ACTOnco, ACTOnco IVD

K210017 · Act Genomics · PZM · Dec 23, 2022 · Pathology

Device Facts

• Record ID: K210017
• Device Name: ACTOnco, ACTOnco IVD
• Applicant: Act Genomics
• Product Code: PZM · Pathology
• Decision Date: Dec 23, 2022
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.6080
• Device Class: Class 2
• Attributes: AI/ML, Real-World Evidence

Real-World Evidence

Submission	Device	Sponsor	RWD Sources	RWE Use Summary	Key Tags
K210017 · Dec 23, 2022	ACTOnco, ACTOnco IVD	Act Genomics	Historical clinical FFPE specimens; Clinical FFPE tumor samples from clinical cases	Historical clinical data was used to evaluate assay invalid rates across 27 tumor types. Clinical FFPE samples were used in a method comparison study to establish analytical accuracy (PPA/NPA) against an externally validated NGS assay.	FFPE clinical specimens; Analytical accuracy; Invalid rates; Retrospective clinical data

Clinical Evidence

Study Design	Population	Comparator	Key Endpoints
Invalid Rates Evaluation; Retrospective analysis of historical clinical data	Patients with 27 different FFPE tumor types; Sample Size: 1526; Number of Sites: 1	Not applicable for this study	Invalid rates (Pre-run and Post-run)
Analytical Accuracy Study; Retrospective method comparison	438 FFPE tumor samples from clinical cases spanning 21 cancer types; Sample Size: 438; Number of Sites: 1	Externally validated NGS assay (Ev-NGS)	PPA and NPA for SNVs, indels, and ERBB2 amplification

AI Performance

Output	Algorithm	Acceptance	Observed	Dev DS	Dev Readers	Test DS	Test Readers
Somatic single nucleotide variants (SNVs)	Amplicon-based targeted NGS pipeline with variant calling plugin	—	PPA: 97.85% (95% CI: 96.89% to 98.52%); NPA: 99.97% (95% CI: 99.97% to 99.98%)	17 ACTOnco runs using a Reference Standard; 18 non-cancer FFPE samples; 20 normal tissue FFPE samples; 18 normal FFPE samples; 20 samples with lower sequencing depth.	—	438 FFPE tumor samples from clinical cases spanning 21 cancer types.	—
Small insertions and deletions (Indels)	Amplicon-based targeted NGS pipeline with variant calling plugin	—	PPA: 97.85% (95% CI: 96.89% to 98.52%); NPA: 99.97% (95% CI: 99.97% to 99.98%)	17 ACTOnco runs using a Reference Standard; 18 non-cancer FFPE samples; 20 normal tissue FFPE samples; 18 normal FFPE samples; 20 samples with lower sequencing depth.	—	438 FFPE tumor samples from clinical cases spanning 21 cancer types.	—
ERBB2 gene amplification	Amplicon-based targeted NGS pipeline with copy number analysis plugin	Observed copy number >= 4	PPA: 93.33% (95% CI: 82.14% to 97.71%); NPA: 100.00% (95% CI: 95.47% to 100.00%)	18 normal FFPE samples; 20 samples with lower sequencing depth; 1 clinical breast cancer sample diluted to 5 levels; 2 FFPE clinical specimens with low tumor purity.	—	126 FFPE samples representing 21 different cancer types compared to HER2 Dual ISH DNA Probe Cocktail test (DISH).	—
Tumor mutational burden (TMB)	Amplicon-based targeted NGS pipeline with TMB calculation module	—	Spearman correlation coefficient of 0.885 compared to WES; Slope: 0.921; Intercept: 1.047	12 single samples; 5 clinical FFPE specimens with TMB score >= 10; 10 samples assessed for TMB across 3 DNA input levels.	—	45 FFPE clinical cases covering 14 cancer types compared to whole exome sequencing (WES).	—

Indications for Use

The ACTOnco IVD assay is an in vitro diagnostic test that uses targeted next generation sequencing of formalin-fixed, paraffin-embedded tumor tissue from patients with solid malignant neoplasms to detect genetic alterations in a broad multi gene panel. The test is intended to provide information on point mutations, small insertions and deletions, ERBB2 gene amplification, and tumor mutational burden 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. ACTOnco IVD is a single-site assay performed at ACT Genomics.

Device Story

ACTOnco IVD is an NGS-based tumor profiling assay performed at a single site. It takes genomic DNA extracted from FFPE tumor tissue as input. The workflow includes library preparation via multiplex PCR amplification of 440 target genes, template preparation via emulsion PCR, and sequencing on the Thermo Fisher Ion GeneStudio S5 Prime System. The software performs signal processing, base calling, read alignment to hg19, and variant calling. It produces a report identifying somatic mutations, ERBB2 amplification, and TMB. Healthcare professionals use these results to inform clinical decision-making in accordance with professional guidelines. The device benefits patients by providing comprehensive genomic profiling to identify potentially actionable genetic alterations.

Clinical Evidence

Bench testing only. Precision/reproducibility study (20 samples, 48 replicates) showed 98.33% call rate for SNVs/Indels. LoD study established sensitivity for various variant types. Method comparison study (438 samples) against a validated NGS assay showed 97.85% PPA and 99.97% NPA for SNVs/Indels. ERBB2 amplification comparison (129 samples) against DISH showed 91.67% PPA and 100% NPA. TMB performance compared to WES (45 samples) showed a Spearman correlation of 0.885.

Technological Characteristics

Targeted amplicon-based NGS assay. DNA extracted from FFPE tissue. Semiconductor sequencing (Ion Torrent). Targets ~1.8Mb of human genome (oncogenes, tumor suppressors, drug metabolism, immune genes). Software includes variant calling, annotation, and TMB calculation. Single-site laboratory workflow. No matched normal required; uses pooled normal baseline.

Indications for Use

Indicated for patients with solid malignant neoplasms to detect genetic alterations (SNVs, indels, ERBB2 amplification, TMB) in 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)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

## I. Background Information

### A. 510(k) Number

K210017

### B. Applicant

ACT Genomics Co., LTD

### C. Proprietary and Established Names

ACTOnco IVD

### D. Regulatory Information

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

## II. Submission/Device Overview

### A. Purpose for submission

New device.

### B. Measurand

Somatic single nucleotide variants, insertions and deletions, ERBB2 gene amplifications 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.

## III. Intended Use/Indications for Use

### A. Intended Use(s)

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The ACTOnco IVD assay is an in vitro diagnostic test that uses targeted next generation sequencing of formalin-fixed, paraffin-embedded tumor tissue from patients with solid malignant neoplasms to detect genetic alterations in a broad multi gene panel. The test is intended to provide information on point mutations, small insertions and deletions, ERBB2 gene amplification, and tumor mutational burden 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. ACTOnco IVD is a single-site assay performed at ACT Genomics.

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

Same as above

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

For prescription use only

For in vitro diagnostic use

# **D. Special Instrument Requirements**

Thermo Fisher Ion GeneStudio S5 Prime System (qualified by ACT Genomics)

# **IV. Device/System Characteristics**

# **A. Device Description**

A description of required equipment, software, reagents, vendors, and storage conditions is provided, and is described in the instructions for use (ACT Genomics manual). ACT Genomics assumes responsibility for the device. The assay system includes a sequencing instrument, reagents (DNA extraction, library preparation and sequencing), software (operation of the sequencing instrument and variant calling), and standard operating procedures (SOPs) for the use of the system. ACT Genomics takes the responsibilities in monitoring the instrument; reagents and consumable materials which will be used in the assay process. The device is an NGS-based tumor profiling assay, which sequences tumor specimens to detect mutations.

# **1. Sample Preparation:**

The ACTOnco IVD assay (referred to as ACTOnco assay hereafter) requires genomic DNA isolated from formalin-fixed, paraffin-embedded (FFPE) tissue specimens. The tumor volume and minimum tumor content needed to obtain sufficient DNA for testing to achieve the stated performance are shown in Table 1 below. If the minimum tumor proportion is less than 50%, the tissue should be macro-dissected to select as much viable tumor as possible and minimize the amount of adjacent non-tumor tissue.

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Table 1. Specimen Handling and Processing FFPE Tissue

| Tissue Type | Volume | Minimum Tumor Proportion | Macrodissection requirements | Limitations | Storage |
| --- | --- | --- | --- | --- | --- |
| Formalin-fixed, paraffin-embedded (FFPE) Tumor tissue blocks or slides. | 5-20 unstained section, 10 microns thick. Total surface area ≥ 125mm² | More than 10% of tumor cells; sections containing >30% viable tumor are preferred. For TMB testing, > 30% tumor cells | Macrodissection will be done if the minimum tumor proportion is less than 50% in order to enrich neoplastic content | Archival FFPE material > 8 years post-resection is not suitable for analysis | Room temperature |

## 2. DNA Extraction:

The ACTOnco assay requires genomic DNA isolated from FFPE tissue. DNA is quantified and concentrated if necessary. Genomic DNA is stored at –80°C until assay initiation. The amount of DNA required to perform the assay is 80 ng, with at least 15% of DNA at a fragment length longer than 500 base pairs. DNA proceeds directly to library preparation.

## 3. Library Preparation:

Sequence libraries are prepared by first amplifying the target region with the custom DNA primers corresponding to all exons and selected introns of 440 target genes. The amplicons are enzymatically treated to partially digest the primers and then ligated to the sequencing adaptor oligonucleotide and one of the 96 oligonucleotide barcodes in the Kit. A second PCR reaction is performed to amplify the barcoded library. Quality and quantity of the barcoded library is checked. The sample should be a smear with an average fragment size peak at 125-300bp ≥ 50%; and total amount ≥ 3ng to ensure subsequent template preparation. The DNA libraries for all samples and controls will then be pooled for the template preparation reaction.

## 4. Template Preparation and Sequencing:

Clonal amplification of the DNA library is performed prior to sequencing. The automated emulsion polymerase chain reaction is performed per protocol. Sequence templates are then be loaded onto chip and sequencing is conducted.

## 5. Data Analysis:

a) Data Management: Sample tracking and archiving of run-associated metadata (barcode number, run name, sample accession number, source (class)) is performed with the following key functions: Tracking sample status through various stages of data analysis; tracking iterations of analysis applied to a give sample; recording versions of databases and algorithms used in analysis; archival of pipeline output files (BAM, VCF) and sequencing run statistics (e.g., total output per chip, and mean depth of coverage).

b) Signal Processing, Base calling, Read Alignment, BAM Generation, and Coverage Statistics: Signal Processing, Base Calling, Read Alignment, BAM Generation, and Coverage statistics are performed by the software. In the sequencing process, the software converts the flow signals to nucleotide bases. Each read is assigned to a barcode in barcode classification. The software then trims barcode and adaptors, and other quality checks. Then, the variant

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caller plugin of the software employs a mapping alignment program to align reads to the human reference genome (hg19). Aligned reads are written to a Binary Alignment Map (BAM) file. The BAM file is then used for further data analysis. The coverage analysis plugin of the software computes mean depth of coverage, uniformity of base coverage, and percent reads on target.

c) Read Alignment Quality check: Following the manufacturer's recommendation, read alignment is used to evaluate the sequencing reaction quality and the corresponding library's quality when an accurate reference is available. Reads are aligned to the sequence of hg19. Any discrepancy in alignment to hg19, whether biological (actual variant) or technical (sequencing error) is listed as a mismatch. Alignment performance metrics are reported using the Alignment Quality (AQ) score, which defines the accuracy of sequencing reads when compared to a reference genome sequence. The aligned length of a read at a given accuracy threshold is defined as the greatest position in the read at which the accuracy in the bases, up to and including the position, meets the accuracy threshold. Accuracy is specified using the Phred -10log10 transformation, where 17 represents an error rate of 2% (98% accuracy). The AQ level will decrease in samples with more variations compared to the reference genome, for example, tumor samples with many SNPs and SNVs. The software defaults at "AQ17 Total Bases". AQ17 is the manufacturer's default setting which provides a long mean read length with some misalignment allowed to accommodate the mismatch in the samples to be detected.

d) Sample QC check: At the sample level, there are three QC metrics. The first metric is the mean depth for each sample, which ensures each sample is sequenced with enough reads. The mean depth for each sample needs to be ≥ 500x. The second metric is the target base coverage (uniformity). Sequence coverage ≥ 100x is required with at least 85% of target regions to meet this coverage metric. The third metric is the sample contamination QC check. Samples are checked for possible contamination through bioinformatic analysis of an SNP profile algorithm to reduce the risk of false-positive calls that could occur as a result of an unexpected contamination event.

e) Mutation calling – single nucleotide variants (SNVs), insertions and deletions (Indels): The analysis pipeline identifies two classes of mutations: (1) SNVs and (2) indels.

i) Analysis of positive and negative controls: At the sample level, sequencing coverage of ≥ 500X is required, and sequence coverage of ≥ 100x is required with at least 85% of target regions.
ii) Filters on sample coverage: Sequencing coverage of ≥ 500X is required for tumor samples.
iii) Filtering for high confidence mutations: Raw SNV and indel calls are subjected to a series of filtering steps to ensure only high-confidence calls are reported. The filter is based on 1) variant frequency that identifies as a possible somatic mutation; 2) a known hotspot mutation; 3) technical characteristics such as total depth, variant read count, and strand bias, 4) background signal observed in in-house normal samples, and 5) not a common artefact.

