CellDx-Tissue

K260235 · Datar Cancer Genetics Private Limited · PZM · May 12, 2026 · Pathology

Device Facts

• Record ID: K260235
• Device Name: CellDx-Tissue
• Applicant: Datar Cancer Genetics Private Limited
• Product Code: PZM · Pathology
• Decision Date: May 12, 2026
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.6080
• Device Class: Class 2
• Attributes: 3rd-Party Reviewed

Indications for Use

The CellDx-Tissue is a qualitative in vitro diagnostic (IVD) test that uses next-generation sequencing of DNA and RNA isolated from formalin-fixed paraffine-embedded (FFPE) tumor tissue from patients previously diagnosed with solid malignant neoplasm to detect tumor gene alterations in a broad multi-gene panel. The test is intended to provide tumor mutation profiling information on somatic mutations, including single nucleotide variants (SNVs), insertions and deletions (indels), one gene amplification, and three fusions. Information provided by the CellDx-Tissue test is intended for use by qualified healthcare professionals in accordance with professional guidelines in oncology. Results from CellDx-Tissue are not intended to be conclusive or prescriptive for the labeled use of any specific therapeutic product. CellDx-Tissue is a single-site assay performed at Datar Cancer Genetics (DCG).

Device Story

CellDx-Tissue is an amplicon-based NGS assay for tumor mutation profiling; inputs are genomic DNA and RNA from FFPE tumor tissue (≥125 mm² surface area, ≥3 mm thickness, ≥20% tumor content). Workflow includes nucleic acid extraction, targeted library synthesis (517-gene panel), clonal amplification via emulsion PCR on Ion Chef System, and semiconductor sequencing on Ion GeneStudio S5 Prime. Bioinformatics pipeline performs base calling, alignment to hg19, and variant calling for SNVs, indels, ERBB2 amplification, and ALK/RET/ROS1 fusions. Performed at a single site (Datar Cancer Genetics) by trained personnel. Output is a clinical report categorizing variants by clinical significance; assists oncologists in identifying potential therapeutic targets; benefits patients by providing comprehensive genomic profiling to guide treatment decisions.

Clinical Evidence

Bench testing only. Performance validated using 2,676 clinical FFPE samples across >25 tumor types. Precision study evaluated 17 specimens with up to 48 replicates, showing 98.69% overall positive call rate. Accuracy established via comparison to orthogonal NGS (SNVs/indels) and FISH (fusions/CNV) across 252 samples, demonstrating high PPA/NPA. LoD studies defined sensitivity thresholds for SNVs, indels, ERBB2 amplification, and fusions. Interference and cross-contamination studies confirmed assay robustness.

Technological Characteristics

Amplicon-based targeted NGS panel (517 genes). Materials: FFPE tumor tissue. Energy: Semiconductor sequencing (hydrogen ion detection). Connectivity: Standalone bioinformatics pipeline on high-performance server. Sterilization: Not applicable (IVD reagents). Software: Rule-based variant calling and annotation pipeline. Standards: ISO 13485, ISO 14971, ISO 15189, IEC 61010-1, ISO/IEC 62304.

Indications for Use

Indicated for patients previously diagnosed with solid malignant neoplasms to detect tumor gene alterations in a broad multi-gene panel using 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 (DEN170058)

Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

## I. Background Information

### A. 510(k) Number

K260235

### B. Applicant

Datar Cancer Genetics Pvt Ltd

### C. Proprietary and Established Names

CellDx - Tissue

### D. Regulatory Information

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

## II. Submission/Device Overview

### A. Purpose for submission

New Device

### B. Measurand

Somatic single nucleotide variants (SNVs), insertions and deletions (Indels), Copy Number Variant (CNV), and select gene fusions, in genomic DNA and RNA isolated from formalin-fixed paraffine-embedded (FFPE) tumor tissue. A complete list of genes included in the assay can be found in Appendix A.

### C. Type of Test

Next-generation sequencing tumor profiling test

## III. Intended Use/Indications for Use

### A. Intended Use(s)

The CellDx-Tissue is a qualitative in vitro diagnostic (IVD) test that uses next-generation sequencing of DNA and RNA isolated from formalin-fixed paraffine-embedded (FFPE) tumor tissue from patients previously diagnosed with solid malignant neoplasm to detect tumor gene

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alterations in a broad multi-gene panel. The test is intended to provide tumor mutation profiling information on somatic mutations, including single nucleotide variants (SNVs), insertions and deletions (indels), one gene amplification, and three fusions.

Information provided by the CellDx-Tissue test is intended for use by qualified healthcare professionals in accordance with professional guidelines in oncology. Results from CellDx-Tissue are not intended to be conclusive or prescriptive for the labeled use of any specific therapeutic product. CellDx-Tissue is a single-site assay performed at Datar Cancer Genetics (DCG).

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

Same as above

# **C. Special Instrument Requirements**

Rx – For prescription use
For *in vitro* diagnostic use

# **D. Special Instrument Requirements**

Thermo Fisher Ion GeneStudio S5 Prime NGS Systems (qualified by Datar Cancer Genetics)

# **IV. Device/System Characteristics**

# **A. Device Description**

The CellDx-Tissue utilizes an amplicon-based sequencing approach to detect a broad range of clinically significant somatic alterations, including SNVs, InDels, and a gene amplification in tumor tissue DNA (ttDNA), and three fusions in tumor tissue RNA (ttRNA). The test requires FFPE tumor tissue specimen having a surface area of 125 mm² or more, a thickness of at least 3 mm, with at least 20% tumor content. Specimens with lower tumor content may be enriched by macro-dissection. A description of required equipment, software, reagents, vendors, and storage conditions is provided in the product labeling (Datar Cancer Genetics Technical Information). The CellDx-Tissue system includes a sequencing instrument, reagents (nucleic acid extraction, library preparation, and sequencing) software (operation of the sequencing instrument and variant calling), and instructions for use (IFU) of the system. All instruments and reagents are qualified by Datar Cancer Genetics.

# **1. Sample Preparation**

The CellDx – Tissue requires genomic DNA and RNA isolated from formalin-fixed paraffin-embedded (FFPE) tissue specimens. The tumor volume and minimum tumor content needed to obtain sufficient DNA and RNA for testing to achieve the stated performance are listed in Table 1. The tumor content of the FFPE specimen is determined by hematoxylin-eosin (HE) staining. If the histopathologist assessed tumor content is < 20%, 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 for Validated Specimen Type

| Tissue Type | Formalin-fixed paraffin-embedded (FFPE) tumor tissue blocks |
| --- | --- |
| Specimen Volume | ≥ 3 mm thick; >125 mm² total surface area |
| Tumor Content | ≥ 20% |
| Macrodissection Requirements | If the minimum tumor content is less than 20%, macrodissection will be done to enrich neoplastic content |
| Limitations | Low (<20%) tumor content, Inadequate tissue volume, Long-term storage degradation. |
| Storage | Room Temperature |

## 2. Nucleic Acid Extraction

DNA extraction is performed using the QIAamp DNA FFPE Advanced UNG Kit (qualified by DCG) as per the manufacturer's instructions for use. The procedure includes Uracil-N-Glycosylase (UNG) treatment to remove deaminated cytosine residues and an RNase A digestion to enhance the purity of the extracted DNA. The yield of extracted DNA is evaluated by real-time PCR. At least 20 ng DNA is required for library synthesis. RNA extraction procedure includes a DNase digestion step to eliminate contaminating DNA. The yield of extracted RNA is determined using the fluorometric assay, based on a standard curve generated from calibration standards. At least 20 ng RNA is required for reverse transcription (cDNA synthesis) and library synthesis. The assay has been validated with extracted DNA and RNA stored at -20°C and -80°C, respectively for up to 28 days.

## 3. Library Synthesis

DNA libraries are synthesized using the Oncomine DNA kit (qualified by DCG). The assay employs an amplicon-based targeted enrichment workflow, in which predefined genomic regions are amplified and converted into sequencing-ready libraries through adapter ligation and barcode incorporation. This approach enables multiplexed sequencing across the 517-gene panel. Library preparation entails the targeted amplification of genomic regions, partial digestion of primer sequences, ligation of barcode adapters, and subsequent clean-up and amplification to produce sequencing-competent libraries.

RNA libraries are synthesized using the Oncomine RNA Kit (qualified by DCG). The assay employs an amplicon-based targeted RNA sequencing workflow, in which input RNA is reverse-transcribed to cDNA and targeted regions are selectively amplified to generate sequencing-ready libraries. The workflow includes cDNA synthesis, targeted amplification, adapter ligation with unique barcodes, and subsequent clean-up steps to prepare multiplexed libraries suitable for NGS. DNA and RNA libraries are quantified using the kit which employs a real-time PCR-based method to measure the concentration of adapter-ligated libraries prior to sequencing. Library concentration results are reviewed against predefined quality acceptance criteria, and all quantitation outputs are retained as part of routine quality assurance and traceability.

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#### 4. Template Preparation and Chip Loading

Template preparation and chip loading are performed on the Ion Chef™ System (qualified by DCG) using the manufacturer's automated workflow for emulsion PCR (ePCR), enrichment, and chip loading. Quantified libraries meeting the predefined quality criteria are pooled and loaded onto the instrument along with the required reagents and consumables supplied in the template preparation kit. The Ion Chef System (qualified by DCG) performs clonal amplification of library molecules on beads through ePCR, followed by automated enrichment to isolate beads containing amplified templates. The instrument then loads the enriched, template-positive beads onto an Ion 550™ sequencing chip (qualified by DCG), producing a sequencing-ready chip for downstream processing on the compatible semiconductor sequencing platform qualified by Datar Cancer Genetics. Instrument checks and onboard controls ensure workflow integrity, and template-prepared chips proceed directly to sequencing.

#### 5. Sequencing

Sequencing is performed on the Ion GeneStudio™ S5 Prime System (qualified by DCG) using the manufacturer's semiconductor sequencing workflow. The sequencing chip generated during template preparation is loaded into the instrument along with the required sequencing reagents once all automated system checks confirm acceptable setup. A predefined run plan specifies the assay configuration, sample assignments, and chip parameters. After sequencing is initiated, the system performs automated nucleotide flows and signal detection to generate base-level data across all loaded libraries. Sequencing data are accepted only when run-level QC metrics and control results meet validated acceptance criteria.

#### 6. Sequencing Data Analysis

##### a. Data Management

Sample tracking, data processing, and archival of sequencing data for the CellDx-Tissue assay are managed through a Laboratory Information Management System (LIMS) integrated with DCG's validated bioinformatics pipeline operating on a high-performance server environment. The system tracks and archives run-associated metadata including barcode identifiers, sequencing run identifiers, sample accession numbers, specimen source, library batch identifiers, and assay modality (DNA or RNA). Key functions include tracking sample status across all stages of data analysis, logging analysis iterations applied to each sample, recording software, algorithm, and database versions used for analysis, and archiving pipeline output files (BAM and VCF) together with sequencing run statistics (e.g., total reads generated, mean depth of coverage, uniformity, and on-target rates). All annotation and population databases are maintained as access-restricted, version-locked static copies in a controlled repository. Any update to software, databases, or internal scripts is managed under a formal change-control process requiring documented impact assessment, regression testing using predefined golden datasets, QA/RA approval, and release prior to clinical use.

