145 | 66-100 | 8.0 | 100% (10/10) |
| Level 2 | | 1022-1168 | 42-67 | 5.2 | 100% (10/10) |
| Level 3 | | 1068-1276 | 29-58 | 3.8 | 100% (10/10) |
| Level 4 | | 872-1196 | 22-36 | 2.8 | 90.0% (9/10) |
| Level 5 | | 937-957 | 22-27 | 2.6 | 20.0% (2/10) |

Table 45: TP53 SNV

| TP53 SNV (Cell Line Dilution Series) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Dilution | AA Change | DP Range | AD Range | Average MAF | Positive Call Rate |
| Level 1 | S241F | 1601-2004 | 196-254 | 12.2 | 100% (10/10) |
| Level 2 | | 1907-2331 | 148-203 | 8.7 | 100% (10/10) |
| Level 3 | | 1730-2136 | 87-154 | 5.9 | 100% (10/10) |
| Level 4 | | 1634-1993 | 57-104 | 4.2 | 100% (10/10) |
| Level 5 | | 1946-2266 | 42-77 | 2.8 | 100% (10/10) |

Table 46: BRCA2 SNV

| BRCA2 SNV (Cell Line Dilution Series) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Dilution | AA Change | DP Range | AD Range | Average MAF | Positive Call Rate |
| Level 1 | S3094* | 1614-1985 | 225-315 | 15.7 | 100% (10/10) |
| Level 2 | | 1822-2188 | 149-217 | 9.4 | 100% (10/10) |
| Level 3 | | 2038-2382 | 123-154 | 6.3 | 100% (10/10) |
| Level 4 | | 1713-2279 | 66-107 | 4.3 | 100% (10/10) |
| Level 5 | | 1973-2086 | 61-63 | 3.1 | 30.0% (3/10) |

Table 47: BRCA1 SNV

| BRCA1 SNV (Cell Line Dilution Series) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Dilution | AA Change | DP Range | AD Range | Average MAF | Positive Call Rate |
| Level 1 | R1443* | 3555-4041 | 357-486 | 11.1 | 100% (10/10) |
| Level 2 | | 3887-4935 | 187-319 | 6.1 | 100% (10/10) |
| Level 3 | | 4168-5324 | 154-256 | 4.1 | 100% (10/10) |
| Level 4 | | 4588-5017 | 140-160 | 3.2 | 20.0% (2/10) |
| Level 5 | | N/A | | | 0% (0/10) |

K192063 - Page 34 of 108

{34}

Table 48: SOX9 Insertion

| SOX9 INS (Cell Line Dilution Series) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Dilution | AA Change | DP Range | AD Range | Average MAF | Positive Call Rate |
| Level 1 | P415Rfs*56 | 3508-3986 | 271-417 | 9.1 | 100% (10/10) |
| Level 2 | | 3071-3873 | 171-243 | 6.1 | 100% (10/10) |
| Level 3 | | 2623-3298 | 143-187 | 5.6 | 60.0% (6/10) |
| Level 4 | | N/A | | | 0% (0/10) |
| Level 5 | | N/A | | | 0% (0/10) |

Table 49: MAML1 Insertion

| MAML1 INS (Cell Line Dilution Series) | | | | | |
| --- | --- | --- | --- | --- | --- |
| Dilution | AA Change | DP Range | AD Range | Average MAF | Positive Call Rate |
| Level 1 | R476Sfs*22 | 2536-2958 | 219-277 | 9.0 | 100% (10/10) |
| Level 2 | | 2884-3303 | 147-225 | 5.7 | 80.0% (8/10) |
| Level 3 | | N/A | | | 0% (0/10) |
| Level 4 | | N/A | | | 0% (0/10) |
| Level 5 | | N/A | | | 0% (0/10) |

Table 50: CTNNA1 Insertion

| CTNNA1 INS (Cell Line Dilution Se…