The thresholds for hotspot variants are ≥ 2% variant frequency and for non-hotspot variants are ≥ 5%. There must be no evidence of strand bias, as determined by a strand bias < 0.9. The total depth which must be ≥ 35 with the variant read count which must be ≥ 5 for hotspot and

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≥ 10 for non-hotspot to be considered as a variant. The variants that had ≥ 5% mutational allele frequency in any in-house normal samples are removed. Using a machine learning model trained from manually reviewed variants, majority of artefacts are removed, while retaining variants at hotspots or on clinically significant genes for manual inspection.

The filtering scheme and threshold are shown in Figure 1 below. The threshold values for the filtering criteria were established based on mutation analysis on replicates of normal FFPE samples, and optimized to reject all false positive SNVs and almost all false positive indel calls from the reference dataset.

Figure 1. Filtering schema and threshold


f) Mutation Annotation: Variants with functional effects of point mutations, frameshifts, in-frame insertion or deletions, splice site, and splice region variants are retained. Common polymorphisms from gnomAD, and in-house normal sample database are removed.

g) Tumor Purity Analysis: The tumor purity of the sequenced sample may be different from the tumor proportion determined by the pathologists using the entire pathology slide if the sample has been subjected to macrodissection. The tumor purity of the sequenced sample is determined by the software, which considers sequencing reads information, allele frequency of single nucleotide polymorphisms, and possible genomic typing. Manual inspection is

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performed to determine tumor purity by considering pathologist-determined tumor proportion and driver mutation mutational allele frequency in cases when a limitation in the software is identified.

h) Copy Number Analysis: Copy number analysis determines the amplification status of the ERBB2 gene by measuring the read coverage of a large segment of the ERBB2 gene relative to the rest of the sequenced region using the software. It includes two steps: (1) Read coverage data is compared to data from a pooled normal baseline and normalized to arrive at a normalized coverage ratio. (2) The observed copy number is determined by assigning the ratio value for the situation when copy number is two, and then determining the copy number based on the ratio at ERBB2 gene. ERBB2 gene is reported as amplification if the observed copy number is ≥ 4.

i) Tumor Mutational Burden (TMB): TMB is calculated based on detected sequence mutations. 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).

## 6. Controls:

a) Positive control: The positive control sample (commercial vendor qualified by ACT Genomics) contains different confirmed mutations, representing a range of mutation allele frequencies. Data generated from the positive control sample is analyzed using the pipeline, and frequencies of the detected mutations are reviewed to determine if (1) the known mutations are among those called, and (2) the observed frequencies for the known mutations match their expected values within a tolerance interval covering 99.9% proportion with 95% confidence. The positive control with expected mutational variant frequency (MAF) are shown in Table 2.

Table 2. Variants in ACTOnco Positive Control

| Variant | Expected MAF |
| --- | --- |
| AKT2_chr19_40743956_G>A | 0.34 |
| BRAF_chr7_140494209_G>A | 0.24 |
| CARD11_chr7_2953038_C>T | 0.44 |
| CTNNB1_chr3_41266133_CCTT>C | 0.1 |
| FGF10_chr5_44310572_T>C | 0.3 |
| FLT3_chr13_28578214_GGA>G | 0.1 |
| KRAS_chr12_25398281_C>T | 0.15 |
| MAP2K1_chr15_66729147_C>T | 0.35 |
| MAP2K1_chr15_66782085_A>G | 0.33 |
| MSH6_chr2_48018236_G>T | 0.3 |
| NRAS_chr1_115256530_G>T | 0.12 |
| PDGFRA_chr4_55138600_G>A | 0.32 |
| RET_chr10_43604493_C>T | 0.3 |
| TET2_chr4_106182946_C>A | 0.28 |
| TSC1_chr9_135802659_C>T | 0.31 |

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**b) Negative control:** The negative control sample uses peripheral blood mononuclear cells (PBMC) from healthy population which are lacking any clinically relevant genetic alterations in target areas. The assay will interrogate 755 hotspot locations and the expected outcome should be negative for a mutational call, that is, result in a wild-type call or no mutation found.

**c) No Template Control:** DNAse, RNAse free distilled water will be used as the no template control sample, and will be included into library preparation to verify that there is no contamination of reagents. The library product of the no template control sample is analyzed by capillary electrophoresis (Fragmented Analyzer) before samples proceed to sequencing. The no template control should have no detectable major peak at 125-300bp and an undetectable concentration.

## 7. Result Reporting:

- ACTOnco results are reported out under one of the two categories: “Cancer Mutations with Evidence of Clinical Significance” or “Cancer Mutations with Potential Clinical Significance”. The two categories are based on the supporting level of clinical evidence.
- Results are reported for point mutations and small insertions and deletions of the 440 gene panel. Refer to Appendix A for a list of genes.
- The ACTOnco does not report mutations in 650 regions among 18431 interrogated target regions (528 regions due to consistently low coverage in 187 genes and 124 regions due to pseudogene in 24 genes). Refer to Appendix B for a list of excluded exons.
- ERBB2 gene is reported as amplification if the observed copy number is ≥ 4.
- TMB reporting: TMB is measured by counting the total number of mutations detected in the consensus coding region (1.12Mb) interrogated by ACTOnco assay. TMB is measured as mutations per megabase (mut/Mb), reported as a number.
- Reporting takes in account the quality metrics listed in the Table 3 below.

## 8. Quality Metrics:

Quality metrics are assessed across the following categories:

- Run (Batch) level: Metrics that are quantified per sequencing run. If the run (batch)-level metrics failed, all the samples in that run will need to be re-sequenced.
- Sample level: Metrics that are quantified per sample. If the sample-level QC failed, the sample will be flagged for inspection or rework of the library or re-sequenced.
- Variant (analyte) level: Metrics that are quantified for individual alteration types and positions, such as sequence coverage. Variants passing analyte-level QC are reported.

**Table 3. Quality Control Metrics and Assay Cutoff**

| Quality Metrics | Level of Qualification | ACTOnco Criteria |
| --- | --- | --- |
| Sequencing Output | Run (batch) level | Total output ≥ 3 Gbp |
| Positive Run Control | Run (batch) level | The observed allele frequencies for the known mutations should be within the 99.9 tolerance interval of 95% confidence of expected value. |
| Negative Run Control | Run (batch) level | The expected outcome should be negative for a mutational call, that is, result in a |

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| Quality Metrics | Level of Qualification | ACTOnco Criteria |
| --- | --- | --- |
| | | wild-type call or no mutation found in the known hotspots. |
| Base Quality (AQ Score) | Run (batch) level | Alignment quality, AQ17 (98% Accuracy) |
| Average Target Coverage | Sample level | ≥ 500X |
| Coverage Uniformity | Sample level | ≥ 85% target regions ≥ 100X coverage |
| Contamination QC | Sample level | HomRate < 0.232 |
| Hotspot SNVs and Indels Calling threshold | Variant/ Analyte level | Mutation coverage (DP) ≥ 35 |
| | | Number of mutant reads (AD) ≥ 5 |
| | Cutoff | Mutation Frequency (VF) ≥ 2% |
| Non-hotspot SNVs and Indels Calling threshold | Variant/ Analyte level | Mutation coverage (DP) ≥ 35 |
| | | Number of mutant reads (AD) ≥ 10 |
| | Cutoff | Mutation Frequency (VF) ≥ 5% |
| ERBB2 Amplification | Variant/ Analyte level | Observed copy number ≥ 4 |
| Calling test failure | Sample level | If a sample with a mean coverage < 500X or coverage uniformity < 85%, the test is deemed “failed” for the sample |

### B. Test Principle

The ACTOnco assay is a custom targeted sequencing platform, utilizing amplicon-based sequencing, to detect point mutations (single nucleotide variants, or SNVs), small insertions and deletions (Indels), ERBB2 gene amplification, and tumor mutational burden (TMB) in tumor specimens. The ACTOnco assay involves target amplification and deep sequencing of all coding exons of 440 genes. The assay uses custom DNA primers corresponding to all exons and selected introns of oncogenes, tumor suppressor genes, drug metabolism genes, and immune-related genes. An overlapping amplicon approach is utilized in which tiled primers are designed to generate multiple overlapping amplicons of the same region to avoid allele dropout. In total, the primers target approximately 1.8Mb of the human genome. Genomic DNA is extracted from FFPE tissue samples.

Sequence libraries are prepared through a multiplex polymerase chain reaction (PCR) amplification step to enrich target sequences. Target sequences are tagged with index oligonucleotide to identify individual sample and adaptor oligonucleotide to anchor the amplicon to complimentary oligonucleotides embedded on the surface of the sequencing bead. Target sequences on the sequencing beads are amplified using emulsion PCR before sequencing. Multiple barcoded sequence libraries are pooled and then sequenced; sequence reads are then aligned to the reference human genome. By comparing the identity of bases from the tumor DNA and the reference human genome, variant alterations are identified in the tumor.

### C. Determination of assay thresholds

#### 1. Requirements on exon coverage:

A power analysis was conducted to estimate the depth or the total number of reads needed to detect a mutation with a true underlying mutation allele frequency (MAF) of 2% or greater, for

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varying levels of power (0.8 to 0.99), assuming a fixed alpha (Type I error rate) of 0.05. The 95% confidence interval (CI) ranges of observed MAF as a function of sequencing depth were also calculated. The study showed that when a mutation is present at 10%, the 95% CI with a sequencing depth of 500X is expected to fall between 7.5% and 13%. When the overall coverage is 100X, the 95% CI for a mutation at 10% is estimated to fall between 5.0% and 17.6%.

To confirm these estimates, empirical data was obtained to measure the range of observed MAF for expected MAF using DNA from 20 normal tissue FFPE samples of unrelated individuals mixed in equimolar parts to create a range of SNPs with expected frequencies as low as 2.5%. A total of 890 common SNPs were considered for this experiment.

A boxplot in Figure 2 shows the observed mutation frequencies for the 890 common SNPs genotyped in the pooled DNA sample binned by their true underlying mutation frequency. The empirical data showed a consistent correlation between expected and observed MAF (Pearson's r = 0.99) with a slope of 0.99 and an intercept of 0.001. The results demonstrated that an observed VF range from 8.3% to 15.6% for an SNP with a true underlying mutation frequency of 10% when the mean depth of the sample was 1056X. This range in values is roughly in line with what the theoretical statistical assessment for a depth of 500X (7.5% to 13.0%). This data provided support for using 5% as the lower limit for reporting mutations detected with a true underlying frequency of 10%. Furthermore, for the variant with an expected MAF equal to 5%, the lower bound of observed variant frequency is 2%, suggesting that an observed MAF of 2% can be used to detect actual 5% variants.

Figure 2. Observed vs Expected Variant Frequency


## 2. Requirements on sample coverage:

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Eighteen normal FFPE samples were profiled using ACTOnco to generate summary statistics across all target exons. The mean coverage across all amplicons was 1102X (640X-1445X, SD=230X). The percentage of >100X amplicon coverage was 92% (90-93%, SD=0.8%). When the amplicons were mapped to the exon level, the mean coverage across all targeted exons was 1121X (647X-1472X, SD=232X). Summary statistics were also computed based on coverage values per exon normalized by per-sample coverage. There were exons that presented with consistently low coverage values. None of the exons of the genes in clinical validation are among those with consistently low coverage. It was determined the low coverage was due to sequence similarity with other loci, poor amplification efficiency, and high GC content. The exons with consistently low coverage were removed from the ACTOnco assay.