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# **b. Signal process, Base Calling, Read Alignment, BAM Generation, and Coverage Statistics**

Primary data processing is performed using Torrent Suite Software (qualified by DCG). During sequencing, raw semiconductor flow signals are converted into nucleotide bases through signal processing and base calling algorithms. Reads are assigned to individual samples through barcode classification, followed by trimming of barcode and adapter sequences and quality filtering. Filtered reads are aligned to the human reference genome (hg19 / GRCh37.p5) using platform-validated alignment algorithms. Aligned reads are written to Binary Alignment Map (BAM) files, which serve as the input for downstream variant calling and copy number analysis. The software also generates coverage statistics including mean depth of coverage, base coverage uniformity, and percentage of reads on target for each sample.

# **c. Read Alignment Quality Check**

Read alignment metrics are used to assess sequencing and library quality. Reads are aligned to hg19, and mismatches arising from biological variation (true variants) or technical sequencing errors are recorded. Alignment performance is summarized using the Alignment Quality (AQ) score derived from a Phred-scaled -10log10 transformation. The manufacturer default setting of AQ17, corresponding to a base accuracy of 98% (2% error rate), is applied. AQ17 allows longer effective read lengths while accommodating mismatches expected in variant-rich tumor samples. Decreases in AQ are expected in samples with higher mutational burden and are evaluated in the context of overall run and sample QC metrics.

# **d. Run - Level and Sample - Level Quality Control Checks**

# **i. Run - Level QC**

A sequencing run is considered valid only if all of the following criteria are met:

(1) The total sequencing output is ≥ 40 million reads per run.
(2) Acceptable alignment quality (AQ17).

Runs failing any of the above criteria are invalid, investigated, and repeated following troubleshooting and corrective actions.

# **ii. Sample – Level QC**

The sequencing performance is evaluated using the following criteria:

(1) For DNA libraries, the mean target depth is ≥ 500x and ≥ 90% of target amplification with ≥ 100 reads.
(2) For RNA Libraries, the total fusion-mapped reads per run is ≥ 500,000 and reads per RNA pool is ≥ 100,000.
(3) The Contamination Score is < 0.120

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Samples that fail QC due to specimen quality, extraction, or library preparation may be re-sequenced, re-prepared, or re-extracted according to the predefined decision rules.

# e. Mutation Calling: Single Nucleotide Variants (SNVs) and Insertions / Deletions (indels)

# i. Analysis of Positive and Negative Controls

Positive and negative controls are analyzed using the same coverage requirements as patient samples (mean depth ≥ 500× and ≥ 100× coverage across ≥ 90% of target regions). Expected variants must be detected in positive controls within validated variant allele frequency (VAF) ranges, and no reportable variants may be detected in negative controls.

# ii. Filters on Sample Coverage

Tumor samples must achieve a minimum mean sequencing depth of ≥ 500× to be eligible for variant calling and reporting.

# iii. Filtering for High-Confidence Mutations

Raw SNV and indel calls are subjected to locked filtering thresholds to ensure only high-confidence somatic variants are reported. Variants are classified as:

(1) Hotspot variants: VAF ≥ 2%, total depth ≥ 40, mutant reads ≥ 8, strand bias ≤ 0.9
(2) Non-hotspot variants: VAF ≥ 5%, total depth ≥ 40, mutant reads ≥ 10, strand bias ≤ 0.85

Variants are annotated using AMP/ASCO/CAP somatic guidelines, HGVS nomenclature, and major public databases (e.g., ClinVar, gnomAD, dbSNP, 1000 Genomes). Population variants with a frequency ≥ 5% in any gnomAD or 1000 Genomes subpopulation are excluded. Filtering thresholds are fixed for clinical use, and any modification requires formal change-control and re-validation.

# f. Mutation Annotation

Variants with functional consequences including missense, nonsense, frameshift, in-frame insertions/deletions, splice-site, and splice-region alterations are retained. Annotation incorporates gene context, predicted functional impact, and clinical relevance. Common polymorphisms present in population databases or internal normal datasets are excluded from somatic reporting.

# g. Tumor Purity Analysis

Tumor purity of the sequenced specimen is estimated bioinformatically using allele frequencies of informative variants and sequencing read distributions. This estimate may differ from the pathologist-reported tumor percentage due to macrodissection or sampling effects. When discrepancies or software limitations are identified, tumor purity is manually reviewed using pathologist estimates and driver mutation VAFs to ensure accurate interpretation of variant calls.

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### h. Copy Number Analysis

Copy number variation analysis is performed for ERBB2 using a variability-corrected informatics baseline derived from normal samples. Amplicon coverage log2 ratios are normalized and adjusted for tumor cellularity to estimate copy number and confidence intervals. ERBB2 amplification is reported as Positive when the observed copy number is ≥ 8.5. Copy numbers ≥ 4 and < 8.5 are reported as Indeterminate / Low-Level Amplification (below the LoD).

### i. Fusion Analysis

RNA-based fusion detection is validated for ALK, RET, and ROS1. Fusions are identified using split-read and discordant read-pair evidence. Clinically reportable fusions are reported only when ≥ 500 fusion-supporting reads are detected and validated against internal positive controls. Fusion calls rely exclusively on RNA-level evidence.

### 7. Controls

a. Positive Control (PC): An external control that consist of a mixture of well-characterized cell line DNA with known somatic variants at defined variant allele frequency (VAF), and RNA with defined fusions (Table 2) and is included in each assay run (batch level).
b. Negative Control (NC): An external control that is derived from genomic DNA and RNA of a known wild-type (WT) material (Table 2) and is included in each assay run (batch level).

Table 2. Positive and Negative Controls Used in the CellDx-Tissue Assay

| Control Type | Qualification |
| --- | --- |
| Positive Control DNA | Qualified by DCG |
| Positive Control RNA | |
| Negative Control DNA | |
| Negative Control RNA | |

c. No-Template Control (NTC): DNAse, RNAse free water is used as the no template control and will be included in each assay run (batch level).

### 8. Results Reporting

The CellDx-Tissue assay report provides structured information on detected genomic alterations, level of clinical significance and their clinical implications. The detected genomic alterations are categorized into Level 2 (Variants with Evidence of Clinical Significance) or Level 3 (Variants with Potential Clinical Significance). The CellDx-Tissue does not report mutations in 217 regions among 13473 interrogated target regions due to low coverage and high GC content. A list of all 517 genes is provided in Appendix A; and a list of excluded regions in the genes or genes with variants that are excluded due

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to challenging regions (e.g., low complexity / repeats) is provided in Appendix B and Appendix C, respectively.

### 9. Quality Metrics

Quality metrics are evaluated across the following categories:

- Run Level: Metrics that evaluate the overall sequencing performance, including instrument functions, reagent performance, data yield, and controls.
- Sample Level: Metrics that evaluate the sequencing quality, library performance, and coverage sufficiency.
- Variant (Analyte) Level: Metrics that are quantified for locus-level coverage and read support. Variants passing analyte-level QC are reported.

Table 3. Quality Control Metrics and Assay Cutoff

| Quality Metric | Frequency | Acceptance Criteria |
| --- | --- | --- |
| Specimen | Sample | Labels intact and Legible, Test Requisition Form(TRF) completely filled, consent signed, No loss of or damage to specimen, Number of FFPE blocks matches TRF, FFPE specimen identifiers match TRF and, LIMS entry |
| Specimen | Sample | >125mm² surface area; ≥3mm thick Tumor content ≥20% (Macro-dissect if <20%) |
| DNA Yield | Sample | >20 ng |
| RNA Yield | Sample | >20 ng |
| Library Yield | Sample | >100 pM |
| Total Data Output | Run | ≥ 40 million reads |
| Positive Control (PC) | Run (Batch) | DNA: All variants detected within expected VAF ranges RNA: All 6 fusions detected. |

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| Quality Metric | Frequency | Acceptance Criteria |
| --- | --- | --- |
| Negative Control (NC) | Run (Batch) | DNA: None of the 890 hotspot mutations should be detected RNA: No fusions detected. |
| Base Quality (AQ Score) | Run | AQ17 (98% accuracy) |
| Mean Target Coverage (DNA) | Sample | ≥ 500x. |
| Coverage Uniformity | Sample | ≥ 90% of target regions with ≥ 100x coverage |
| Contamination Score | Sample | ≤ 0.120 |
| Mean Target Coverage (RNA) | Sample | Total mapped fusion reads ≥ 500,000 Pool 1, Pool 2 (each): >100,000 reads |
| SNVs and Indels Calling | Variant | Mutation coverage (total depth) ≥ 40 Number of mutant reads (variant read count) ≥ 8 hotspot variants / ≥ 10 non-hotspot variants Variant Allele Fraction (VAF) ≥ 0.02 hotspot variants / ≥ 0.05 non-hotspot variants Strand bias < 0.9 hotspot variants / < 0.85 non-hotspot variants |
| *ERBB2* Amplification | Variant | CN ≥8.5 is Positive CN ≥4 to <8.5 is Indeterminate / Low Level Amplification CN <4 is Negative |
| *ALK*, *RET*, and *ROS1* Fusions | Variant | Positive where supporting read counts are ≥500 |

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| Quality Metric | Frequency | Acceptance Criteria |
| --- | --- | --- |
| Test Failure Criteria | Sample | DNA mean coverage < 500x, or Coverage uniformity < 90%, or Contamination score >0.120, or Fusion mapped reads <500,000, or Pool reads <100,000 |
| Reprocessed Specimen | Sample | All above QC criteria met |
| Report | Sample | Patient ID matches LIMS entry Accreditation Logos match standard template Signing authority signatures present |

### B. Principles of Operation

The CellDx-Tissue utilizes an amplicon-based sequencing approach to detect a broad range of clinically significant somatic alterations, including small nucleotide variants (SNVs), insertions and deletions (Indels), and ERBB2 gene amplification in tumor tissue DNA (ttDNA); and ALK, RET, and ROS1 fusions in tumor tissue RNA (ttRNA). The test requires FFPE tumor tissue specimen having a surface area of more than 125 mm², a thickness of at least 3 mm, with at least 20% tumor content. Specimens with lower tumor content may be enriched by macro-dissection. The assay involves target enrichment and deep sequencing of specific regions across a comprehensive panel of 517 genes, including oncogenes, tumor suppressor genes, and other clinically relevant genes. DNA and RNA primers are designed to target all coding exons, selected introns and targeted RNA sequences including chimeric fusion reads. Sequence libraries are prepared using DNA and RNA through a multiplex polymerase chain reaction (PCR) amplification step to enrich the target sequences. The target sequences are tagged with unique barcode oligonucleotides for individual sample identification and adaptor oligonucleotides that facilitate anchoring to the sequencing platform. These target sequences are then clonally amplified on microscopic beads using emulsion PCR before sequencing. Multiple barcoded sequence libraries are subsequently pooled and loaded onto a semiconductor sequencing chip. The resulting sequence reads are then aligned to the reference human genome (hg19), to identify gene variants.