Sequence coverage was further evaluated to establish minimum criteria for the analysis and reporting of variants. A 100X minimum coverage threshold per exon is required based on the power calculation, which showed 100X coverage was necessary to call mutations with true underlying mutation frequency of 10% or greater, with 95% power at an alpha level of 0.05. Of the remaining exons across all genes, 99.1% (97.7-99.7%, SD=0.5%) were sequenced to a depth of 100X or greater. The distribution of the mean coverage value and median coverage value for the targeted exon were shown in the below Figure 3.

**Figure 3. Distribution of mean and median coverage values for targeted regions of ACTOnco test using 18 samples with higher coverage. Dashed line indicates 100X coverage.**


A second set of 20 samples with lower sequencing depth was evaluated using the same analysis. The mean coverage across all amplicons was 572X (471X-637X, SD=46X). The percentage of >100X amplicon coverage was 91.1% (87.9-92.7%, SD=1.1%). When the amplicons were mapped to the exon level, the mean coverage across all targeted exons was 616X (495X-695X, SD=54X). With the same remaining exons across all genes, 97.8% (97.1-98.9%, SD=0.5%) were sequenced to a depth of 100X or greater. The distribution of the mean coverage value and median coverage value for the targeted exon were shown in the below Figure 4.

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Figure 4. Distribution of mean and median coverage values for targeted regions of ACTOnco test using 20 samples with lower coverage. Dashed line indicates 100X coverage.


Based on the two studies above, the threshold of a minimum of 85% of amplicon must have 100X was established for the ACTOnco test.

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

Variant calling parameters such as sequence coverage (allele depth, AO), variant coverage (variant read, DP) and strand bias (SB) were assessed as filters for specificity while maintaining the ability to detect true positive calls. Thresholds were established to ensure specificity is maintained at targeted MAF levels for reporting. Mutation thresholds were established at 2% and 5% for sequence mutations based on a categorization with somatic hotspots and non-hotspots positions.

The cutoffs for AO, DP, and SB for hotspot variants and non-hotspot variants were established using two development studies. The first study was designed to select the best parameter cutoff combination using a dataset of 17 ACTOnco run using a Reference Standard. Three parameters each were evaluated with multiple cutoffs to generate a total of 6888 combinations. A list of 3844 positive variants and 9146 negative variants was used to evaluate the performance of each parameter combination. The cutoff combination which generates the best positive predictive value was selected. The second study aimed to determine the cutoff for variant read count. The study used 18 non-cancer FFPE samples and evaluate a total of 858 hotspot variants and 2359 non-hotspot variants at different variant read cutoffs. The lowest read count which can filter out >99.5% of noise variants (5 for hotspot variants, 10 reads for non-hotspot variants) was selected as the cutoff.

### D. Substantial Equivalence Information:

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

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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):**

**Table 4. Similarities between the predicate and subject devices**

| Characteristic | Predicate MSK-IMPACT (DEN170058) | Subject Device ACTOnco IVD |
| --- | --- | --- |
| 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 ACTOnco IVD assay is an in vitro diagnostic test that uses targeted next generation sequencing of formalin-fixed, paraffin-embedded tumor tissue from patients with solid malignant neoplasms to detect genetic alterations in a broad multi gene panel. The test is intended to provide information on point mutations, small insertions and deletions, ERBB2 amplification, and tumor mutational burden 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. ACTOnco IVD is a single-site assay performed at ACT Genomics. |
| **Specimen Types** | Formalin-fixed, paraffin-embedded (FFPE) tumor tissue with matched 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 |
| **Assay cut-off** | MSK-IMPACT does not report mutations below 2% for known hotspot mutations and 5% for non-hotspot mutations. | Same |
| **Laboratory / Test Environment** | Single-site assay | Same |

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**Table 5. Differences between the predicate and subject devices**

| Characteristic | Predicate MSK-IMPACT (DEN170058) | Subject Device ACTOnco IVD |
| --- | --- | --- |
| Differences | | |
| **Sequencing Instrument** | Illumina HiSeq™ 2500 Sequencer | Thermo Fisher Ion GeneStudio™ S5 Prime System |
| **Target Enrichment Technology** | Hybrid Capture | Amplicon |
| **Genes on Panel** | 468 | 440 |
| **Black List** | 73 exons | 650 amplicons within 199 genes excluded from reporting SNV/ indels due to pseudo gene or consistently low coverage (≤ 35x). |
| **Variant Type** | Intended to provide information on somatic mutations (point mutations and small insertions and deletions), and microsatellite instability. | Intended to provide information on somatic mutations (point mutations, small insertions and deletions), ERBB2 gene amplification, and TMB. |
| **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%) | - Based on ≥ 500x target coverage; - 100x for ≥85% target regions; - hotspot mutation calling threshold (mutation coverage (DP) ≥ 35, mutant reads (AD) ≥ 5, strand bias (SB) < 0.9, mutation frequency (VF) ≥ 2%), and non-hotspot mutation threshold (DP ≥ 35, AD ≥ 10, SB < 0.9, VF ≥ 5%). |
| **Controls** | - Positive control - Negative control - No template control (NTC) - Matched normal | - Positive control - Negative control - No template control (NTC) - Normalized to a baseline established using pooled normal samples |
| **Clinical Evidence Curation** Oncopanel results are reported under one of these two categories: • “Cancer Mutations with Evidence of | 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. | A variant interpretation summary is generated, which includes biologic impact, variant specific effect and therapeutic relevance curated from professional guidelines and recommendations, therapeutic labeling, disease specific expert and advocacy group recommendations, and medical literature. Classification criteria were developed by ACT Genomics with the reference of AMP guideline, to communicate |

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| Characteristic | Predicate MSK-IMPACT (DEN170058) | Subject Device ACTOnco IVD |
| --- | --- | --- |
| Differences | | |
| Clinical Significance” or • “Cancer Mutations with Potential Clinical Significance.” | OncoKB undergoes periodic updates through the review of new information by a panel of experts. | the level of clinical evidence available for individual mutations in the test report. ACT Genomics undergoes periodic updates through the review of new information by medical informatics scientists and scientific content management team. |

### E. Standards/Guidance Documents Referenced:

The following FDA guidance documents were consulted:

1. (1) eCopy Program for Medical Device Submissions; Guidance for Industry and Food and Drug Administration Staff (December 3, 2015);
2. (2) Refuse to Accept Policy for 510(k)s; Guidance for Industry and Food and Drug Administration Staff (February 21, 2019);
3. (3) Guidance for the Content of Premarket Submissions for Software Contained in Medical Devices; Guidance for Industry and FDA Staff (May 11, 2005);
4. (4) Format for Traditional and Abbreviated 510(k)s – Guidance for Industry and FDA Staff (August 12, 2005);
5. (5) Off-The-Shelf Software Use in Medical Devices; Guidance for Industry, FDA Reviewers, and Compliance (September 9, 1999);
6. (6) User Protocol for Evaluation of Qualitative Test Performance; Approved Guideline - Second Edition. CLSI EP12-A2
7. (7) Evaluation Of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline - Second Edition. CLSI EP17-A2
8. (8) Interference Testing in Clinical Chemistry. CLSI EP07 3rd Edition
9. (9) Evaluation Of Precision of Quantitative Measurement Procedures; Approved Guideline - Third Edition. CLSI EP05
10. (10) Medical Devices - Applications of Risk Management to Medical Devices. ISO 14971: 2019
11. (11) Medical Device Software - Software Life Cycle Processes. IEC 62304 Edition 1.1 2015-06 Consolidated Version

### F. Performance Characteristics:

#### 1. Analytical Performance- General

The ACTOnco assay is a targeted NGS panel with 440 genes. The targeted regions of interest in ACTOnco are designed to detect SNVs, deletions up to 19bp and insertions up to 12bp in length of the targeted genes, as well as ERBB2 amplifications, 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 indicated

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with this assay. In addition, the assay was evaluated for performance regarding the panel wide quality metrics.

# **a) Invalid Rates**

The invalid rates across multiple tumor types obtained from historical data was evaluated with 1526 FFPE clinical specimens tested with the ACTOnco assay. The data shows the separate invalid rates for the different steps involved in the assay workflow including the percentage of specimens with insufficient tumor, DNA integrity and yield, the percentage with failed library prep and the percentage that failed the sequencing run per cancer type (Table 6). The overall (Pre and Post-run) invalid rates ranged from 0% to 26% principally due to insufficient tumor and insufficient DNA yield related to the specimen source. The data shows that the DNA extraction is valid across tumor types and interference effects from different specimens are not significant across different tumor types supporting the performance of the pan-tumor specimen handling.

**Table 6. Invalid Rates for 27 FFPE Tumor Types.**

| Cancer type | Number of sample received | Pre-Run Invalids | Pre-Run Invalids | Pre-Run Invalids | Post-Run Invalids | Overall Invalid rate (Pre + Post) |
| --- | --- | --- | --- | --- | --- | --- |
| | | Insufficient Tumor (< 10%) | Insufficient DNA Integrity (DNA CE < 15%) | Insufficient DNA Yield (< 80ng) | Sequencing Failure (Target base coverage at 100x <85%) | |
| Adenocarcinoma | 13 | 1 | 0 | 1 | 0 | 15.38% |
| Brain cancer | 29 | 0 | 0 | 3 | 0 | 10.34% |
| Breast cancer | 160 | 10 | 7 | 9 | 0 | 16.25% |
| Cervical cancer | 22 | 0 | 3 | 0 | 0 | 13.64% |
| Cholangiocarcinoma | 65 | 6 | 0 | 1 | 0 | 10.77% |
| Colon cancer | 158 | 19 | 6 | 2 | 0 | 17.09% |
| Esophageal cancer | 31 | 1 | 1 | 0 | 0 | 6.45% |
| Gastric cancer | 44 | 5 | 1 | 1 | 0 | 15.91% |
| Gastrointestinal stromal tumor | 9 | 0 | 2 | 0 | 0 | 22.22% |
| Glioblastoma Multiforme | 39 | 0 | 0 | 1 | 0 | 2.56% |
| Head and Neck Cancer | 20 | 0 | 0 | 1 | 0 | 5.00% |
| Hepatocellular carcinoma | 31 | 1 | 3 | 1 | 0 | 16.13% |
| Kidney cancer | 16 | 1 | 0 | 1 | 0 | 12.50% |
| Lung cancer | 303 | 36 | 5 | 28 | 0 | 22.77% |
| Melanoma | 9 | 0 | 0 | 0 | 0 | 0.00% |
| Neuroendocrine carcinoma | 18 | 1 | 1 | 0 | 0 | 11.11% |
| Oral cancer | 20 | 1 | 0 | 0 | 0 | 5.00% |
| Ovarian cancer | 140 | 15 | 6 | 0 | 0 | 15.00% |
| Pancreatic cancer | 142 | 23 | 2 | 12 | 0 | 26.06% |
| Peritoneal cancer | 10 | 0 | 0 | 1 | 0 | 10.00% |
| Prostate cancer | 21 | 0 | 0 | 2 | 0 | 9.52% |
| Sarcoma | 80 | 4 | 3 | 2 | 0 | 11.25% |
| Small bowel cancer | 9 | 0 | 0 | 2 | 0 | 22.22% |
| Thymic cancer | 13 | 2 | 1 | 0 | 0 | 23.08% |

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| Thyroid cancer | 9 | 0 | 1 | 0 | 0 | 11.11% |
| --- | --- | --- | --- | --- | --- | --- |
| Unknown primary | 25 | 5 | 0 | 0 | 0 | 20.00% |
| Urothelial cancer | 25 | 3 | 1 | 0 | 0 | 16.00% |
| Uterine cancer | 65 | 2 | 2 | 2 | 2 | 12.31% |
| Total | 1526 | 136 | 45 | 70 | 2 | 16.58% |

## 2. Precision

### a) Precision Panel

The within lab precision (between-run and within-run) of the ACTOnco assay was assessed using 20 FFPE clinical samples (12 single samples across 10 tumor types and 8 mixed samples across 4 tumor types) to represent different tumor types, different variants, and the range of frequencies. Extracted DNA from each of the 20 samples was tested in duplicate by 2 different operators on 2 instruments across 2 non- consecutive start days using 3 kit lots, yielding 48 replicates per sample. For each replicate tested, all observed mutations which passed the ACTOnco assay QC metrics were reported and assessed for precision. The single samples or mixed samples with known mutations corresponding to FDA level 2 variants (mutations with evidence of clinical significance) and level 3 variants (mutation with evidence of potential clinical significance) are shown in Table 7 and Table 8, respectively. Among the 12 single samples, one sample does not contain any variants with FDA level 2 or level 3 and is not listed in Table 7.

Table 7. Summary of variants by tissue type within 12 single samples

| Sample | Tissue type | Mutation type | Gene/Exon | cDNA change | Amino acid change | Approximate MAF |
| --- | --- | --- | --- | --- | --- | --- |
| Sample 1 | Colon adenocarcinoma | SNV | BRAF exon15 | 140453136_A>T | p.V600E | 29.06% |
| Sample 1 | Colon adenocarcinoma | SNV | GRIN2A exon4 | 10032000_C>A | p.G275* | 50.66% |
| Sample 1 | Colon adenocarcinoma | DEL | KDM5C exon23 | 53223787_CCA>C | p.C1190fs | 58.95% |
| Sample 1 | Colon adenocarcinoma | INS | KMT2D exon34 | 49432235_C>CG | p.S2969fs | 24.81% |
| Sample 1 | Colon adenocarcinoma | DEL | MAP3K1 exon15 | 56179358_CAG>C | p.E1225fs | 27.16% |
| Sample 1 | Colon adenocarcinoma | SNV | RECQL4 exon5 | 145741926_G>A | p.R193* | 19.53% |
| Sample 1 | Colon adenocarcinoma | DEL | RNF43 exon9 | 56435160_AC>A | p.G659fs | 70.63% |
| Sample 1 | Colon adenocarcinoma | DEL | SPOP exon8 | 47685274_TA>T | p.F225fs | 28.11% |
| Sample 1 | Colon adenocarcinoma | SNV | TP53 exon11 | 7572929_A>G | p.*394Rext*9 | 28.98% |
| Sample 1 | Colon adenocarcinoma | SNV | TP53 exon5 | 7578554_A>G | p.Y126H | 19.00% |
| Sample 2 | Kidney cancer | SNV | NSD1 exon19 | 176709523_C>T | p.R1984* | 8.28% |
| Sample 2 | Kidney cancer | SNV | SETD2 exon3 | 47162711_T>A | p.K1139* | 19.24% |
| Sample 3 | Tumor of exocrine pancreas | SNV | IDH1 exon4 | 209113112_C>G | p.R132P | 36.71% |

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| Sample | Tissue type | Mutation type | Gene/Exon | cDNA change | Amino acid change | Approximate MAF |
| --- | --- | --- | --- | --- | --- | --- |
| Sample 3 | Tumor of exocrine pancreas | SNV | KRAS exon2 | 25398284_C>T | p.G12D | 32.13% |
| Sample 3 | Tumor of exocrine pancreas | SNV | TP53 exon7 | 7577539_G>A | p.R248W | 53.06% |
| Sample 4 | Liver cancer | SNV | ARID2 exon5 | 46211626_A>T | p.K198* | 30.76% |
| Sample 4 | Liver cancer | DEL | ARID2 exon15 | 46245542_TC>T | p.P1213fs | 16.36% |
| Sample 4 | Liver cancer | SNV | TP53 exon6 | 7578224_T>A | p.R209* | 40.45% |
| Sample 5 | Cholangiocarcinoma | SNV | KRAS exon2 | 25398284_C>T | p.G12D | 20.98% |
| Sample 5 | Cholangiocarcinoma | SNV | TP53 exon6 | 7578190_T>C | p.Y220C | 31.86% |
| Sample 6 | Colon adenocarcinoma | DEL | B2M exon2 | 45007740_GGA>G | p.R65fs | 17.84% |
| Sample 6 | Colon adenocarcinoma | SNV | CTNNB1 exon3 | 41266137_C>T | p.S45F | 35.38% |
| Sample 6 | Colon adenocarcinoma | SNV | ERBB2 exon19 | 37880220_T>C | p.L755S | 18.69% |
| Sample 6 | Colon adenocarcinoma | SNV | GNAS exon6 | 57480483_C>T | p.R160C | 17.13% |
| Sample 6 | Colon adenocarcinoma | SNV | KRAS exon2 | 25398285_C>T | p.G12S | 17.30% |
| Sample 6 | Colon adenocarcinoma | SNV | MLH1 exon4 | 37045935_C>T | p.T117M | 66.43% |
| Sample 6 | Colon adenocarcinoma | DEL | NBN exon10 | 90967511_CT>C | p.R466fs | 32.29% |
| Sample 6 | Colon adenocarcinoma | DEL | PIK3R1 exon3 | 67569265_GC>G | p.P129fs | 16.39% |
| Sample 6 | Colon adenocarcinoma | SNV | RAF1 exon7 | 12645694_A>T | p.S259T | 17.54% |
| Sample 7 | Endometrial cancer | SNV | AKT1 exon4 | 105243045_A>G | p.W80R | 25.22% |
| Sample 7 | Endometrial cancer | DEL | EZH2 exon10 | 148515024_TC>T | p.G395fs | 25.03% |
| Sample 7 | Endometrial cancer | SNV | KRAS exon2 | 25398284_C>T | p.G12D | 40.10% |
| Sample 7 | Endometrial cancer | DEL | MAP2K4 exon4 | 11998922_CA>C | p.K143fs | 24.62% |
| Sample 7 | Endometrial cancer | SNV | PAX5 exon2 | 37020768_A>C | p.V26G | 27.27% |
| Sample 7 | Endometrial cancer | SNV | PIK3CA exon2 | 178916876_G>A | p.R88Q | 25.20% |
| Sample 7 | Endometrial cancer | SNV | PIK3CA exon8 | 178928079_G>A | p.E453K | 28.15% |
| Sample 7 | Endometrial cancer | DEL | TP53 exon8 | 7577143_CAGT>C | p.L265del | 27.08% |
| Sample 8 | Urinary system cancer | SNV | ERBB2 exon8 | 37868208_C>A | p.S310Y | 21.48% |
| Sample 9 | Endometrial cancer | SNV | ARID1A exon18 | 27101135_C>T | p.Q1473* | 44.33% |
| Sample 9 | Endometrial cancer | DEL | KDM6A exon17 | 44928975_AG>A | p.A694fs | 43.77% |

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| Sample | Tissue type | Mutation type | Gene/Exon | cDNA change | Amino acid change | Approximate MAF |
| --- | --- | --- | --- | --- | --- | --- |
| Sample 9 | Endometrial cancer | SNV | PIK3CA exon2 | 178916890_C>T | p.R93W | 44.63% |
| Sample 9 | Endometrial cancer | SNV | PIK3CA exon10 | 178936092_A>G | p.E545G | 45.45% |
| Sample 9 | Endometrial cancer | SNV | PTEN exon5 | 89692905_G>A | p.R130Q | 88.18% |
| Sample 9 | Endometrial cancer | DEL | RNF43 exon9 | 56435160_AC>A | p.G659fs | 48.78% |
| Sample 11 | Lung cancer | DEL | CDKN2A exon2 | 21971147_TGGGCTCCGCGCCGTGGA>T | p.L65fs | 18.53% |
| Sample 11 | Lung cancer | DEL | CDKN2A exon2 | 21971176_TCCGCCACTCGGGCG>T | p.S56fs | 6.41% |
| Sample 11 | Lung cancer | SNV | PIK3CA exon21 | 178952085_A>G | p.H1047R | 19.21% |
| Sample 11 | Lung cancer | INS | TP53 exon6 | 7578186_C>CT | p.P222fs | 26.73% |
| Sample 12 | Skin cancer | SNV | BRAF exon15 | 140453136_A>T | p.V600E | 52.96% |
| Sample 12 | Skin cancer | SNV | CDKN2A exon2 | 21971179_G>C | p.A60G | 46.91% |
| Sample 12 | Skin cancer | SNV | ERBB4 exon14 | 212537975_G>A | p.R544W | 24.89% |
| Sample 12 | Skin cancer | SNV | PIK3R1 exon13 | 67591106_A>G | p.K567E | 16.22% |
| Sample 12 | Skin cancer | SNV | RAC1 exon2 | 6426893_C>T | p.P29L | 34.31% |
| Sample 12 | Skin cancer | SNV | SMARCA4 exon26 | 11141519_C>T | p.Q1166* | 27.56% |

Table 8. Summary of variants by tissue type within 8 mixed sample

| Sample | Tissue type | Mutation type | Gene/Exon | cDNA change | Amino Acid change | Approximate MAF |
| --- | --- | --- | --- | --- | --- | --- |
| Sample 13 | Lung cancer | SNV | CTNNB1 exon3 | 41266101_C>T | p.S33F | 11.72% |
| Sample 13 | Lung cancer | SNV | EGFR exon18 | 55241708_G>C | p.G719A | 10.32% |
| Sample 13 | Lung cancer | SNV | EGFR exon20 | 55249071_C>T | p.T790M | 11.41% |
| Sample 13 | Lung cancer | SNV | EGFR exon21 | 55259515_T>G | p.L858R | 11.17% |
| Sample 13 | Lung cancer | SNV | SMARCA4 exon32 | 11169037_A>T | p.K1511* | 13.94% |
| Sample 13 | Lung cancer | SNV | TP53 exon8 | 7577106_G>C | p.P278A | 11.51% |
| Sample 14 | Lung cancer | SNV | B2M exon1 | 45003746_T>C | p.M1? | 7.48% |
| Sample 14 | Lung cancer | INS | B2M exon2 | 45007890_G>GT | p.K114* | 7.43% |
| Sample 14 | Lung cancer | SNV | BRAF exon15 | 140453136_A>T | p.V600E | 12.41% |
| Sample 14 | Lung cancer | SNV | EGFR exon18 | 55241708_G>C | p.G719A | 5.41% |
| Sample 14 | Lung cancer | SNV | EGFR exon20 | 55249071_C>T | p.T790M | 6.04% |
| Sample 14 | Lung cancer | SNV | EGFR exon21 | 55259469_G>A | p.V843I | 6.40% |

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| Sample | Tissue type | Mutation type | Gene/Exon | cDNA change | Amino Acid change | Approximate MAF |
| --- | --- | --- | --- | --- | --- | --- |
| Sample 14 | Lung cancer | SNV | EGFR exon21 | 55259515_T>G | p.L858R | 6.37% |
| Sample 14 | Lung cancer | SNV | SMARCA4 exon32 | 11169037_A>T | p.K1511* | 6.99% |
| Sample 14 | Lung cancer | SNV | TP53 exon8 | 7577106_G>C | p.P278A | 5.43% |
| Sample 15 | Breast cancer | SNV | PIK3CA exon21 | 178952085_A>G | p.H1047R | 16.57% |
| Sample 15 | Breast cancer | SNV | TP53 exon7 | 7577539_G>A | p.R248W | 17.03% |
| Sample 16 | Breast cancer | SNV | PIK3CA exon21 | 178952085_A>G | p.H1047R | 11.32% |
| Sample 16 | Breast cancer | SNV | TP53 exon7 | 7577539_G>A | p.R248W | 10.29% |
| Sample 16 | Breast cancer | SNV | TP53 exon7 | 7577556_C>T | p.C242Y | 33.94% |
| Sample 17 | Skin cancer | SNV | ARID1B exon3 | 157222648_C>T | p.Q626* | 5.96% |
| Sample 17 | Skin cancer | SNV | BRAF exon15 | 140453136_A>T | p.V600E | 7.95% |
| Sample 17 | Skin cancer | SNV | BRAF exon11 | 140481397_C>A | p.V471F | 20.06% |
| Sample 17 | Skin cancer | SNV | CARD11 exon6 | 2979559_C>T | p.D230N | 19.56% |
| Sample 17 | Skin cancer | SNV | FANCA exon27 | 89833576_G>C | p.S858R | 7.10% |
| Sample 17 | Skin cancer | SNV | NF1 exon12 | 29533315_C>T | p.R440* | 23.72% |
| Sample 17 | Skin cancer | SNV | NOTCH4 exon4 | 32188899_G>A | p.Q219* | 5.58% |
| Sample 17 | Skin cancer | SNV | NRAS exon3 | 115256529_T>C | p.Q61R | 8.71% |
| Sample 17 | Skin cancer | DEL | TP53 exon4 | 7579546_CG>C | p.P47fs | 6.82% |
| Sample 18 | Skin cancer | SNV | BRAF exon15 | 140453136_A>T | p.V600E | 2.02% |
| Sample 18 | Skin cancer | SNV | BRAF exon11 | 140481397_C>A | p.V471F | 5.56% |
| Sample 18 | Skin cancer | MNV | DNMT3A exon16 | 25466800_GG>AA | p.R635W | 26.56% |
| Sample 18 | Skin cancer | SNV | EZH2 exon16 | 148508728_A>T | p.Y646N | 24.10% |
| Sample 18 | Skin cancer | SNV | NF1 exon12 | 29533315_C>T | p.R440* | 7.87% |
| Sample 18 | Skin cancer | SNV | NRAS exon3 | 115256529_T>A | p.Q61L | 28.18% |
| Sample 18 | Skin cancer | SNV | NRAS exon3 | 115256529_T>C | p.Q61R | 2.31% |
| Sample 18 | Skin cancer | SNV | TP53 exon8 | 7577099_C>T | p.R280K | 36.85% |
| Sample 19 | Urinary system cancer | INS | ARID1A exon9 | 27092804_A>AC | p.Q944fs | 12.90% |
| Sample 19 | Urinary system cancer | SNV | RXRA exon10 | 137328351_C>T | p.S427F | 9.84% |
| Sample 20 | Urinary system cancer | INS | ARID1A exon9 | 27092804_A>AC | p.Q944fs | 8.05% |
| Sample 20 | Urinary system cancer | SNV | RXRA exon10 | 137328351_C>T | p.S427F | 6.52% |

### b) Panel-wide precision for SNVs and Indels

The precision was assessed for all detected mutations across 48 replicates for each sample. The positive and negative call rates were calculated based on the total number of mutations along with the two-sided 95% CI. Table 9 summarizes the positive and negative call rates rolled up (mutational variant type, operator, instrument, reagent lot, and days). The overall positive call rate was 98.3% across all samples and replicates (35,308/ 35,906, 98.2%-98.5% CI). The

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overall negative call rate was 99.997 % across all samples and replicates (723,628/ 723,648, 99.996%-99.998% CI).