### C. Determination of Assay Thresholds

#### 1. Requirements on Exon Coverage

A power analysis was conducted to estimate the minimum sequencing depth (total number of reads) needed to detect a mutation with a true underlying variant allele fraction (VAF) of 0.02 or greater, for varying levels of statistical 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 VAF as a function of sequencing depth were also calculated. This study showed that when

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a mutation is present at 0.1 VAF, the 95% CI with a sequencing depth of 500X is expected to fall between 0.075 and 0.13. When the overall coverage is 100X, the 95% CI for a mutation at 10% VAF is estimated to fall between 0.05 and 0.176.

To confirm these estimates, empirical data was obtained to measure the range of observed VAF for expected VAF, utilizing DNA from 20 formalin-fixed, paraffin-embedded (FFPE) normal tissue specimens from unrelated individuals. Equimolar parts of the DNA from these specimens were mixed to create five secondary specimen pools with a range of genetic variants (SNPs) having expected frequencies as low as 2%. A total of 863 common SNPs were considered for this study.

The observed variant frequencies for these common SNPs genotyped in the pooled normal samples, binned by their true underlying variant frequency is shown in Figure 1. The empirical data demonstrated a strong correlation between expected and observed VAF (Pearson's r = 0.99), with a slope of 0.99 and an intercept of 0.013. For SNPs with a true underlying variant fraction of 0.1, the observed variant fraction ranged from 0.069 to 0.138 when the mean coverage of the specimen was 1151X. This range is consistent with the theoretical statistical assessment for a depth of 500X (0.075 to 0.13). This data supports using 0.05 as the lower limit for reporting mutations detected with a true underlying frequency of 10%.


Figure 1. Observed vs. Expected Variant Allele Frequency for the CellDx-Tissue Assay

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## 2. Requirements on Sample Coverage

Twenty FFPE normal (diploid) specimens were profiled using CellDx-Tissue to generate summary statistics across all targeted exons. The mean coverage across all amplicons at optimal depth was 2087X (Range: 1420X-3369X, SD = 481X). The percentage of amplicons with >100X coverage was 99.3% (Range: 98.0-99.9%, SD = 0.5%). When mapped to the exon level, the mean coverage across all targeted exons was 2087X (Range: 1420X-3371X, SD = 482X). Summary statistics were also computed on coverage values per exon normalized by per-sample coverage. Exons with consistently low coverage (median normalized coverage < 0.05) were excluded from SNV/indel reporting, primarily due to high GC content (Appendix B), with the exception of two TERT promoter amplicons where variant calling parameters were relaxed with medical justification.

Sequence coverage was further evaluated to establish minimum criteria for the analysis and reporting of variants. Based on the power calculations, a minimum 100X coverage threshold per exon is required to call mutations with a true underlying mutation frequency of 10% or greater, with 95% power at an alpha level of 0.05 (Figure 2).


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Figure 2. Distribution of Mean and Medium Coverage Values for the Targeted Regions of the CellDx-Tissue Assay using High Coverage Samples. The dashed blue line indicates 100X coverage.

A second set of 20 samples were evaluated at lower sequencing depth. The mean coverage across all amplicons was 471X (Range: 284X-663X, SD = 101X). The percentage of amplicons with >100X coverage was 92.6% (Range: 86.6-96.2%, SD = 2.4%). When mapped to the exon level, the mean coverage across all targeted exons was 471X (Range: 284X-662X, SD = 101X). With the same remaining exons across all genes, 94.1% (Range: 88.0-97.7%, SD = 2.42%) were sequenced to a depth of 100X or greater. The distribution of these mean and medium coverage values for the targeted exons is shown in Figure 3.

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**Figure 3. Distribution of Mean and Medium Coverage Values for Targeted Regions of the CellDx – Tissue Assay with Low Coverage Samples.** The dashed blue line indicates 100X coverage.

Based on these two studies, the threshold of $\geq 90\%$ of amplicons achieving 100X coverage was established as a critical quality metric for the CellDx-Tissue assay.

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### 3. Requirements on Variant Calling Thresholds

Variant calling parameters such as sequence coverage (Alternate Allele Observation, AO), variant coverage (Coverage Depth, 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 VAF levels for reporting, particularly at 0.02 and 0.05 for hotspot and non-hotspot categories, respectively.

The cutoffs for AO, DP, and SB for hotspot variants and non-hotspot variants were established using two key development studies. The first study was designed to select the optimal parameter cutoff combination utilizing a dataset of 20 CellDx-Tissue runs using a Reference Standard. This involved evaluating multiple cutoffs for each of the three parameters, generating a total of 7,650 combinations. A list of 3,723 positive variants and 4,630 negative variants was used to evaluate the performance of each parameter combination, with the goal of achieving a Positive Predictive Value (PPV) greater than 96%. The relationship between these parameters and PPV, highlighting the selected cutoff combination: AO = 8, DP = 40, SB = 0.90 for hotspot variants; and AO = 10, DP = 40, SB = 0.85 for non-hotspot variants is illustrated in Figure 3.

The second study aimed to determine the optimal cutoff for Coverage Depth (DP) and minimum variant read count. This study used ten non-cancer FFPE samples and evaluated a total of 902 hotspot variants and 6,418 non-hotspot variants at different Coverage Depth cutoffs. The lowest Coverage Depth (DP) that effectively filtered out >99.7% of noise variants were selected, corresponding to a minimum of 8 reads for hotspot variants and 10 reads for non-hotspot variants.

### 4. Tumor-Only vs. Matched-Normal Germline Filtering

A study was conducted to demonstrate analytical equivalence between the CellDx-Tissue tumor-only (T/O) germline filtering approach and a matched tumor–normal (T/N) reference method, as required under 21 CFR 866.6080(b)(1)(iv). The study utilized 52 prospectively selected matched peripheral blood and tumor specimens representing 16 solid tumor types (including bladder / urinary tract (n = 1), bowel (n = 10), breast (n = 13), cervix (n = 2), esophagus / stomach (n = 1), head and neck (n = 3), liver (n = 1), lung (n = 6), ovary / fallopian tube (n = 5), pancreas (n = 3), peritoneum (n = 1), prostate (n = 1), testis (n = 1), thymus (n = 1), uterus (n = 2), and unknown primary (n = 1), and diverse ethnic representation (65% Asian, 27% White/Caucasian, 6% African American and 2% unspecified ethnicity from the US.

The T/O pipeline employed an in-silico filtering strategy that excluded variants with population frequency ≥5% in gnomAD (v2.1.1) or 1000 genome (v Phase 3v5), while the reference method used variants called using torrent variant caller (TVC, v5.18) with total white blood cell (WBC) genomic DNA (gDNA) as the germline control. Clinically actionable variants were defined as those with ClinVar (v 20250323) pathogenic/likely pathogenic classification or OncoKB (v 4.27) Level 1/2 oncogenic annotation with population frequency ≤0.1%. A total of 345 clinically actionable variants were evaluated, including germline pathogenic alterations in BRCA2, CHEK2, ATM, MSH2, PALB2, and RAD51D.

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The study demonstrated 100% PPA (345/345; 95% CI: 98.9% - 100%) and 100% NPA (2,745/2,745; 95% CI: 99.9% - 100%), with zero false negatives (FN) for actionable germline variants. Twenty-one (6.1%) actionable variants had VAFs between 0.02 -0.05, confirming assay sensitivity near the LoD. This analysis concluded the T/O filtering strategy is analytically equivalent to matched T/N subtraction.

### D. Substantial Equivalence Information

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

MSK-IMPACT

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

DEN170058

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

| Characteristics | Predicate Device: MSK-IMPACT | Subject Device: CellDx-Tissue |
| --- | --- | --- |
| **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 CellDx-Tissue is a qualitative in vitro diagnostic (IVD) test that uses next-generation sequencing of DNA and RNA isolated from formalin-fixed paraffine-embedded (FFPE) tumor tissue from patients previously diagnosed with solid malignant neoplasm to detect tumor gene alterations in a broad multi-gene panel. The test is intended to provide tumor mutation profiling information on somatic mutations, including single nucleotide variants (SNVs), insertions and deletions (indels), one gene amplification, and three fusions. |

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| Characteristics | Predicate Device: MSK-IMPACT | Subject Device: CellDx-Tissue |
| --- | --- | --- |
| **Similarities** | | |
| **Indications for Use** | 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. | Information provided by the CellDx-Tissue test is intended for use by qualified healthcare professionals in accordance with professional guidelines in oncology. Results from CellDx-Tissue are not intended to be conclusive or prescriptive for the labeled use of any specific therapeutic product. CellDx-Tissue is a single-site assay performed at Datar Cancer Genetics (DCG). |
| **Specimen Type** | Formalin-fixed, paraffin-embedded (FFPE) tumor tissue matched with normal specimens from patients with solid malignant neoplasms | Formalin-fixed, paraffin-embedded (FFPE) tumor tissue from patients with solid malignant neoplasms. |
| **Target Population** | Patients with solid malignant neoplasms | Same |
| **Test Environment** | Single Site Assay | Same |
| **Assay Cut-Off** | Does not report mutations below 2% for known hotspot mutations and 5% for non-hotspot mutations | Same |
| **Report Format** | Results are reported under one of the following categories: • Variants with Evidence of Clinical Significance • Variants with Potential Clinical Significance | Same |

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| Characteristics | Predicate Device: MSK-IMPACT | Subject Device: CellDx-Tissue |
| --- | --- | --- |
| **Similarities** | | |
| **Clinical Evidence Curation** | - Uses OncoKB, knowledge base - Classification criteria were developed by MSK to communicate the level of clinical evidence available for individual mutations in the test report. | Variant calls are organized into Variant with Evidence of Clinical Significance or Variant with Potential Clinical Significance depending on the designated cancer type. |
| **Differences** | | |
| **Genes on Panel** | 468 (6,357 exons) | 517 (6,597 exons) |
| **Variant Types** | Somatic Variants including point mutations and small insertions and deletions, and microsatellite instability | Somatic Variants including point mutations and small insertions and deletions, *ERBB2* amplification, and three fusions (*ALK*, *RET*, and *ROS1*) |
| **Black List** | 73 exons | 157 exons |
| **Analyte** | DNA | DNA and RNA |
| **Minimum Tumor Content** | 10% (20% preferred; 25% for MSI) | > 20% |
| **Nucleic Acid Input** | 50 ng – 250 ng | DNA: 20 ng – 50 ng RNA: 20 ng – 50 ng |
| **Sequencing Instrument** | Illumina HiSeq 2500 Sequencer | Thermo Fisher Ion GeneStudio S5 Prime System |
| **Technology** | Hybrid Capture | Amplicon |
| **Sequencing Chemistry** | Sequencing by Synthesis (Fluorescent Detection) | Sequencing by Synthesis (Hydrogen Ion Detection) |
| **Controls** | - Positive Control - Negative Control - No Template Control (NTC) - Matched Normal | - Positive Control - Negative Control - No Template Control (NTC) |

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| Characteristics | Predicate Device: MSK-IMPACT | Subject Device: CellDx-Tissue |
| --- | --- | --- |
| **Differences** | | |
| **Germline Filtering** | Matched normal specimen analysis | Computational, population database filtering |
| **Coverage Requirements** | ≥ 200x mean; 100x for ≥ 98% target regions | ≥ 500x; 100x for ≥ 90% target regions |
| **Variant Calling Thresholds** | Hotspot: • Mutant reads (AD) ≥ 8 • Mutation coverage (DP) ≥ 20 • Mutation frequency (VAF) ≥ 0.02 Non-hotspot • Mutant reads (AD) ≥ 10, • Mutation coverage (DP) ≥ 20 • Mutation frequency (VAF) ≥ 0.05 | Hotspot: • Mutant reads (AD) ≥ 8, • Mutation coverage (DP) ≥ 40 • Strand bias (SB) < 0.9 • Mutation frequency (VAF) ≥ 0.02 Non-hotspot • Mutant reads (AD) ≥ 10, • Mutation coverage (DP) ≥ 40 • Strand bias (SB) < 0.85 • Mutation frequency (VAF) ≥ 0.05 |
| **Contamination QC** | Percent heterozygous sites at fingerprint SNPs < 55%; Average MAF at homozygous fingerprint SNPs < 2% | Estimated contamination score < 0.120, Contamination score estimated from the signal derived from reference reads at homozygous alternate sites. |
| **Criteria for Calling “Failed” Samples** | If a sample presents with mean coverage across all exons < 50x and no mutations are detected due to the low overall coverage, the test is deemed “failed” for the sample | If a sample presents with mean coverage across all exons < 500x for DNA and <500,000 reads for RNA, the test is deemed “failed” for the sample. |

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## **E. Standards/Guidance Documents Referenced**

The following FDA guidance documents and standards were consulted:

1. 1. Medical Devices – Quality Management Systems: Requirements for Regulatory Purposes. ISO 13485: 2016
2. 2. Medical Devices – Application of Risk Management to Medical Devices. ISO 14971: 2019
3. 3. Medical Laboratories – Requirements for Quality and Competence. ISO 15189: 2022
4. 4. Medical Devices – Symbols to be used with Information to be Supplies by the Manufacturer – Part 1: General Requirements. ISO 15223-1: 2021
5. 5. Medical Device Software – Software Life Cycle Processes. ISO / IEC 62304: 2006
6. 6. Safety Requirements for Electrical Equipment for Measurement, Control, and Laboratory Use – Part 1: General Reequipments. IEC 61010-1: 2010
7. 7. Electrical Equipment for Measurement, Control and Laboratory Use – EMC Requirements – Part 1: General Requirements. EN 61326-1: 2013
8. 8. Evaluation of Stability of In Vitro Diagnostic Reagents; Approved Guideline - CLSI EP25-A
9. 9. Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline - Second Edition. CLSI EP17-A2
10. 10. Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline - Third Edition. CLSI EP05-A3
11. 11. User Protocol for Evaluation of Qualitative Test Performance; Approved Guideline - Second Edition. CLSI EP12-A2

## **F. Performance Characteristics**

### **1. Analytical Performance – General**

The CellDx – Tissue assay is a targeted NGS panel with 517 genes. The targeted regions of interest in CellDx – Tissue are designed to detect SNVs, insertions up to 20 bp and deletions up to 30 bp in length of the targeted genes, as well as *ERBB2* amplifications, *ALK*, *RET*, and *ROS1* fusions. For SNVs and indels, a representative approach to validation of the targeted genes in the panel was submitted with data representing variant types for SNVs and indels, and at the gene levels for amplifications and fusions indicated with this assay. In addition, the assay was evaluated for performance regarding the panel-wide quality metrics.

#### ***a. Invalid Rates***

Multiple factors can influence the overall robustness and performance of complex molecular tests, including pre-analytical factors and overall sample quality. If key in-process or automated data quality metrics are not met, the CellDx – Tissue assay supports repeat samples through the workflow once. Performance throughout the verification and validation of the device was tracked, and a summary of the rates for first pass (no repeat) and overall pass (allowing for a single repeat) are presented in

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Table 4. Data were aggregated for clinical cases from >25 tumor types. Resulting pass rates by tumor type across the workflow are shown in Table 5.

**Table 4. Acceptability Rates of CellDx – Tissue Assay**

| Clinical FFPE Samples | Acceptability Rate (n/N) (95% CI) |
| --- | --- |
| First Pass | 81.8% (2190/2676) (80.33%–83.25%) |
| After Repeat Test | 93.1% (2492/2676) (92.10%–94.02%) |

**Table 5. Comparability of Tumor Invalid Rates for the CellDx – Tissue Assay**

| Organ | Number of Samples Failed | | | | Invalid Rate (n/N) (%) |
| --- | --- | --- | --- | --- | --- |
| | Pre-analytical | Pre-run | Post-Run | | |
| | Low Tumor Purity^{1} | Low Nucleic Acid Yield^{2} | Library Failure^{3} | Data QC Failure | |
| Adrenal Gland | 0 | 0 | 0 | 0 | (0/2) 0% |
| Ampulla of Vater | 1 | 0 | 0 | 0 | (1/33) 3% |
| Biliary Tract | 0 | 0 | 0 | 0 | (0/7) 0% |
| Bladder/Urinary Tract | 0 | 0 | 0 | 2 | (2/20) 10% |
| Bone | 0 | 0 | 0 | 0 | (0/2) 0% |
| Bowel | 6 | 4 | 2 | 35 | (47/479) 10% |
| Breast | 3 | 5 | 2 | 3 | (13/317) 4% |
| Cervix | 0 | 0 | 0 | 0 | (0/41) 0% |
| CNS/Brain | 0 | 1 | 1 | 3 | (5/67) 7% |
| Esophagus/Stomach | 0 | 0 | 0 | 9 | (9/115) 8% |
| Head and Neck | 12 | 3 | 0 | 17 | (32/220) 15% |
| Kidney | 0 | 0 | 0 | 0 | (0/18) 0% |
| Liver | 0 | 0 | 0 | 0 | (0/12) 0% |
| Lung | 2 | 3 | 0 | 38 | (43/695) 6% |
| Ovary/Fallopian Tube | 0 | 1 | 0 | 3 | (4/73) 5% |
| Ovary/Fallopian Tube, Uterus | 0 | 0 | 0 | 0 | (0/3) 0% |
| Pancreas | 2 | 0 | 0 | 2 | (4/27) 15% |
| Peritoneum | 0 | 0 | 0 | 0 | (0/2) 0% |

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| Organ | Number of Samples Failed | | | | Invalid Rate (n/N) (%) |
| --- | --- | --- | --- | --- | --- |
| | Pre-analytical | Pre-run | Post-Run | | |
| | Low Tumor Purity^{1} | Low Nucleic Acid Yield^{2} | Library Failure^{3} | Data QC Failure | |
| Pleura | 0 | 1 | 0 | 1 | (2/2) 100% |
| Prostate | 0 | 0 | 0 | 6 | (6/217) 3% |
| Skin | 0 | 0 | 5 | 7 | (12/51) 24% |
| Soft Tissue | 1 | 0 | 0 | 0 | (1/18) 6% |
| Testis | 0 | 0 | 0 | 1 | (1/2) 50% |
| Thymus | 0 | 0 | 0 | 0 | (0/4) 0% |
| Thyroid | 0 | 0 | 0 | 0 | (0/88) 0% |
| Unknown Primary | 0 | 0 | 0 | 0 | (0/2) 0% |
| Uterus | 0 | 0 | 0 | 2 | (2/155) 1% |
| Vulva/Vagina | 0 | 0 | 0 | 0 | (0/4) 0% |
| **Total** | **27** | **18** | **10** | **129** | **(184/2676) 6.9%** |

$^{1}$ < 20 %

$^{2}$ < 20 ng

$^{3}$ No amplification

### b. Invalid Rates Observed in Method Comparison

Invalid rates were infrequent across all tested biomarkers and were consistently within the acceptance criteria for the CellDx-Tissue assay workflow. A summary of the invalid rates observed during the method comparison study is shown in Table 6.

Table 6. Invalid Rates Observed in the Method Comparison Study.

| Category | DNA | RNA | Total |
| --- | --- | --- | --- |
| 1st Pass Valid | 233 | 221 | 454 |
| After Repeat Test Valid | 10 | 23 | 33 |
| Low Nucleic Acid Yield (<20 ng) | 14 | 6 | 17 |
| Library Failure | 1 | 0 | 1 |
| Invalid (Sequencing QC Failure) | 7 | 5 | 12 |
| Total Invalid | 22 | 11 | 33 |
| Total Attempts | 265 | 252 | 517 |
| **Total Invalid (%)** | **8.30%** | **4.4%** | **6.38%** |

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## 2. Precision

### a. Precision Panel

The within lab precision (between-run and within-run) of the CellDx – Tissue assay was assessed using 17 FFPE clinical samples originating from eight primary tumor types (Bowel (n = 5, 29.4%), Lung (n = 4, 23.5%), Prostate (n = 2, 11.8%), CNS (n = 1, 5.9%), Breast (n = 1, 5.9%), Ovary (n = 2, 11.8%), Esophagus (n=1, 5.9%) and Thyroid (n = 1, 5.9%) to represent different variants and a range of frequencies. Extracted DNA and RNA from each of the 17 samples was tested in duplicate by two different operators on multiple sequencing instruments across ≥3 non-consecutive days using two reagent lots, yielding 24-48 replicates per sample. For each replicate tested, all observed variants which passed the CellDx – Tissue assay QC metrics were reported and assessed for precision.

### b. Panel-Wide Precision

Precision was accessed for each variant across all replicates. The positive call rates (PCR) and negative call rates (NCR) were calculated along with the two-sided 95% confidence intervals.

Table 7 summarizes the positive call rate (PCR) and negative call rate (NCR) stratified by mutation type (SNV, insertions, and deletions) and variant allele frequency (VAF). An overall PCR of 98.69% across all samples and replicates (2871/2909; 95% CI: 98.21% – 99.05%; Average VAF range: 2.0 – 85.52), with an increase in PCR at higher VAFs observed, and an overall NCR of 100% (533539/533539; 95% CI: 99.99, 100.00).

The positive call rates for selected individual sequence mutations assessed in the precision study, along with the VAF range, mean SD, and percent CV per variant are presented in Appendix D. A total of 48 SNVs and 15 indels (3 insertions, 15 deletions) are provided.