**Table 9. Correct Calls for SNVs, Insertions and Deletions**

| Mutational Variant Type | Operator | Instrument | Reagent lot | Days | Number of correct calls* | Number Attempted | Call rate, % (95%CI) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| All | All | All | All | All | 35308 | 35906 | 98.3 (98.2-98.5) |
| Deletion | All | All | All | All | 1996 | 2010 | 99.3 (98.8-99.6) |
| Insertion | All | All | All | All | 512 | 528 | 97.0 (95.1-98.1) |
| MNV | All | All | All | All | 963 | 991 | 97.2 (95.9-98.0) |
| SNV | All | All | All | All | 31837 | 32377 | 98.3 (98.2-98.5) |
| Negative (WT) | All | All | All | All | 723628 | 723648 | 99.997 (99.996-99.998) |

* Correct calls may be positive (mutation present) or negative (wild type present). A correct call was defined as the same mutational variant call with each of the observations of a sample with that variant.

In addition to the mutations with FDA level (as listed in Table 7 and Table 8), other mutations identified in each specimen in any of the test replicates were evaluated (n = 751) with 48 replicates each. Amongst the 751 mutations, 662 mutations had 100% correct calls, 37/751 mutations had ≥ 90% correct calls, 32/751 mutations had ≥ 50% correct calls, and only 5/751 mutations had < 50% correct calls. Note the ≤ 50% correct calls were for variants where the mean MAF was at their respective cutoff (2 or 5%). There were 15/751 mutations with 100% correct call but the total observations were not 48 due to loss of observations due to assay QC.

The coefficient of variance (CV) for the MAF was also calculated for all replicates. Two hundred and eighty-three (283) of the 386 (73%) mutations in the single samples had a percent CV ≤ 10%, 88 of 386 (23%) were between 10 and 20%, and 15 of 386 (4%) were > 20%. In the mixed samples, 133/365 (37%) had a percent CV ≤ 10%, 195/365 (53%) were between 10 and 20%, and 37/365 (10%) were > 20%.

Positive call rates for known mutations with FDA level 2 or level 3 listed in Table 7 and Table 8 are summarized in Table 10 and Table 11, respectively.

**Table 10. Summary of the Positive Call Rate within the 12 Single Samples**

| Gene/ Exon | Mutation (cDNA/Protein Changes) | NC range* | MAF range | MAF Mean | MAF Median | MAF SD | MAF %CV | Positive /Total Calls | Positive Call Rate (two-sided 95% CI) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| BRAF exon15 | 140453136_A>T p.V600E | 0.93-0.96 | 0.255 - 0.331 | 0.29 | 0.29 | 0.02 | 6.5 | 48/48 | 100.0% (92.6%,100.0%) |
| KRAS exon2 | 25398285_C>T p.G12S | 0.93-0.96 | 0.149 - 0.203 | 0.17 | 0.17 | 0.01 | 7.19 | 48/48 | 100.0% (92.6%,100.0%) |
| BRAF exon15 | 140453136_A>T p.V600E | 0.91-0.95 | 0.475 - 0.568 | 0.53 | 0.53 | 0.02 | 3.62 | 48/48 | 100.0% (92.6%,100.0%) |

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| Gene/ Exon | Mutation (cDNA/Protein Changes) | NC range* | MAF range | MAF Mean | MAF Median | MAF SD | MAF %CV | Positive /Total Calls | Positive Call Rate (two-sided 95% CI) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| GRIN2A exon4 | 10032000_C>A p.G275* | 0.93-0.96 | 0.48 - 0.534 | 0.51 | 0.51 | 0.01 | 2.68 | 48/48 | 100.0% (92.6%,100.0%) |
| KDM5C exon23 | 53223787_CCA>C p.C1190fs | 0.93-0.96 | 0.531 - 0.649 | 0.59 | 0.59 | 0.02 | 4.14 | 48/48 | 100.0% (92.6%,100.0%) |
| KMT2D exon34 | 49432235_C>CG p.S2969fs | 0.93-0.96 | 0.222 - 0.275 | 0.25 | 0.25 | 0.01 | 5.38 | 48/48 | 100.0% (92.6%,100.0%) |
| MAP3K1 exon15 | 56179358_CAG>C p.E1225fs | 0.93-0.96 | 0.22 - 0.313 | 0.27 | 0.28 | 0.02 | 8.1 | 48/48 | 100.0% (92.6%,100.0%) |
| RECQL4 exon5 | 145741926_G>A p.R193* | 0.93-0.96 | 0.173 - 0.222 | 0.2 | 0.2 | 0.01 | 5.68 | 48/48 | 100.0% (92.6%,100.0%) |
| RNF43 exon9 | 56435160_AC>A p.G659fs | 0.93-0.96 | 0.58 - 0.885 | 0.71 | 0.7 | 0.07 | 9.38 | 48/48 | 100.0% (92.6%,100.0%) |
| SPOP exon8 | 47685274_TA>T p.F225fs | 0.93-0.96 | 0.228 - 0.329 | 0.28 | 0.28 | 0.02 | 8.77 | 48/48 | 100.0% (92.6%,100.0%) |
| TP53 exon11 | 7572929_A>G p.*394Rext*9 | 0.93-0.96 | 0.222 - 0.348 | 0.29 | 0.29 | 0.03 | 9.77 | 48/48 | 100.0% (92.6%,100.0%) |
| TP53 exon5 | 7578554_A>G p.Y126H | 0.93-0.96 | 0.151 - 0.224 | 0.19 | 0.19 | 0.01 | 7.77 | 48/48 | 100.0% (92.6%,100.0%) |
| NSD1 exon19 | 176709523_C>T p.R1984* | 0.9-0.95 | 0.055 - 0.115 | 0.08 | 0.08 | 0.01 | 15.54 | 48/48 | 100.0% (92.6%,100.0%) |
| SETD2 exon3 | 47162711_T>A p.K1139* | 0.9-0.95 | 0.15 - 0.246 | 0.19 | 0.19 | 0.02 | 11.78 | 48/48 | 100.0% (92.6%,100.0%) |
| IDH1 exon4 | 209113112_C>G p.R132P | 0.93-0.96 | 0.294 - 0.428 | 0.37 | 0.37 | 0.03 | 7.85 | 48/48 | 100.0% (92.6%,100.0%) |
| KRAS exon2 | 25398284_C>T p.G12D | 0.93-0.96 | 0.259 - 0.369 | 0.32 | 0.32 | 0.02 | 5.6 | 48/48 | 100.0% (92.6%,100.0%) |
| TP53 exon7 | 7577539_G>A p.R248W | 0.93-0.96 | 0.481 - 0.58 | 0.53 | 0.53 | 0.02 | 4.68 | 48/48 | 100.0% (92.6%,100.0%) |
| ARID2 exon5 | 46211626_A>T p.K198* | 0.91-0.96 | 0.244 - 0.358 | 0.31 | 0.3 | 0.03 | 8.32 | 48/48 | 100.0% (92.6%,100.0%) |
| ARID2 exon15 | 46245542_TC>T p.P1213fs | 0.91-0.96 | 0.096 - 0.238 | 0.16 | 0.17 | 0.02 | 13.66 | 48/48 | 100.0% (92.6%,100.0%) |
| TP53 exon6 | 7578224_T>A p.R209* | 0.91-0.96 | 0.338 - 0.456 | 0.4 | 0.4 | 0.03 | 6.25 | 48/48 | 100.0% (92.6%,100.0%) |
| KRAS exon2 | 25398284_C>T p.G12D | 0.92-0.95 | 0.171 - 0.246 | 0.21 | 0.21 | 0.02 | 8.1 | 48/48 | 100.0% (92.6%,100.0%) |
| TP53 exon6 | 7578190_T>C p.Y220C | 0.92-0.95 | 0.225 - 0.418 | 0.32 | 0.32 | 0.05 | 14.64 | 48/48 | 100.0% (92.6%,100.0%) |
| B2M exon2 | 45007740_GGA>G p.R65fs | 0.93-0.96 | 0.116 - 0.208 | 0.18 | 0.18 | 0.02 | 9.46 | 48/48 | 100.0% (92.6%,100.0%) |
| CTNNB1 exon3 | 41266137_C>T p.S45F | 0.93-0.96 | 0.315 - 0.396 | 0.35 | 0.35 | 0.02 | 5.25 | 48/48 | 100.0% (92.6%,100.0%) |
| ERBB2 exon19 | 37880220_T>C p.L755S | 0.93-0.96 | 0.15 - 0.24 | 0.19 | 0.19 | 0.02 | 9.04 | 48/48 | 100.0% (92.6%,100.0%) |
| GNAS exon6 | 57480483_C>T p.R160C | 0.93-0.96 | 0.136 - 0.208 | 0.17 | 0.17 | 0.02 | 9.41 | 48/48 | 100.0% (92.6%,100.0%) |
| MLH1 exon4 | 37045935_C>T p.T117M | 0.93-0.96 | 0.618 - 0.726 | 0.66 | 0.66 | 0.02 | 3.6 | 48/48 | 100.0% (92.6%,100.0%) |
| NBN exon10 | 90967511_CT>C p.R466fs | 0.93-0.96 | 0.029 - 0.675 | 0.32 | 0.32 | 0.09 | 28.74 | 46/47 | 97.9% (88.9%,99.6%) |