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Table 7. Precision Positive (PCR) and Negative (NCR) Call Rates

| Variant Type | VAF Level (%) | Unique Mutations | PCR (%) (n/N) | NCR (%) (n/N) | AF Range (%) | Mean Allele Depth/ FSR Range | Mean Loci Depth Rang |
| --- | --- | --- | --- | --- | --- | --- | --- |
| All | AF ≥ 0 | 63 | 98.69 (2871/2909) | 100 (533539/533539) | 2.00 - 85.52 | 46 - 1455 | 440 - 1984 |
| | AF ≥ 2.0 | 63 | 98.69 (2871/2909) | 100 (533539/533539) | 2.00 - 85.52 | 46 - 1455 | 440 - 1984 |
| | AF ≥ 5.0 | 60 | 98.99 (2758/2786) | 100 (533539/533539) | 3.20 - 85.52 | 46 - 1455 | 440 - 1978 |
| | AF ≥ 10.0 | 53 | 99.23 (2451/2470) | 100 (450769/450769) | 7.43 - 85.52 | 82 - 1455 | 440 - 1978 |
| | AF ≥ 15.0 | 48 | 99.15 (2211/2230) | 100 (450769/450769) | 15.44 - 85.52 | 142 - 1455 | 440 - 1978 |
| Variants with Evidence of Clinical Significance | AF ≥ 0 | 10 | 99.56 (455/457) | 100 (365377/365377) | 2.26 - 79.57 | 74 - 1455 | 604 - 1978 |
| | AF ≥ 2.0 | 10 | 99.56 (455/457) | 100 (365377/365377) | 2.26 - 79.57 | 74 - 1455 | 604 - 1978 |
| | AF ≥ 5.0 | 9 | 100 (411/411) | 100 (324483/324483) | 20.07 - 79.57 | 290 - 1455 | 604 - 1978 |
| | AF ≥ 10.0 | 9 | 100 (411/411) | 100 (324483/324483) | 20.07 - 79.57 | 290 - 1455 | 604 - 1978 |
| | AF ≥ 15.0 | 9 | 100 (411/411) | 100 (324483/324483) | 20.07 - 79.57 | 290 - 1455 | 604 - 1978 |
| Hotspot Variants | AF ≥ 0 | 24 | 97.99 (1073/1095) | 100 (409829/409829) | 2.00 - 60.77 | 46 - 1092 | 737 - 1984 |
| | AF ≥ 2.0 | 24 | 97.99 (1073/1095) | 100 (409829/409829) | 2.00 - 60.77 | 46 - 1092 | 737 - 1984 |
| | AF ≥ 5.0 | 21 | 98.77 (960/972) | 100 (409829/409829) | 3.20 - 60.77 | 46 - 1092 | 737 - 1978 |
| | AF ≥ 10.0 | 17 | 99.25 (791/797) | 100 (409829/409829) | 15.44 - 60.77 | 214 - 1092 | 810 - 1978 |
| | AF ≥ 15.0 | 17 | 99.25 (791/797) | 100 (409829/409829) | 15.44 - 60.77 | 214 - 1092 | 810 - 1978 |

N/A: Not Applicable; FSR: Fusion Supporting Reads

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| Variant Type | VAF Level (%) | Unique Mutations | PCR (%) (n/N) | NCR (%) (n/N) | AF Range (%) | Mean Allele Depth/ FSR Range | Mean Loci Depth Rang |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Non-hotspot Variants | AF ≥ 0 | 39 | 99.12 (1798/1814) | 100 (407205/407205) | 5.04 - 85.52 | 63 - 1455 | 440 - 1977 |
| | AF ≥ 2.0 | 39 | 99.12 (1798/1814) | 100 (407205/407205) | 5.04 - 85.52 | 63 - 1455 | 440 - 1977 |
| | AF ≥ 5.0 | 39 | 99.12 (1798/1814) | 100 (407205/407205) | 5.04 - 85.52 | 63 - 1455 | 440 - 1977 |
| | AF ≥ 10.0 | 36 | 99.22 (1660/1673) | 100 (324435/324435) | 7.43 - 85.52 | 82 - 1455 | 440 - 1977 |
| | AF ≥ 15.0 | 31 | 99.09 (1420/1433) | 100 (324435/324435) | 17.53 - 85.52 | 142 - 1455 | 440 - 1977 |
| SNVs | AF ≥ 0 | 48 | 99.13 (2172/2191) | 100 (449879/449879) | 2.00 - 79.57 | 46 - 1455 | 449 - 1984 |
| | AF ≥ 2.0 | 48 | 99.13 (2172/2191) | 100 (449879/449879) | 2.00 - 79.57 | 46 - 1455 | 449 - 1984 |
| | AF ≥ 5.0 | 45 | 99.56 (2059/2068) | 100 (449879/449879) | 3.20 - 79.57 | 46 - 1455 | 449 - 1978 |
| | AF ≥ 10.0 | 41 | 99.84 (1891/1894) | 100 (408049/408049) | 10.33 - 79.57 | 101 - 1455 | 449 - 1978 |
| | AF ≥ 15.0 | 39 | 99.83 (1795/1798) | 100 (408049/408049) | 15.44 - 79.57 | 142 - 1455 | 449 - 1978 |
| Insertions | AF ≥ 0 | 3 | 100 (144/144) | 100 (85248/85248) | 7.43 - 46.99 | 82 - 571 | 758 - 1430 |
| | AF ≥ 2.0 | 3 | 100 (144/144) | 100 (85248/85248) | 7.43 - 46.99 | 82 - 571 | 758 - 1430 |
| | AF ≥ 5.0 | 3 | 100 (144/144) | 100 (85248/85248) | 7.43 - 46.99 | 82 - 571 | 758 - 1430 |
| | AF ≥ 10.0 | 3 | 100 (144/144) | 100 (85248/85248) | 7.43 - 46.99 | 82 - 571 | 758 - 1430 |
| | AF ≥ 15.0 | 1 | 100 (48/48) | 100 (42720/42720) | 35.92 - 46.99 | 571 - 571 | 1375 - 1375 |
| Deletions | AF ≥ 0 | 12 | 96.69 (555/574) | 100 (254204/254204) | 3.54 - 85.52 | 69 - 578 | 440 - 1699 |
| | AF ≥ 2.0 | 12 | 96.69 (555/574) | 100 (254204/254204) | 3.54 - 85.52 | 69 - 578 | 440 - 1699 |
| | AF ≥ 5.0 | 12 | 96.69 (555/574) | 100 (254204/254204) | 3.54 - 85.52 | 69 - 578 | 440 - 1699 |
| | AF ≥ 10.0 | 9 | 96.30 (416/432) | 100 (170736/170736) | 9.28 - 85.52 | 108 - 578 | 440 - 1699 |
| | AF ≥ 15.0 | 8 | 95.83 (368/384) | 100 (170736/170736) | 15.64 - 85.52 | 269 - 578 | 440 - 1699 |

N/A: Not Applicable; FSR: Fusion Supporting Reads

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| Variant Type | VAF Level (%) | Unique Mutations | PCR (%) (n/N) | NCR (%) (n/N) | AF Range (%) | Mean Allele Depth/ FSR Range |
| --- | --- | --- | --- | --- | --- | --- |
| *ERBB2* Amplification | 1 | 100 (120/120) | - | N/A | N/A | N/A |
| *ALK* Fusion | 1 | 100 (45/45) | - | N/A | 7121-30568 | N/A |
| *ROS1* Fusion | 1 | 95.0 (38/40) | - | N/A | 504-2328 | N/A |
| *RET* Fusion | 1 | 100 (46/46) | - | N/A | 740-175619 | N/A |

N/A: Not Applicable; FSR: Fusion Supporting Reads

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

The precision was calculated for each individual specimen as shown in Table 8. Results from the precision studies were combined across all reportable genes for each specimen. The positive and negative call rates for sequenced mutations (SNVs and indels) in each sample were calculated based on the total number of mutations along with the two-sided 95% CI.

**Table 8. Positive and Negative Call Rates per Sample**

| Sample ID | Cancer Type | Unique Mutations Detected* | PCR Per Mutation | PCR (%) (n/N) (95% CI) | NCR (%) (n/N) (95% CI) |
| --- | --- | --- | --- | --- | --- |
| Sample 1 | Ovary/ Fallopian Tube | 4 | 45/48 for 1*** | 98.43 189/192 (95.5, 99.5) | 100 42672/42672 (99.99, 100) |
| | | | 48/48 for 3 | | |
| Sample 2 | Thyroid | 1 | 48/48 for 1 | 100 48/48 (92.6, 100.0) | 100 42672/42672 (99.99, 100) |
| Sample 3 | Prostate | 1 | 48/48 for 1 | 100 48/48 (92.6, 100.0) | 100 42672/42672 (99.99, 100) |
| Sample 4 | Bowel | 6 | 25/31 for 1** | 94.09, 175/186, (88.0, 95.5) | 100, 42672/42672, (99.99,100) |
| | | | 26/31 for 1** | | |
| | | | 31/31 for 4** | | |

*Across the 48 Replicates

**Reduction in the number of replicates due to QC failure

***Variant with VAF at LoD

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| Sample ID | Cancer Type | Unique Mutations Detected* | PCR Per Mutation | PCR (%) (n/N) (95% CI) | NCR (%) (n/N) (95% CI) |
| --- | --- | --- | --- | --- | --- |
| Sample 5 | Lung | 1 | 46/48 for 1*** | 95.83, 46/48, (85.8, 99.5) | 100, 42720/42720, (100, 100) |
| Sample 6 | Bowel | 16 | 42/48 for 2*** | 98.04, 753/768, (96.8, 98.8) | 99.99, 42522/42528, (99.97,99.99) |
| | | | 45/48 for 1*** | | |
| | | | 48/48 for 13 | | |
| Sample 7 | Lung | 1 | 47/48 for 1 | 97.91, 47/48, (89.1, 99.6) | 99.91, 42634/42672, (99.88,99.94) |
| Sample 8 | Prostate | 2 | 46/46 for 2** | 100, 92/92, (96.0, 100) | 100, 40940/40940, (99.99,100) |
| Sample 9 | Lung | 6 | 48/48 for 6 | 100, 288/288, (98.7, 100) | 100, 42623/42624, (99.99,100) |
| Sample 10 | Bowel | 12 | 45/48 for 1*** | 99.3, 572/576, (98.7, 100) | 100, 42432/42432, (99.99,100) |
| | | | 47/48 for 1*** | | |
| | | | 48/48 for 10 | | |
| Sample 11 | CNS/ Brain | 4 | 48/48 for 4 | 100, 192/192, (98.0, 100) | 99.94, 42646/42672, (99.91,99.96) |
| Sample 12 | Bowel | 3 | 44/46 for 1**,*** | 97.1, 134/138, (98.0, 100) | 100, 40801/40802, (99.99,100) |
| | | | 44/46 for 1** | | |
| | | | 46/46 for 1** | | |
| Sample 13 | Breast | 6 | 47/48 for 1*** | 99.65, 287/288, (98.1, 99.9) | 100 42623/42624, (99.99, 100) |
| | | | 48/48 for 5 | | |

*Across the 48 Replicates

**Reduction in the number of replicates due to QC failure

***Variant with VAF at LoD

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# **d. Precision for *ERBB2* Amplification**

Precision of *ERBB2* amplification was evaluated using 15 samples. Three out of the 15 samples were tested at two DNA input levels using two different operators on two instruments across three non-consecutive days using two kit lots, yielding 12 replicates per sample per input level. The remaining samples were tested in duplicate by two different operators on six instruments across four non-consecutive days using two kit lots, yielding 24 replicates per sample per input level. Of the 15 samples, four contained *ERBB2* amplifications and the remaining samples contained no *ERBB2* amplifications. The CellDx-Tissue reports an *ERBB2* amplification when the observed copy number for the gene is determined by the test to be 8.5 copies or more. The tumor purity, input, mean observed copy number (CN), coefficient of variation (%CV), positive and negative call rates along with the 95% CIs for each sample are summarized in Table 9. The call rates were 100% for both amplification and no amplification groups.