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| Gene/ Exon | Mutation (cDNA/Protein Changes) | NC range* | MAF range | MAF Mean | MAF Median | MAF SD | MAF %CV | Positive /Total Calls | Positive Call Rate (two-sided 95% CI) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| PIK3R1 exon3 | 67569265_GC>G p.P129fs | 0.93-0.96 | 0.122 - 0.213 | 0.16 | 0.16 | 0.02 | 13.2 | 48/48 | 100.0% (92.6%,100.0%) |
| RAF1 exon7 | 12645694_A>T p.S259T | 0.93-0.96 | 0.14 - 0.234 | 0.18 | 0.17 | 0.02 | 11.01 | 48/48 | 100.0% (92.6%,100.0%) |
| AKT1 exon4 | 105243045_A>G p.W80R | 0.93-0.96 | 0.215 - 0.309 | 0.25 | 0.25 | 0.02 | 7.93 | 48/48 | 100.0% (92.6%,100.0%) |
| EZH2 exon10 | 148515024_TC>T p.G395fs | 0.93-0.96 | 0.202 - 0.297 | 0.25 | 0.26 | 0.02 | 8.5 | 48/48 | 100.0% (92.6%,100.0%) |
| KRAS exon2 | 25398284_C>T p.G12D | 0.93-0.96 | 0.359 - 0.436 | 0.4 | 0.4 | 0.02 | 3.98 | 48/48 | 100.0% (92.6%,100.0%) |
| MAP2K4 exon4 | 11998922_CA>C p.K143fs | 0.93-0.96 | 0.208 - 0.285 | 0.25 | 0.25 | 0.02 | 8.08 | 48/48 | 100.0% (92.6%,100.0%) |
| PAX5 exon2 | 37020768_A>C p.V26G | 0.93-0.96 | 0.225 - 0.312 | 0.27 | 0.27 | 0.02 | 7.26 | 48/48 | 100.0% (92.6%,100.0%) |
| PIK3CA exon2 | 178916876_G>A p.R88Q | 0.93-0.96 | 0.22 - 0.285 | 0.25 | 0.25 | 0.02 | 6.41 | 48/48 | 100.0% (92.6%,100.0%) |
| PIK3CA exon8 | 178928079_G>A p.E453K | 0.93-0.96 | 0.23 - 0.331 | 0.28 | 0.28 | 0.02 | 8.51 | 48/48 | 100.0% (92.6%,100.0%) |
| TP53 exon8 | 7577143_CAGT>C p.L265del | 0.93-0.96 | 0.223 - 0.334 | 0.27 | 0.27 | 0.02 | 8.32 | 48/48 | 100.0% (92.6%,100.0%) |
| ERBB2 exon8 | 37868208_C>A p.S310Y | 0.91-0.95 | 0.177 - 0.257 | 0.21 | 0.21 | 0.02 | 7.97 | 48/48 | 100.0% (92.6%,100.0%) |
| ARID1A exon18 | 27101135_C>T p.Q1473* | 0.9-0.96 | 0.329 - 0.519 | 0.44 | 0.44 | 0.04 | 8.71 | 48/48 | 100.0% (92.6%,100.0%) |
| KDM6A exon17 | 44928975_AG>A p.A694fs | 0.9-0.96 | 0.386 - 0.496 | 0.44 | 0.44 | 0.02 | 5.68 | 48/48 | 100.0% (92.6%,100.0%) |
| PIK3CA exon2 | 178916890_C>T p.R93W | 0.9-0.96 | 0.4 - 0.5 | 0.45 | 0.45 | 0.02 | 4.99 | 48/48 | 100.0% (92.6%,100.0%) |
| PIK3CA exon10 | 178936092_A>G p.E545G | 0.9-0.96 | 0.344 - 0.533 | 0.45 | 0.46 | 0.05 | 10.12 | 48/48 | 100.0% (92.6%,100.0%) |
| PTEN exon5 | 89692905_G>A p.R130Q | 0.9-0.96 | 0.842 - 0.916 | 0.88 | 0.88 | 0.02 | 1.79 | 48/48 | 100.0% (92.6%,100.0%) |
| RNF43 exon9 | 56435160_AC>A p.G659fs | 0.9-0.96 | 0.397 - 0.6 | 0.49 | 0.48 | 0.04 | 8.83 | 48/48 | 100.0% (92.6%,100.0%) |
| CDKN2A exon2 | 21971147_TGGG CTCCGCGCCGT GGA>T p.L65fs | 0.93-0.96 | 0.129 - 0.239 | 0.19 | 0.18 | 0.03 | 13.8 | 48/48 | 100.0% (92.6%,100.0%) |
| CDKN2A exon2 | 21971176_TCCG CCACTCGGGCG >T p.S56fs | 0.93-0.96 | 0.035 - 0.097 | 0.06 | 0.07 | 0.02 | 26.64 | 34/46 | 73.9% (59.7%,84.4%) |
| PIK3CA exon21 | 178952085_A>G p.H1047R | 0.93-0.96 | 0.167 - 0.235 | 0.19 | 0.19 | 0.01 | 7.74 | 48/48 | 100.0% (92.6%,100.0%) |
| TP53 exon6 | 7578186_C>CT p.P222fs | 0.93-0.96 | 0.192 - 0.351 | 0.27 | 0.26 | 0.03 | 12.67 | 48/48 | 100.0% (92.6%,100.0%) |
| CDKN2A exon2 | 21971179_G>C p.A60G | 0.91-0.95 | 0.349 - 0.562 | 0.47 | 0.47 | 0.05 | 9.86 | 48/48 | 100.0% (92.6%,100.0%) |
| ERBB4 exon14 | 212537975_G>A p.R544W | 0.91-0.95 | 0.199 - 0.299 | 0.25 | 0.25 | 0.03 | 10.36 | 48/48 | 100.0% (92.6%,100.0%) |
| PIK3R1 exon13 | 67591106_A>G p.K567E | 0.91-0.95 | 0.109 - 0.214 | 0.16 | 0.16 | 0.02 | 13.94 | 48/48 | 100.0% (92.6%,100.0%) |

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| Gene/ Exon | Mutation (cDNA/Protein Changes) | NC range* | MAF range | MAF Mean | MAF Median | MAF SD | MAF %CV | Positive /Total Calls | Positive Call Rate (two-sided 95% CI) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| RAC1 exon2 | 6426893_C>T p.P29L | 0.91-0.95 | 0.264 - 0.429 | 0.34 | 0.34 | 0.03 | 10.09 | 48/48 | 100.0% (92.6%,100.0%) |
| SMARCA 4 exon26 | 11141519_C>T p.Q1166* | 0.91-0.95 | 0.199 - 0.354 | 0.28 | 0.28 | 0.03 | 11.19 | 48/48 | 100.0% (92.6%,100.0%) |

* NC Range = Normalized coverage range

**Table 11. Summary of the Positive call rate within the 8 mixed samples**

| Gene/ Exon | Mutation (cDNA/Protein Changes) | NC range* | MAF range | MAF Mean | MAF Median | MAF SD | MAF %CV | Positive /Total Calls | Positive Call Rate (two-sided 95% CI) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| EGFR exon18 | 55241708_G>C p.G719A | 0.92-0.97 | 0.077 - 0.128 | 0.103 | 0.102 | 0.009 | 8.833 | 48/48 | 100.0% (92.6%,100.0%) |
| EGFR exon20 | 55249071_C>T p.T790M | 0.92-0.97 | 0.08 - 0.145 | 0.114 | 0.113 | 0.015 | 12.981 | 48/48 | 100.0% (92.6%,100.0%) |
| EGFR exon21 | 55259515_T>G p.L858R | 0.92-0.97 | 0.093 - 0.132 | 0.112 | 0.113 | 0.009 | 7.835 | 48/48 | 100.0% (92.6%,100.0%) |
| BRAF exon15 | 140453136_A>T p.V600E | 0.93-0.96 | 0.086 - 0.179 | 0.124 | 0.125 | 0.016 | 12.885 | 48/48 | 100.0% (92.6%,100.0%) |
| EGFR exon18 | 55241708_G>C p.G719A | 0.93-0.96 | 0.038 - 0.069 | 0.054 | 0.054 | 0.007 | 12.511 | 48/48 | 100.0% (92.6%,100.0%) |
| EGFR exon20 | 55249071_C>T p.T790M | 0.93-0.96 | 0.038 - 0.087 | 0.060 | 0.059 | 0.013 | 20.804 | 48/48 | 100.0% (92.6%,100.0%) |
| EGFR exon21 | 55259469_G>A p.V843I | 0.93-0.96 | 0.042 - 0.087 | 0.064 | 0.065 | 0.009 | 14.278 | 45/48 | 93.8% (83.2%,97.9%) |
| EGFR exon21 | 55259515_T>G p.L858R | 0.93-0.96 | 0.051 - 0.085 | 0.064 | 0.062 | 0.008 | 12.098 | 48/48 | 100.0% (92.6%,100.0%) |
| PIK3CA exon21 | 178952085_A>G p.H1047R | 0.92-0.96 | 0.129 - 0.203 | 0.166 | 0.166 | 0.019 | 11.415 | 48/48 | 100.0% (92.6%,100.0%) |
| PIK3CA exon21 | 178952085_A>G p.H1047R | 0.93-0.97 | 0.083 - 0.142 | 0.113 | 0.113 | 0.014 | 12.424 | 48/48 | 100.0% (92.6%,100.0%) |
| BRAF exon15 | 140453136_A>T p.V600E | 0.92-0.96 | 0.053 - 0.112 | 0.080 | 0.078 | 0.012 | 15.603 | 48/48 | 100.0% (92.6%,100.0%) |
| NRAS exon3 | 115256529_T>C p.Q61R | 0.92-0.96 | 0.052 - 0.121 | 0.087 | 0.087 | 0.016 | 18.283 | 48/48 | 100.0% (92.6%,100.0%) |
| BRAF exon15 | 140453136_A>T p.V600E | 0.91-0.95 | 0.01 - 0.032 | 0.020 | 0.019 | 0.005 | 25.844 | 23/48 | 47.9% (34.5%,61.7%) |
| NRAS exon3 | 115256529_T>A p.Q61L | 0.91-0.95 | 0.225 - 0.326 | 0.282 | 0.283 | 0.020 | 7.236 | 48/48 | 100.0% (92.6%,100.0%) |
| NRAS exon3 | 115256529_T>C p.Q61R | 0.91-0.95 | 0.012 - 0.044 | 0.023 | 0.023 | 0.005 | 22.366 | 36/48 | 75.0% (61.2%,85.1%) |
| CTNNB1 exon3 | 41266101_C>T p.S33F | 0.92-0.97 | 0.088 - 0.161 | 0.117 | 0.116 | 0.018 | 14.984 | 48/48 | 100.0% (92.6%,100.0%) |
| SMARCA 4 exon32 | 11169037_A>T p.K1511* | 0.92-0.97 | 0.114 - 0.17 | 0.139 | 0.138 | 0.014 | 10.168 | 48/48 | 100.0% (92.6%,100.0%) |
| TP53 exon8 | 7577106_G>C p.P278A | 0.92-0.97 | 0.072 - 0.156 | 0.115 | 0.119 | 0.021 | 18.195 | 48/48 | 100.0% (92.6%,100.0%) |
| B2M exon1 | 45003746_T>C p.M1? | 0.93-0.96 | 0.044 - 0.109 | 0.075 | 0.076 | 0.014 | 18.508 | 46/48 | 95.83% (86.02%,98.84%) |
| B2M exon2 | 45007890_G>GT p.K114* | 0.93-0.96 | 0.04 - 0.117 | 0.074 | 0.075 | 0.020 | 26.994 | 42/48 | 87.5% (75.3%,94.1%) |