**Table 9. Summary of the *ERBB2* Gene Amplification Precision**

| Sample ID | Cancer Type | *ERBB2* Status | Tumor Purity (%) | Input (ng) | Mean CN | CN CV (%) | PCR % (n/N) (95% CI) | NCR % (n/N) (95% CI) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample 1 | Breast | Positive | 58 | 10 | 16.3 | 11.0 | 100 (24/24) (85.7, 100) | - |
| | | | | 20 | 16.4 | 7.0 | 100 (24/24) (85.7, 100) | - |
| Sample 2 | Bowel | Positive | 21 | 10 | 8.06 | 6.3 | 100 (12/12) (75.8, 100) | - |
| | | | | 20 | 7.82 | 4.9 | 100 (12/12) (75.8, 100) | - |
| Sample 3 | Esophagus | Positive | 49 | 10 | 15.57 | 10.2 | 100 (12/12) (75.8, 100) | - |
| | | | | 20 | 15.34 | 7.7 | 100 (12/12) (75.8, 100) | - |
| Sample 4 | Ovarian / Fallopian Tube | Positive | 55 | 10 | 14.54 | 7.1 | 100 (12/12) (75.8, 100) | - |
| | | | | 20 | 14.02 | 7.0 | 100 (12/12) (75.8, 100) | - |

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| Sample ID | Cancer Type | ERBB2 Status | Tumor Purity (%) | Input (ng) | Mean CN | CN CV (%) | PCR % (n/N) (95% CI) | NCR % (n/N) (95% CI) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample 5 | CNS | Negative | 92 | 10 | 1.98 | 5.1 | - | 100 (24/24) (86.2, 100) |
| | | | | 20 | 1.98 | 4.6 | - | 100 (24/24) (86.2, 100) |
| Sample 6 | Bowel | Negative | 37 | 10 | 1.34 | 15.2 | - | 100 (24/24) (86.2, 100) |
| | | | | 20 | 1.32 | 14.9 | - | 100 (24/24) (86.2, 100) |
| Sample 7 | Lung | Negative | 28 | 10 | 1.97 | 8.8 | - | 100 (24/24) (86.2, 100) |
| | | | | 20 | 1.93 | 8.8 | - | 100 (24/24) (86.2, 100) |
| Sample 8 | Prostate | Negative | 22 | 10 | 1.79 | 18.6 | - | 100 (24/24) (86.2, 100) |
| | | | | 20 | 1.75 | 13.7 | - | 100 (22/22) (85.1, 100) |
| Sample 9 | Lung | Negative | 20 | 10 | 1.28 | 15.1 | - | 100 (24/24) (86.2, 100) |
| | | | | 20 | 1.28 | 16.4 | - | 100 (24/24) (86.2, 100) |
| Sample 10 | Bowel | Negative | 29 | 10 | 1.54 | 5.7 | - | 100 (24/24) (86.2, 100) |
| | | | | 20 | 1.54 | 10.3 | - | 100 (24/24) (86.2, 100) |
| Sample 11 | Lung | Negative | 45 | 10 | 1.58 | 11.8 | - | 100 (24/24) (86.2, 100) |
| | | | | 20 | 1.62 | 9.2 | - | 100 (24/24) (86.2, 100) |

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| Sample ID | Cancer Type | ERBB2 Status | Tumor Purity (%) | Input (ng) | Mean CN | CN CV (%) | PCR % (n/N) (95% CI) | NCR % (n/N) (95% CI) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample 12 | Bowel | Negative | 39 | 10 | 2.87 | 24.2 | - | 100 (7/7) (59.0, 100) |
| | | | | 20 | 2.70 | 8.0 | - | 100 (24/24) (86.2, 100) |
| Sample 13 | Prostate | Negative | 36 | 10 | 1.98 | 6.6 | - | 100 (24/24) (86.2, 100) |
| | | | | 20 | 2.08 | 5.7 | - | 100 (24/24) (86.2, 100) |
| Sample 14 | Thyroid | Negative | 22 | 10 | 1.99 | 2.9 | - | 100 (24/24) (86.2, 100) |
| | | | | 20 | 1.99 | 3.3 | - | 100 (24/24) (86.2, 100) |
| Sample 15 | Ovary | Negative | 56 | 10 | 1.52 | 13.7 | - | 100 (24/24) (86.2, 100) |
| | | | | 20 | 1.58 | 9.1 | - | 100 (24/24) (86.2, 100) |

# **e. Precision for ALK, RET, and ROS1 Fusions**

The precision of RNA fusions was evaluated across different samples with varying tumor purities. The results are summarized in Table 10.

**Table 10. Summary of ALK, RET, and ROS1 Fusions**

| Fusion | Tumor Purity (%) | LoD Level | Mean Supporting Reads (Range) | PCR (%) n/N (95% CI) | NCR (%) n/N (95% CI) |
| --- | --- | --- | --- | --- | --- |
| ALK | 20 | 1x* | 15062 (7121-30568) | 100 45/45 (92.1, 100) | 100 609/609 (99.4, 100) |

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| Fusion | Tumor Purity (%) | LoD Level | Mean Supporting Reads (Range) | PCR (%) n/N (95% CI) | NCR (%) n/N (95% CI) |
| --- | --- | --- | --- | --- | --- |
| *RET* | 25 | 1.8x* | 24438 (740-175619) | 100 46/46 (92.3, 100) | 100 608/608 (99.4, 100) |
| *ROS1* | 45 | 0.8x** | 1273 (504-2328) | 95 38/40 (90.8, 100) | 100 614/614 (99.4, 100) |

# *f. Analysis of Source of Variance*

The Average Positive Agreement (APA) and Average Negative Agreement (ANA) was assessed to analyze the imprecision caused by different sources of variance. Data analysis is presented stratified by variant type and present for 1) overall, 2) instrument to instrument, 3) operator to operator, and 4) day to day (Table 11).

**Table 11. Reproducibility of the CellDx – Tissue Assay**

| Alteration Type | Metric | Overall (%) (95% CI) | Inter-Instrument (%) (95% CI) | Inter-Operator (%) (95% CI) | Inter-Day (%) (95% CI) |
| --- | --- | --- | --- | --- | --- |
| Variants with Evidence of Clinical Significance | APA | 99.56 (98.42, 99.88) | 99.56 (95.19, 100) | 99.57 (98.34, 100) | 99.57 (96.74, 100) |
| | ANA | 100 (100, 100) | 100 (100, 100) | 100 (100, 100) | 100 (100, 100) |
| Hotspot Variants | APA | 97.99 (96.98, 98.67) | 97.94 (95.27, 99.44) | 97.95 (96.21, 98.74) | 97.99 (95.31, 98.99) |
| | ANA | 100 (100, 100) | 100 (100, 100) | 100 (100, 100) | 100 (100, 100) |
| Non-hotspot Variants | APA | 99.12 (98.57, 99.46) | 99.11 (97.62, 99.82) | 99.11 (98.27, 99.55) | 99.12 (97.76, 99.66) |
| | ANA | 100 (100, 100) | 100 (100, 100) | 100 (100, 100) | 100 (100, 100) |
| SNVs | APA | 99.13 (98.65, 99.44) | 99.11 (97.61, 99.72) | 99.12 (98.33, 99.50) | 99.13 (97.88, 99.61) |
| | ANA | 100 (100, 100) | 100 (100, 100) | 100 (100, 100) | 100 (100, 100) |

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| Alteration Type | Metric | Overall (%) (95% CI) | Inter-Instrument (%) (95% CI) | Inter-Operator (%) (95% CI) | Inter-Day (%) (95% CI) |
| --- | --- | --- | --- | --- | --- |
| Insertions (All) | APA | 100 (97.40, 100) | 100 (86.20, 100) | 100 (94.93, 100) | 100 (90.36, 100) |
| | ANA | 100 (100, 100) | 100 (100, 100) | 100 (100, 100) | 100 (100, 100) |
| Insertions (1 – 5 bp) | APA | 100 (97.40, 100) | 100 (86.20, 100) | 100 (94.93, 100) | 100 (90.36, 100) |
| | ANA | 100 (100, 100) | 100 (100, 100) | 100 (100, 100) | 100 (100, 100) |
| Insertions (5 – 10 bp) | APA | N/A | N/A | N/A | N/A |
| | ANA | 100 (100, 100) | 100 (100, 100) | 100 (100, 100) | 100 (100, 100) |
| Insertions (11 – 30 bp) | APA | N/A | N/A | N/A | N/A |
| | ANA | 100 (100, 100) | 100 (100, 100) | 100 (100, 100) | 100 (100, 100) |
| Deletions (All) | APA | 96.69 (94.89, 97.87) | 96.69 (89.77, 98.37) | 96.69 (93.71, 98.10) | 96.69 (92.13, 98.51) |
| | ANA | 100 (100, 100) | 100 (100, 100) | 100 (100, 100) | 100 (100, 100) |
| Deletions (1 – 5 bp) | APA | 96.39 (94.43, 97.68) | 96.38 (88.89, 98.22) | 96.38 (93.14, 97.92) | 96.39 (91.44, 98.37) |
| | ANA | 100 (100, 100) | 100 (100, 100) | 100 (100, 100) | 100 (100, 100) |
| Deletions (5 – 10 bp) | APA | N/A | N/A | N/A | N/A |
| | ANA | 100 (100, 100) | 100 (100, 100) | 100 (100, 100) | 100 (100, 100) |
| Deletions (11 – 30 bp) | APA | 100 (92.59, 100) | 100 (67.56, 100) | 100 (86.20, 100) | 100 (75.75, 100) |
| | ANA | 100 (100, 100) | 100 (100, 100) | 100 (100, 100) | 100 (100, 100) |

N/A: Not Available / Applicable

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| Alteration Type | Metric | Overall (%) (95% CI) | Inter-Instrument (%) (95% CI) | Inter-Operator (%) (95% CI) | Inter-Day (%) (95% CI) |
| --- | --- | --- | --- | --- | --- |
| *ERBB2* Amplification | APA | 100 (92.59, 100) | 100 (67.56, 100) | 100 (86.20, 100) | 100 (75.75, 100) |
| | ANA | 100 (99.25, 100) | 100 (95.68, 100) | 100 (98.51, 100) | 100 (97.06, 100) |
| *ALK* Fusion | APA | 100 (92.13, 100) | 100 (60.97, 100) | 100 (79.61, 100) | 100 (74.12, 100) |
| | ANA | 100 (99.20, 100) | 100 (94.65, 100) | 100 (97.64, 100) | 100 (96.87, 100) |
| *RET* Fusion | APA | 100 (92.29, 100) | 100 (64.57, 100) | 100 (79.61, 100) | 100 (75.75, 100) |
| | ANA | 100 (99.20, 100) | 100 (94.65, 100) | 100 (97.64, 100) | 100 (96.87, 100) |
| *ROS1* Fusion | APA | 95.00 (83.50, 98.62) | 91.67 (43.65, 96.99) | 95.83 (77.19, 100) | 95.00 (72.25, 100) |
| | ANA | 100 (99.20, 100) | 100 (94.65, 100) | 100 (97.64, 100) | 100 (96.87, 100) |

N/A: Not Available / Applicable

### g. Lot-to-Lot Precision

The performance of the CellDx – Tissue assay was assessed across two unique kit lots by determining the concordance of variant calls in 17 FFPE tissue samples. The two unique lots were used to process 372 test cases in duplicate for a total of 744 observations. All batches were sequenced on the same instrument. Table 12. lists the APA and ANA used to assess lot to lot performance. The overall panel-wide variants (SNVs, Insertions and Deletions) APA is 98.68% and the ANA is 100%. ERBB2 amplification, ALK, and RET fusions had a concordance of 100%; and ROS1 fusions demonstrated a concordance of 93.75%.

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**Table 12. Lot-to-Lot Precision of CellDx – Tissue Assay**

| Variant Type | Performance | Between Lot 1& Lot 2 (%) (95% CI) |
| --- | --- | --- |
| Variants with Evidence of Clinical Significance | APA | 99.57 (98.34, 100) |
| | ANA | 100 (100, 100) |
| Panel-Wide (SNVs + Insertions + Deletions) | APA | 98.68 (97.97, 99.16) |
| | ANA | 100 (100, 100) |
| Hotspot SNVs (Including Clinically Significant Variants) | APA | 97.95 (96.21, 98.74) |
| | ANA | 100 (100, 100) |
| Non-hotspot SNVs (Including Clinically Significant Variants) | APA | 99.11 (98.27, 99.55) |
| | ANA | 100 (100, 100) |
| SNVs (Hotspot + Non-hotspot) | APA | 99.12 (98.33, 99.50) |
| | ANA | 100 (100, 100) |
| Insertions | APA | 100 (94.93, 100) |
| | ANA | 100 (100, 100) |
| Deletions | APA | 96.69 (93.71, 98.10) |
| | ANA | 100 (100, 100) |
| *ERBB2* Amplification | APA | 100 (86.20, 100) |
| | ANA | 100 (98.51, 100) |

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| Variant Type | Performance | Between Lot 1& Lot 2 (%) (95% CI) |
| --- | --- | --- |
| ALK Fusion | APA | 100 (85.13, 100) |
| | ANA | 100 (98.41, 100) |
| RET Fusion | APA | 100 (85.69, 100) |
| | ANA | 100 (98.41, 100) |
| ROS1 Fusion | APA | 93.75 (76.39, 99.11) |
| | ANA | 100 (98.41, 100) |

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

The LoD of the CellDx – Tissue assay for SNVs and indels is defined as the lowest concentration with ≥ 95% of replicates for a variant are reliably detected. The LoD of the CellDx – Tissue assay for ERBB2 amplification is determined as the minimum tumor purity required for robust reporting of amplification status and incorporated the clinical reporting framework where CN ≥ 8.5 is Positive, CN ≥ 4 to < 8.5 is Indeterminate / Low-Level Amplification (below LoD), and CN < 4 is Negative. The LoD of the CellDx – tissue assay for ALK, RET, and ROS1 fusions is determined as the minimum tumor purity required for robust reporting of fusion status. The recommended nucleic acid input for the CellDx – Tissue assay is 20 ng of DNA and 20 ng of RNA recovered from tissue with a minimum of 20% viable tumor nuclei. Details of the data are discussed and shown below.

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

The analytical sensitivity of the CellDx – Tissue assay for SNVs, insertions, and deletions was evaluated by assessing 10 clinical FFPE specimens from seven different cancer types with 29 SNVs, 1 insertion, and 2 deletions. Each sample was diluted to at least five dilution levels and tested in 10 replicates per level using two reagent lots. The call rate was determined for each variant and the LoD was approximated between the call rate that was below 95% and the highest call rate (100%). A summary of the estimation of the LoD range for a set of representative variants is shown in Table 13. The established VAF range for each variant type (Hotspot SNVs, Non-hotspot SNVs, insertions, and deletions) is shown in Table 14.

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Table 13. Estimation of LoD Range for Representative Variants

| Tumor Type | Variant Type | Level | Gene | AA Change | Avg DP | Avg AD | VAF Range | Mean VAF (%) | PCR (%) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Lung | SNV | 3 | TP53 | p.R175H | 1869 | 68 | 0.026-0.048 | 3.7 | 100 |
| Lung | SNV | 5 | TET2 | c.3954+3T>G | 1426 | 63 | 0.024-0.098 | 4.3 | 100 |
| Lung | DEL | 5 | EGFR | p.E746_A750del | 1939 | 69 | 0.026-0.058 | 3.5 | 100 |
| Breast | SNV | 2 | GPS2 | c.318-6C>T | 1886 | 123 | 0.056-0.078 | 6.5 | 100 |
| Breast | SNV | 4 | PIK3CA | p.H1047R | 1678 | 58 | 0.022-0.049 | 3.5 | 100 |
| Uterus | SNV | 2 | TP53 | p.R273C | 1955 | 75 | 0.024-0.053 | 3.8 | 100 |
| Uterus | SNV | 2 | CTLA4 | p.A54T | 2000 | 132 | 0.054-0.078 | 6.6 | 100 |
| Uterus | SNV | 3 | CTNNB1 | p.G34E | 1959 | 74 | 0.026-0.045 | 3.7 | 100 |
| Head and Neck | SNV | 2 | NRAS | p.A59T | 2000 | 107 | 0.044-0.071 | 5.4 | 100 |
| Head and Neck | SNV | 3 | DICER1 | c.3269+6C>T | 1921 | 128 | 0.045-0.13 | 6.8 | 100 |
| Head and Neck | SNV | 4 | TP53 | p.G262V | 1885 | 75 | 0.026-0.047 | 3.9 | 100 |
| Head and Neck | SNV | 3 | NF2 | p.R196* | 659 | 48 | 0.059-0.104 | 6.7 | 100 |
| Head and Neck | SNV | 4 | EP300 | c.3728+5G>A | 1271 | 56 | 0.035-0.052 | 4.0 | 100 |
| Head and Neck | SNV | 4 | FAT1 | p.R1205* | 1919 | 118 | 0.05-0.078 | 6.1 | 100 |

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| Tumor Type | Variant Type | Level | Gene | AA Change | Avg DP | Avg AD | VAF Range | Mean VAF (%) | PCR (%) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Head and Neck | SNV | 4 | *LATS1* | p.Q953* | 1603 | 56 | 0.028-0.047 | 3.4 | 100 |
| Head and Neck | SNV | 3 | *PHF6* | p.Y301* | 938 | 52 | 0.041-0.077 | 5.7 | 100 |
| Bowel | SNV | 2 | *TP53* | p.R273H | 1925 | 127 | 0.052-0.077 | 6.6 | 100 |
| Esophagus / Stomach | INS | 4 | *ARID1A* | p.A1136Gfs*57 | 1503 | 92 | 0.042-0.088 | 6.2 | 100 |
| Esophagus / Stomach | SNV | 3 | *TP53* | p.R209* | 1924 | 165 | 0.064-0.16 | 8.5 | 100 |
| Esophagus / Stomach | SNV | 3 | *POT1* | p.E456* | 1281 | 71 | 0.032-0.107 | 5.5 | 100 |
| Lung | SNV | 1 | *TP53* | p.R248W | 1797 | 124 | 0.043-0.094 | 6.9 | 100 |
| Uterus | SNV | 2 | *FANCA* | c.2015-8C>T | 1765 | 101 | 0.04-0.066 | 5.7 | 100 |
| Uterus | SNV | 2 | *TP53* | p.Y220C | 1287 | 85 | 0.055-0.083 | 6.6 | 100 |
| Uterus | SNV | 2 | *PPP2R1A* | p.P179R | 1708 | 57 | 0.022-0.05 | 3.4 | 100 |
| Prostate | SNV | 5 | *MLH1* | p.Q346H | 1781 | 82 | 0.033-0.056 | 4.1 | 100 |
| Prostate | SNV | 5 | *AR* | p.L702H | 1579 | 83 | 0.044-0.072 | 5.4 | 100 |
| Lung | INS | 2 | *ARID1A* | p.P1468Lfs*13 | 938 | 88 | 0.076-0.122 | 9.5 | 100 |
| Lung | SNV | 5 | *TCF7L2* | c.1162-12C>T | 1720 | 134 | 0.039-0.144 | 8.0 | 100 |
| Lung | SNV | 3 | *TP53* | p.Y126* | 1911 | 151 | 0.029-0.154 | 7.2 | 100 |
| Lung | SNV | 4 | *FANCE* | c.855+6T>A | 1029 | 64 | 0.026-0.095 | 5.2 | 100 |
| Lung | SNV | 2 | *KMT2C* | p.E1333* | 891 | 105 | 0.045-0.214 | 11.5 | 100 |
| Lung | SNV | 3 | *KMT2D* | c.840-10G>A | 1861 | 147 | 0.025-0.153 | 7.3 | 100 |

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**Table 14. Analysis Sensitivity (LoD VAF) for Representative SNVs and Indels**

| Variant | Established VAF Range (%) | Tumor Purity (%) | Average Allele Depth | Number of Variants in Clinical Cases in the Established Range |
| --- | --- | --- | --- | --- |
| Hotspot SNVs | 2.2 – 9.4 | 7 | 57 – 124 | 10 |
| Non-hotspot SNVs | 2.4 – 21.4 | 4 – 24 | 63 – 105 | 19 |
| Insertions | 4.2 – 8.8 | 6 | 92 | 1 |
| Deletions | 2.6 – 12.2 | 4 – 24 | 69 – 88 | 2 |
| Homopolymer Context | 4.2 – 8.8 | 6 | 92 | 1 |
| Non-Homopolymer Context | 2.6 – 12.2 | 4 – 24 | 69 – 88 | 2 |

# **b. LoD – Amplifications and Fusions (Tumor Purity)**

# **i. ERBB2 Amplification**

The analytical sensitivity of *ERBB2* was assessed by testing one clinical breast cancer sample diluted with normal FFPE DNA to five detection levels. Each dilution level was run in 10 replicates per level per reagent lot (two lots), resulting in a total of 50 observations. The cutoff for *ERBB2* detection is an observed copy number $\geq 8.5$. The data summarized in Table 15 showed 100% call rates for samples with a tumor purity of 15%.

**Table 15. Analysis Sensitivity for *ERBB2* Amplification by the CellDx-Tissue Assay**

| Dilution Level | Tumor Purity (%) | Mean Observed Copy Number (Mean $\pm$ SD) | Observed Copy Number Range | Positive Call Rate (%) (n/N) |
| --- | --- | --- | --- | --- |
| 1 | 60 | 30.2 $\pm$ 0.8 | 29–32 | 100 (10/10) |
| 2 | 30 | 15.0 $\pm$ 0.7 | 14–16 | 100 (10/10) |
| 3 | 15 | 8.5 $\pm$ 0.2 | 8–11 | 100 (10/10) |

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| Dilution Level | Tumor Purity (%) | Mean Observed Copy Number (Mean ± SD) | Observed Copy Number Range | Positive Call Rate (%) (n/N) |
| --- | --- | --- | --- | --- |
| 4 | 8 | 5.1 ± 0.9 | 4–7 | 20 (2/10) |
| 5 | 4 | 3.15 ± 0.1 | 3–3 | 0 (0/10) |

# ii. *ALK*, *RET*, and *ROS1* RNA Fusions

The analytical sensitivity of *ALK*, *RET*, and *ROS1* was assessed by testing four clinical (three lung and one bowel) cancer samples. *ALK* and *RET* clinical samples were diluted with normal FFPE DNA to five dilution levels. *ROS1* clinical samples were diluted to nine dilution levels. Each dilution level was run in 10 replicates per level (across two lots), resulting in a total of 50 observations for *ALK* and *RET* and 90 observations for *ROS1*. The data is summarized in Table 16 – Table 19.