{23}

| Gene/ Exon | Mutation (cDNA/Protein Changes) | NC range* | MAF range | MAF Mean | MAF Median | MAF SD | MAF %CV | Positive /Total Calls | Positive Call Rate (two-sided 95% CI) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| SMARCA 4 exon32 | 11169037_A>T p.K1511* | 0.93-0.96 | 0.042 - 0.095 | 0.070 | 0.069 | 0.009 | 13.278 | 47/48 | 97.9% (89.1%,99.6%) |
| TP53 exon8 | 7577106_G>C p.P278A | 0.93-0.96 | 0.028 - 0.096 | 0.054 | 0.052 | 0.014 | 26.180 | 29/48 | 60.4% (46.3%,73.0%) |
| TP53 exon7 | 7577539_G>A p.R248W | 0.92-0.96 | 0.12 - 0.194 | 0.170 | 0.171 | 0.014 | 8.243 | 48/48 | 100.0% (92.6%,100.0%) |
| TP53 exon7 | 7577539_G>A p.R248W | 0.93-0.97 | 0.071 - 0.133 | 0.103 | 0.103 | 0.012 | 11.914 | 48/48 | 100.0% (92.6%,100.0%) |
| TP53 exon7 | 7577556_C>T p.C242Y | 0.93-0.97 | 0.296 - 0.386 | 0.339 | 0.340 | 0.016 | 4.753 | 48/48 | 100.0% (92.6%,100.0%) |
| ARID1B exon3 | 157222648_C>T p.Q626* | 0.92-0.96 | 0.039 - 0.079 | 0.060 | 0.061 | 0.011 | 18.421 | 37/48 | 77.1% (63.5%,86.7%) |
| BRAF exon11 | 140481397_C>A p.V471F | 0.92-0.96 | 0.167 - 0.228 | 0.201 | 0.200 | 0.015 | 7.521 | 48/48 | 100.0% (92.6%,100.0%) |
| CARD11 exon6 | 2979559_C>T p.D230N | 0.92-0.96 | 0.164 - 0.234 | 0.196 | 0.192 | 0.018 | 9.182 | 48/48 | 100.0% (92.6%,100.0%) |
| FANCA exon27 | 89833576_G>C p.S858R | 0.92-0.96 | 0.051 - 0.099 | 0.071 | 0.071 | 0.011 | 15.549 | 48/48 | 100.0% (92.6%,100.0%) |
| NF1 exon12 | 29533315_C>T p.R440* | 0.92-0.96 | 0.18 - 0.287 | 0.237 | 0.239 | 0.026 | 11.096 | 48/48 | 100.0% (92.6%,100.0%) |
| NOTCH4 exon4 | 32188899_G>A p.Q219* | 0.92-0.96 | 0.029 - 0.077 | 0.056 | 0.056 | 0.009 | 16.549 | 39/48 | 81.3% (68.1%,89.8%) |
| TP53 exon4 | 7579546_CG>C p.P47fs | 0.92-0.96 | 0.049 - 0.098 | 0.068 | 0.066 | 0.012 | 17.066 | 47/48 | 97.9% (89.1%,99.6%) |
| BRAF exon11 | 140481397_C>A p.V471F | 0.91-0.95 | 0.044 - 0.079 | 0.056 | 0.053 | 0.008 | 14.586 | 37/48 | 77.08% (63.46%,86.69%) |
| DNMT3A exon16 | 25466800_GG>A A p.R635W | 0.91-0.95 | 0.217 - 0.308 | 0.266 | 0.270 | 0.022 | 8.136 | 48/48 | 100.0% (92.6%,100.0%) |
| EZH2 exon16 | 148508728_A>T p.Y646N | 0.91-0.95 | 0.213 - 0.264 | 0.241 | 0.242 | 0.012 | 4.814 | 48/48 | 100.0% (92.6%,100.0%) |
| NF1 exon12 | 29533315_C>T p.R440* | 0.91-0.95 | 0.051 - 0.107 | 0.079 | 0.080 | 0.012 | 15.487 | 48/48 | 100.0% (92.6%,100.0%) |
| TP53 exon8 | 7577099_C>T p.R280K | 0.91-0.95 | 0.305 - 0.422 | 0.369 | 0.368 | 0.024 | 6.570 | 48/48 | 100.0% (92.6%,100.0%) |
| ARID1A exon9 | 27092804_A>AC p.Q944fs | 0.93-0.96 | 0.101 - 0.169 | 0.129 | 0.127 | 0.015 | 11.318 | 48/48 | 100.0% (92.6%,100.0%) |
| RXRA exon10 | 137328351_C>T p.S427F | 0.93-0.96 | 0.066 - 0.131 | 0.098 | 0.101 | 0.015 | 15.049 | 48/48 | 100.0% (92.6%,100.0%) |
| ARID1A exon9 | 27092804_A>AC p.Q944fs | 0.93-0.96 | 0.056 - 0.106 | 0.081 | 0.079 | 0.014 | 17.269 | 48/48 | 100.0% (92.6%,100.0%) |
| RXRA exon10 | 137328351_C>T p.S427F | 0.93-0.96 | 0.035 - 0.118 | 0.065 | 0.064 | 0.015 | 23.236 | 43/48 | 89.6% (77.8%,95.5%) |

* NC Range = Normalized coverage range

### c) Per-Specimen Precision for SNVs and Indels

Precision was calculated for each individual specimen as shown in Table 12 (single samples) and Table 13 (mixed samples). Results from the precision studies were combined across all reportable genes for each specimen. The positive and negative call rates for sequence mutations (SNVs, MNVs, insertions and deletions) in each sample were calculated based on the total number of mutations along with the two-sided 95% CI.

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**Table 12. Positive and negative call rates per sample (Single sample)**

| Sample ID | Total No. unique mutations detected across 48 replicates | Positive Call Rate per Mutation | Positive call rate (two-sided 95% CI) | Negative call rate (two-sided 95% CI) |
| --- | --- | --- | --- | --- |
| Sample 1 | 66 | 28/48 for 1* 45/48 for 1* 46/46 for 1** 48/48 for 63 | 3143/3166 99.27% (98.91%, 99.51%) | 36192/36192 100.00% (99.99%, 100.00%) |
| Sample 2 | 34 | 48/48 for 34 | 1632/1632 100% (99.76%, 100%) | 36239/36240 99.99% (99.98%, 99.99%) |
| Sample 3 | 14 | 48/48 for 14 | 672/672 100% (99.43%, 100%) | 36192/36192 100.00% (99.99%, 100.00%) |
| Sample 4 | 52 | 48/48 for 52 | 2496/2496 100% (99.84%, 100%) | 36230/36240 99.97% (99.95%, 99.99%) |
| Sample 5 | 17 | 48/48 for 17 | 816/816 100% (99.53%, 100%) | 36192/36192 100.00% (99.99%, 100.00%) |
| Sample 6 | 45 | 46/46 for 1** 46/47 for 1*, ** 48/48 for 43 | 2156/2157 99.95% (99.73%, 99.99%) | 36192/36192 100.00% (99.99%, 100.00%) |
| Sample 7 | 35 | 47/47 for 1** 48/48 for 34 | 1679/1679 100% (99.77%, 100%) | 36192/36192 100.00% (99.99%, 100.00%) |
| Sample 8 | 13 | 5/47 for 1*, ** 48/48 for 12 | 581/623 93.25% (91.01%, 94.97%) | 36192/36192 100.00% (99.99%, 100.00%) |
| Sample 9 | 49 | 24/48 for 1* 32/48 for 1 33/48 for 1 46/48 for 2 48/48 for 44 | 2293/2352 97.49% (96.77%, 98.05%) | 36140/36144 99.99% (99.97%, 99.99%) |
| Sample 10 | 11 | 48/48 for 11 | 528/528 100% (99.27%, 100%) | 36240/36240 100.00% (99.99%, 100.00%) |
| Sample 11 | 16 | 34/46 for 1*, ** 48/48 for 15 | 754/766 98.43% (97.28%, 99.1%) | 36192/36192 100.00% (99.99%, 100.00%) |
| Sample 12 | 34 | 20/20 for 1** 24/24 for 1** 48/48 for 32 | 1580/1580 100% (99.75%, 100%) | 36192/36192 100.00% (99.99%, 100.00%) |

* Samples with mean MAF below or closed to the Cutoff

** Sample that yielded a reduction in number of replicates due to failure of QC of the data

**Table 13. Positive and negative call rates per sample (Mixed Sample)**

| Sample ID | Total No. unique mutations detected across 48 replicates | Positive Call Rate per Mutation | Positive call rate (two-sided 95% CI) | Negative call rate (two-sided 95% CI) |
| --- | --- | --- | --- | --- |
| Sample 13 | 30 | 43/48 for 1 48/48 for 29 | 1435/1440 99.65% (99.19%, 99.85%) | 36093/36096 99.99% (99.98%, 99.99%) |
| Sample 14 | 52 | 21/48 for 1* 26/48 for 1* 29/48 for 1* 36/48 for 1* 39/48 for 1 42/48 for 1 43/48 for 2 44/48 for 1 | 2375/2496 95.15% (94.24%, 95.93%) | 36047/36048 99.99% (99.98%, 99.99%) |

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| Sample ID | Total No. unique mutations detected across 48 replicates | Positive Call Rate per Mutation | Positive call rate (two-sided 95% CI) | Negative call rate (two-sided 95% CI) |
| --- | --- | --- | --- | --- |
| | | 45/48 for 1 46/48 for 3 47/48 for 3 48/48 for 36 | | |
| Sample 15 | 22 | 48/48 for 22 | 1056/1056 100% (99.64%,100%) | 36192/36192 100.00% (99.99%,100.00%) |
| Sample 16 | 32 | 46/48 for 1 47/48 for 1 48/48 for 30 | 1533/1536 99.8% (99.43%, 99.93%) | 36192/36192 100.00% (99.99%,100.00%) |
| Sample 17 | 78 | 26/48 for 2* 31/48 for 1* 37/47 for 1** 37/48 for 1* 38/48 for 1* 39/48 for 1* 44/44 for 1** 46/48 for 4 47/47 for 4** 47/48 for 3 48/48 for 59 | 3623/3735 97% (96.4%, 97.5%) | 36144/36144 100.00% (99.99%,100.00%) |
| Sample 18 | 81 | 23/48 for 1* 36/48 for 1* 37/48 for 1* 39/48 for 1 40/48 for 2* 41/48 for 1 43/47 for 1** 45/45 for 1** 45/48 for 1 46/47 for 1** 46/48 for 2 47/48 for 5 48/48 for 63 | 3786/3883 97.5% (96.96%, 97.95%) | 36096/36096 100.00% (99.99%,100.00%) |
| Sample 19 | 32 | 39/45 for 1*, ** 42/42 for 1** 47/48 for 1 31/31 for 1** 48/48 for 28 | 1503/1510 99.54% (99.05%, 99.78%) | 36239/36240 99.99% (99.98%,99.99%) |
| Sample 20 | 38 | 3/40 for 1*, ** 17/17 for 1** 21/48 for 1* 33/48 for 1* 39/48 for 1* 42/48 for 1 43/48 for 2 44/48 for 1 45/48 for 1* 46/46 for 1** 46/48 for 3 47/48 for 1 48/48 for 24 | 1667/1783 93.49% (92.25%, 94.55%) | 36240/36240 100.00% (99.99%,100.00%) |

* Samples with mean MAF below or closed to the Cutoff

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** Sample that yielded a reduction in number of replicates due to failure of QC of the data

#### d) Precision for ERBB2 amplification

Precision of ERBB2 amplification was evaluated across the same set of 20 samples (12 single and 8 mixed). Each sample was tested in duplicate by 2 different operators on 2 instruments across 2 non- consecutive start days using 3 kit lots, yielding 48 replicates per sample. Within the 20 samples, 3 samples contained ERBB2 amplification and the rest of the samples contained no ERBB2 amplification. The assay reports ERBB2 amplification when the observed copy number for the gene is greater than or equal to 4 copies of the observed copy number determined by the assay. The mean observed copy number (CNV), observed copy number range, standard deviation (SD), coefficient of variation (%CV), positive call rate along with 95% CI for each sample with ERBB2 amplification are summarized in Table 14. The call rates were 100% for both amplification and no amplification groups.

**Table 14. Summary of the ERBB2 gene amplification precision**

| Sample ID | Sample Type | Standardized Cancer Type | Gene | No. replicates | CNV Status | Mean Observed CNV | Observed copy number range | SD (%CV) | No. Positive Calls | Positive Call Rate (95% CI) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample 3 | Single | Pancreas Adenocarcinoma | ERBB2 | 48 | Amp* | 8.03 | (7.5, 8.5) | 0.19 (2.37) | 48 | 100.0% (92.6%, 100.0%) |
| Sample 18 | Mixed | Breast Invasive ductal carcinoma | ERBB2 | 48 | Amp* | 14.31 | (13, 16) | 0.65 (4.50) | 48 | 100.0% (92.6%, 100.0%) |
| Sample 19 | Mixed | Breast Invasive ductal carcinoma | ERBB2 | 48 | Amp* | 31.16 | (28.5, 32.5) | 0.97 (3.11) | 48 | 100.0% (92.6%, 100.0%) |

*Amp = amplification.

#### e) Precision for TMB

Precision of TMB was evaluated across 12 single samples with TMB scores ranging from 0.7 to 45.8. Specimens spanned the range of TMB scores and included 10 different tumor types. The tumor purity for these samples ranged from 32.5% to 72.7%. The mean TMB score, SD, %CV and mean target region coverage for each sample are shown in Table 15. The calculated %CV for samples with TMB scores >5 are less than 10% except sample 9, which contains the highest percentage of SNVs with low MAF (<10%) in the whole sample sets. Overall, the data supports a repeatable TMB reporting by the ACTOnco assay.

**Table 15. TMB Precision Data**

| Sample ID | Tumor Type | Number of Valid Results | Mean Report Tumor Purity (%) | Mean TMB score | SD | %CV | Mean Target Region Coverage |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Sample 1 | Colon adenocarcinoma | 48 | 72.667 | 39.248 | 1.629 | 4.151 | 768.771 |
| Sample 2 | Kidney cancer | 47 | 33.043 | 10.702 | 0.870 | 8.131 | 833.511 |
| Sample 3 | Tumor of exocrine pancreas | 48 | 51.792 | 1.931 | 0.895 | 46.352 | 731.438 |

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| Sample ID | Tumor Type | Number of Valid Results | Mean Report Tumor Purity (%) | Mean TMB score | SD | %CV | Mean Target Region Coverage |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Sample 4 | Liver cancer | 48 | 37.771 | 26.417 | 2.380 | 9.008 | 835.354 |
| Sample 5 | Cholangiocarcinoma | 48 | 44.500 | 2.808 | 1.100 | 39.178 | 765.250 |
| Sample 6 | Colon adenocarcinoma | 48 | 34.600 | 28.706 | 1.317 | 4.587 | 796.667 |
| Sample 7 | Endometrial cancer | 48 | 60.000 | 18.810 | 1.194 | 6.347 | 854.021 |
| Sample 8 | Urinary system cancer | 48 | 52.125 | 2.417 | 0.999 | 41.321 | 869.063 |
| Sample 9 | Endometrial cancer | 48 | 66.771 | 18.331 | 3.186 | 17.381 | 728.229 |
| Sample 10 | Breast cancer | 37 | 32.514 | 1.159 | 0.686 | 59.144 | 840.054 |
| Sample 11 | Lung cancer | 39 | 38.974 | 4.472 | 0.678 | 15.166 | 838.769 |
| Sample 12 | Skin cancer | 48 | 52.854 | 13.033 | 1.014 | 7.782 | 881.792 |

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

The LoD of the ACTOnco assay for SNVs and indels is defined as the MAF at which 95% of replicates for a variant type are reliably detected. The LoDs of the ACTOnco assay for ERBB2 amplification and TMB is determined as the minimum tumor purity required for robust reporting of amplification status and TMB scores, respectively. The recommended DNA input for the ACTOnco assay is 80 ng of total DNA, with a minimum tumor purity of 30% for TMB reporting.

#### a) LoD – SNVs and Indels:

The LoD of the ACTOnco assay for SNVs and indels was evaluated by assessing 10 clinical FFPE specimens from 8 different cancer types with 15 SNVs, 5 insertions and 11 deletions. Each sample was diluted to 5 dilution levels and tested in 5 replicates per level using two reagent lots. The call rate was determined for each variant and the LoD/C95 was approximated between the call rate that was below 95% and the highest call rate (100%). Table 16 is a summary of the estimation of the LoD range for each variant along with the cutoff within the entire panel evaluated in the study. The established MAF range for each variant type (Hotspot SNVs, Non-hotspot SNVs, insertions and deletion) is shown in Table 17.

Table 16. Estimation of the LoD range for the representative variants

| Variant Type | Variant ID | Gene Exon | RGT Lot | Mean MAF Range | Dilution Levels | Call Rates (n/N) | LoD Approx (MAF) | Assay cutoff |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| SNV | EGFR_chr7_55259515_T>G | EGFR 21 | A | 0.027 to 0.211 | Level 4 Level 5 | 100% (5/5) 100% (5/5) | 0.027 to 0.041 | 0.02 |
| SNV | EGFR_chr7_55259515_T>G | EGFR 21 | B | 0.022 to 0.201 | Level 4 Level 5 | 100% (5/5) 60% (3/5) | 0.022 to 0.045 | 0.02 |
| SNV | BRCA1_chr17_41244716_A>T | BRCA1 10 | A | 0.021 to 0.332 | Level 3 Level 4 | 100% (5/5) 0% (0/5) | 0.041 to 0.079 | 0.05 |
| SNV | BRCA1_chr17_41244716_A>T | BRCA1 10 | B | 0.019 to 0.315 | Level 3 Level 4 | 100% (5/5) 0% (0/5) | 0.037 to 0.087 | 0.05 |
| SNV | BRAF_chr7_140453136_A>T | BRAF 15 | A | 0.013 to 0.259 | Level 3 Level 4 | 100% (5/5) 60% (3/5) | 0.022 to 0.043 | 0.02 |

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| Variant Type | Variant ID | Gene Exon | RGT Lot | Mean MAF Range | Dilution Levels | Call Rates (n/N) | LoD Approx (MAF) | Assay cutoff |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| SNV | BRAF_chr7_140453136_A>T | BRAF 15 | B | 0.011 to 0.272 | Level 3 Level 4 | 100% (5/5) 40% (2/5) | 0.019 to 0.044 | 0.02 |
| SNV | NRAS_chr1_115256530_G>T | NRAS 3 | A | 0.035 to 0.395 | Level 4 Level 5 | 100% (5/5) 100% (5/5) | 0.035 to 0.066 | 0.02 |
| SNV | NRAS_chr1_115256530_G>T | NRAS 3 | B | 0.039 to 0.415 | Level 4 Level 5 | 100% (5/5) 100% (5/5) | 0.039 to 0.047 | 0.02 |
| SNV | PIK3CA_chr3_178952085_A>G | PIK3CA 21 | A | 0.006 to 0.209 | Level 2 Level 3 | 100% (5/5) 20% (1/5) | 0.016 to 0.036 | 0.02 |
| SNV | PIK3CA_chr3_178952085_A>G | PIK3CA 21 | B | 0.005 to 0.201 | Level 2 Level 3 | 100% (5/5) 0% (0/5) | 0.015 to 0.040 | 0.02 |
| SNV | EGFR_chr7_55249071_C>T | EGFR 20 | A | 0.015 to 0.240 | Level 2 Level 3 | 100% (5/5) 40% (2/5) | 0.018 to 0.044 | 0.02 |
| SNV | EGFR_chr7_55249071_C>T | EGFR 20 | B | 0.012 to 0.237 | Level 2 Level 3 | 100% (5/5) 33% (1/3) | 0.019 to 0.062 | 0.02 |
| SNV | KRAS_chr12_25398284_C>T | KRAS 2 | A | 0.026 to 0.326 | Level 4 Level 5 | 100% (5/5) 100% (5/5) | 0.026 to 0.048 | 0.02 |
| SNV | KRAS_chr12_25398284_C>T | KRAS 2 | B | 0.030 to 0.337 | Level 4 Level 5 | 100% (5/5) 100% (5/5) | 0.030 to 0.039 | 0.02 |
| INS | KIT_chr4_55593640_T>TTT ACAT AGACCC | KIT 11 | A | 0.005 to 0.283 | Level 1 Level 2 | 100% (5/5) 80% (4/5) | 0.066 to 0.283 | 0.05 |
| INS | KIT_chr4_55593640_T>TTT ACAT AGACCC | KIT 11 | B | 0.004 to 0.321 | Level 2 Level 3 | 100% (5/5) 0% (0/5) | 0.030 to 0.063 | 0.05 |
| INS | ERBB2_chr17_37880981_A>AGCATACGTGATG | ERBB2 20 | A | 0.005 to 0.177 | Level 1 Level 2 | 100% (5/5) 60% (3/5) | 0.029 to 0.177 | 0.02 |
| INS | ERBB2_chr17_37880981_A>AGCATACGTGATG | ERBB2 20 | B | 0.005 to 0.174 | Level 2 Level 3 | 100% (5/5) 20% (1/5) | 0.011 to 0.027 | 0.02 |
| INS | KMT2D_chr12_49432655_G>GA | KMT2D 34 | A | 0.008 to 0.375 | Level 2 Level 3 | 100% (5/5) 0% (0/5) | 0.027 to 0.064 | 0.05 |
| INS | KMT2D_chr12_49432655_G>GA | KMT2D 34 | B | 0.009 to 0.356 | Level 2 Level 3 | 100% (5/5) 0% (0/4) | 0.028 to 0.071 | 0.05 |
| INS | MUC16_chr19_9019605_C>CTGG | MUC16 22 | A | 0.009 to 0.446 | Level 2 Level 3 | 100% (5/5) 0% (0/5) | 0.022 to 0.061 | 0.05 |
| INS | MUC16_chr19_9019605_C>CTGG | MUC16 22 | B | 0.007 to 0.454 | Level 1 Level 2 | 100% (5/5) 40% (5/2) | 0.052 to 0.454 | 0.05 |
| DEL | EGFR_chr7_55242464_AGG AATTA AGAGAAGC>A | EGFR 19 | A | 0.005 to 0.420 | Level 2 Level 3 | 100% (5/5) 80% (4/5) | 0.028 to 0.066 | 0.02 |
| DEL | EGFR_chr7_55242464_AGG AATTA AGAGAAGC>A | EGFR 19 | B | 0.007 to 0.411 | Level 2 Level 3 | 100% (5/5) 40% (2/5) | 0.023 to 0.073 | 0.02 |
| DEL | BRCA1_chr17_41222948_T TCTTCTGGGGTCAGGCC AG>T | BRCA1 15 | A | 0.046 to 0.209 | Level 2 Level 3 | 100% (4/4) 0% (0/2) | 0.046 to 0.118 | 0.05 |
| DEL | BRCA1_chr17_41222948_T TCTTCTGGGGTCAGGCC AG>T | BRCA1 15 | B | 0.072 to 0.298 | Level 3 Level 4 | 100% (1/1) 100% (1/1) | 0.072 to 0.087 | 0.05 |
| DEL | KMT2D_chr12_49434491_A G>A | KMT2D 31 | A | 0.012 to 0.644 | Level 3 Level 4 | 100% (5/5) 20% (1/5) | 0.042 to 0.112 | 0.05 |
| DEL | KMT2D_chr12_49434491_A G>A | KMT2D 31 | B | 0.017 to 0.390 | Level 3 Level 4 | 100% (5/5) 80% (4/5) | 0.055 to 0.099 | 0.05 |

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| Variant Type | Variant ID | Gene Exon | RGT Lot | Mean MAF Range | Dilution Levels | Call Rates (n/N) | LoD Approx (MAF) | Assay cutoff |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| DEL | PTEN_chr10_89692782_CT>C | PTEN 5 | A | 0.014 to 0.284 | Level 2 Level 3 | 100% (5/5) 0% (0/5) | 0.039 to 0.107 | 0.05 |
| DEL | PTEN_chr10_89692782_CT>C | PTEN 5 | B | 0.021 to 0.258 | Level 2 Level 3 | 100% (5/5) 20% (1/5) | 0.046 to 0.105 | 0.05 |
| SNV | TP53_chr17_7578524_G>A | TP53 5 | A | 0.034 to 0.260 | Level 4 Level 5 | 100% (5/5) 0% (0/5) | 0.034 to 0.067 | 0.05 |
| SNV | TP53_chr17_7578524_G>A | TP53 5 | B | 0.024 to 0.284 | Level 3 Level 4 | 100% (5/5) 60% (3/5) | 0.057 to 0.090 | 0.05 |
| SNV | PTEN_chr10_89692904_C>G | PTEN 5 | A | 0.051 to 0.680 | Level 4 Level 5 | 100% (5/5) 100% (5/5) | 0.051 to 0.102 | 0.05 |
| SNV | PTEN_chr10_89692904_C>G | PTEN 5 | B | 0.051 to 0.672 | Level 4 Level 5 | 100% (5/5) 100% (5/5) | 0.051 to 0.088 | 0.05 |
| SNV | KRAS_chr12_25398285_C>G | KRAS 2 | A | 0.008 to 0.085 | Level 2 Level 3 | 100% (5/5) 20% (1/5) | 0.017 to 0.032 | 0.02 |
| SNV | KRAS_chr12_25398285_C>G | KRAS 2 | B | 0.006 to 0.086 | Level 2 Level 3 | 100% (5/5) 0% (0/5) | 0.015 to 0.034 | 0.02 |
| SNV | RB1_chr13_48953788_T>C | RB1 - | A | 0.013 to 0.222 | Level 2 Level 3 | 100% (5/5) 40% (2/5) | 0.049 to 0.107 | 0.05 |
| SNV | RB1_chr13_48953788_T>C | RB1 - | B | 0.016 to 0.237 | Level 2 Level 3 | 100% (5/5) 20% (1/5) | 0.046 to 0.102 | 0.05 |
| SNV | TP53_chr17_7577568_C>T | TP53 7 | A | 0.006 to 0.524 | Level 2 Level 3 | 100% (5/5) 0% (0/5) | 0.025 to 0.067 | 0.05 |
| SNV | TP53_chr17_7577568_C>T | TP53 7 | B | 0.007 to 0.527 | Level 2 Level 3 | 100% (5/5) 0% (0/5) | 0.024 to 0.065 | 0.05 |
| SNV | BRCA2_chr13_32910983_G>A | BRCA2 11 | A | 0.024 to 0.252 | Level 2 Level 3 | 100% (5/5) 40% (2/5) | 0.043 to 0.092 | 0.05 |
| SNV | BRCA2_chr13_32910983_G>A | BRCA2 11 | B | 0.015 to 0.259 | Level 2 Level 3 | 100% (5/5) 40% (2/5) | 0.050 to 0.093 | 0.05 |
| SNV | BRCA2_chr13_32930651_G>A | BRCA2 15 | A | 0.012 to 0.518 | Level 3 Level 4 | 100% (5/5) 0% (0/5) | 0.026 to 0.058…