**Table 16. Analysis Sensitivity for *ALK* Amplification by the CellDx-Tissue Assay**

| Dilution Level | Tumor Purity (%) | Fusion Read Counts | Mean Fusion Read Range | Positive Call Rate (%) (n/N) |
| --- | --- | --- | --- | --- |
| 1 | 20 | 4868 | 1640-8989 | 100 (10/10) |
| 2 | 15 | 1048 | 603-1544 | 80 (8/10) |
| 3 | 11 | 1104 | 734-1506 | 30 (3/10) |
| 4 | 8 | 1014 | 642-1409 | 30 (3/10) |
| 5 | 6 | 931 | 740-1049 | 30 (3/10) |

**Table 17. Analysis Sensitivity for *RET* Amplification by the CellDx-Tissue Assay**

| Dilution Level | Tumor Purity (%) | Fusion Read Counts | Mean Fusion Read Range | Positive Call Rate (%) (n/N) |
| --- | --- | --- | --- | --- |
| 1 | 25 | 11534 | 5788-19474 | 100 (10/10) |
| 2 | 19 | 7321 | 3996-10730 | 100 (7/7) |
| 3 | 14 | 4282 | 1366-8416 | 100 (10/10) |
| 4 | 11 | 7604 | 2120-16136 | 100 (7/7) |

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| Dilution Level | Tumor Purity (%) | Fusion Read Counts | Mean Fusion Read Range | Positive Call Rate (%) (n/N) |
| --- | --- | --- | --- | --- |
| 5 | 8 | 6095 | 600-18193 | 86 (6/7) |

**Table 18. Analysis Sensitivity for *ROS1* Amplification by the CellDx-Tissue Assay**

| Dilution Level | Tumor Purity (%) | Fusion Read Counts | Mean Fusion Read Range | Positive Call Rate (%) (n/N) |
| --- | --- | --- | --- | --- |
| 1 | 70 | 80775 | 28692-130814 | 100 (10/10) |
| 2 | 53 | 41636 | 28054-58784 | 100 (10/10) |
| 3 | 39 | 24685 | 5883-39701 | 100 (10/10) |
| 4 | 30 | 46067 | 9422-123494 | 100 (10/10) |
| 5 | 22 | 9000 | 5861-14743 | 100 (10/10) |
| 6 | 12 | 11498 | 4925-33280 | 100 (9/9) |
| 7 | 9 | 5692 | 1785-13236 | 100 (10/10) |
| 8 | 7 | 4393 | 1015-13653 | 100 (10/10) |
| 9 | 5 | 4346 | 1605-7058 | 100 (10/10) |
| 10* | 20 | 39,875 | 34,176 - 49,210 | 100 (5/5) |
| 11* | 10 | 30,333 | 22,285 - 42,599 | 100 (5/10) |
| 12* | 7 | 1,853 | 1,158 - 2,641 | 100 (5/5) |
| 13* | 5 | 1,599 | 892 - 3,587 | 100 (5/5) |
| 14* | 4 | 942 | 715 - 1,431 | 70 (7/10) |
| 15* | 3 | 883 | 554 - 1,141 | 30 (3/10) |
| 16* | 2 | - | - | 0 (0/0) |

*Samples and dilution levels are from a Supplemental Study to determine the tumor purity LoD for *ROS1*.

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**Table 19. Analysis Sensitivity Summary of RNA Fusion**

| Gene | Tumor Purity Range (%) | Fusion Read Count Range | LoD Tumor Purity (%) | LoD Supporting Reads |
| --- | --- | --- | --- | --- |
| *ALK* | 20 – 6 | 4868-931 | 20 | >1640 |
| *RET* | 25 – 8 | 11533–6095 | 14 | >1366 |
| *ROS1** | 20 – 2 | 39875-883 | 5 | >892 |

\*Supplemental LoD Study

#### **4. Linearity/Assay Reportable Range**

Not applicable

#### **5. Traceability (Controls, Calibrators, or Methods)**

##### ***a. Traceability***

The CellDx-Tissue assay is not traceable to any known standard. Controls and quality metrics are described in the device description section.

##### ***b. Stability***

Reagent stability is based on the manufacturer expiration dating, verified by Datar Cancer Genetics. Stability of the reagents is monitored through the use of consistent controls.

##### ***c. Expected Values (Controls, Calibrators, or Methods)***

The external positive control (commercial vendor qualified by Datar Cancer Genetics) contains different confirmed mutations, representing a range of mutation allele frequencies. A negative external control (commercial vendor qualified by Datar Cancer Genetics) contains a non-cancerous cell line derived DNA /RNA with no variants of interest. Both external controls are processed from library preparation through sequencing to serve as an end-to-end control to demonstrate assay performance. Failure of either external control to meet the quality control metrics will result in all test samples on the run be flagged and re-sequenced.

#### **6. Analytical Specificity**

##### ***a. Cut-Off / False Positive Range (Limit of Blank)***

Non-cancerous FFPE tissues were evaluated for analytical specificity to access the risk of false positives in normal tissue when detecting SNVs, indels, amplifications, and fusions using the CellDx – Tissue assay. A total of 37 normal or benign-adjacent tissue were processed across two reagent lots, multiple operators, and days. Ninety (90) false positive events were detected for SNVs and indels for a false positive rate (FPR) of 0.000054 (95 CI: 0.000043 - 0.000066). None of the detected variants were classified as Variants with Evidence of Clinical Significance (Level 2), while four were classified as

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Variants with Potential Evidence of Clinical Significance (Level3). The FPR was determined to be 0 (95 CI: 0 - 3.09) for *ERBB2* amplifications, and *ALK*, *RET*, and *ROS1* fusion genes.

# **b. Necrotic Tissue and Tumor Block Age**

The impact of necrosis on the performance of the CellDx – Tissue assay was evaluated by assessing the invalid rates for both DNA and RNA libraries, defined as the proportion of libraries that failed to meet the predefined quality control thresholds. All 378 FFPE DNA and 377 FFPE RNA samples were evaluated for necrosis over a range of 0 - 80 % and invalid rates were examined. The assay performance was assessed and compared to the predicate device. The DNA and RNA invalid rates ranged from 0 to 10.52% and 0 to 1.78%, respectively (Table 20). The PPA for overall variant (SNVs, insertions, and deletions) detected ranged from 97.8% to 100%, while an NPA of 99.5% was observed (Table 21).

**Table 20. Necrotic Tissue Invalid Rate for the CellDx-Tissue Assay**

| Cancer Types | Sample Count | Necrotic Tissue Content (%) | Invalid Rate (%) (n/N) |
| --- | --- | --- | --- |
| DNA | | | |
| 23 | 281 | 0 – 5 | 1.78 (5/281) |
| 15 | 72 | 5 – 20 | 0 (0/72) |
| 12 | 19 | 21 – 40 | 10.52 (2/19) |
| 4 | 6 | 41 – 80 | 0 (0/6) |
| RNA | | | |
| 23 | 281 | 0 – 5 | 1.42 (4/281) |
| 15 | 71 | 5 – 20 | 1.40 (1/71) |
| 12 | 19 | 21 – 40 | 0 (0/19) |
| 4 | 6 | 41 – 80 | 0 (0/6) |

**Table 21. Concordance of Overall Variant (SNV + Insertions + Deletions) Detection**

| Necrotic Tissue Content (%) | Sample Count | PPA (%) (95 CI) (n/N) | NPA (%) (95 CI) (n/N) |
| --- | --- | --- | --- |
| 0 – 5 | 86 | 97.8 (93.8, 99.3) (135/138) | 99.6 (99.4, 99.7) (13140/13197) |

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| Necrotic Tissue Content (%) | Sample Count | PPA (%) (95 CI) (n/N) | NPA (%) (95 CI) (n/N) |
| --- | --- | --- | --- |
| >5 – 20 | 28 | 100 (91.0, 100) (39/39) | 99.2 (98.9, 99.4) (4268/4303) |
| 21 – 40 | 8 | 100 (77.2, 100) (13/13) | 99.8 (99.3, 99.9) (1224/1227) |
| 41 – 53 | 1 | 100 (34.2, 100) (2/2) | 99.3 (96.4, 99.9) (153/153) |
| Overall | 123 | 98.4 (95.5, 99.5) (189/192) | 99.5 (99.4, 99.6) (18784,18880) |

### c. Interfering Substances

The impact of interfering substances on the performance of the CellDx – Tissue assay was assessed by processing DNA and RNA from FFPE samples tested in the presence of each interfering substance at varying amounts (Table 22). The samples were evaluated for concordance of variant call when compared to samples processed without the interfering substances. Replicates for six test cases were analyzed for six experimental and one baseline condition. Performance was evaluated across nine samples x seven conditions x five replicates. Analysis of all variant types tested (SNVs, indels, amplifications, and fusions showed no effect of exogenous and endogenous interferent for all conditions (Table 23 – Table 25).

Table 22. Endogenous and Exogenous Interfering Substances Tested

| Type | Substance | Concentration Spiked | |
| --- | --- | --- | --- |
| | | At Extraction | At Library Preparation |
| Exogenous | Ethanol | 3x | 5% |
| Exogenous | Index Adaptor | - | 3x |
| Exogenous | Proteinase K | 3x | 0.04 mg/mL |
| Exogenous | Wash Buffer | 3x | 5% |
| Endogenous | Melanin | 0.2 mg/mL | - |
| Endogenous | Hemoglobin | 2 mg/mL | - |

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**Table 23. Interfering Substances Concordance by Test Condition for SNVs and Indels**

| Test Condition | PCR (%) (n/N) (95% CI) | NCR (%) (n/N) (95% CI) |
| --- | --- | --- |
| Ethanol | 99.2 (119/120) (95.4, 99.9) | 100 (106080/106080) (99.99, 100) |
| Adaptor | 98.8 (115/120) (90.6, 98.2) | 100 (106080/106080) (99.99, 100) |
| Proteinase K | 94.9 (112/118) (89.3, 97.6) | 100 (104312/104312) (99.99, 100) |
| Wash Buffer | 98.3 (118/120) (94.1, 99.5) | 100 (106080/106080) (99.99, 100) |
| Melanin | 100 (110/110) (96.6, 100.0) | 100 (97240/97240) (99.99, 100) |
| Hemoglobin | 98.3 (118/120) (94.1, 99.5) | 100 (106080/106080) (99.99, 100) |

**Table 24. Interfering Substances Concordance by Test Condition for *ERBB2***

| Test Condition | PCR (%) (n/N) (95% CI) | NCR (%) (n/N) (95% CI) |
| --- | --- | --- |
| Ethanol | 100 (10/10) (69.2, 100.0) | 100 (35/35) (90.1, 100.0) |
| Adaptor | 100 (10/10) (69.2, 100.0) | 100 (35/35) (90.1, 100.0) |
| Proteinase K | 100 (10/10) (69.2, 100.0) | 100 (35/35) (90.1, 100.0) |
| Wash Buffer | 100 (10/10) (69.2, 100.0) | 100 (35/35) (90.1, 100.0) |
| Melanin | 100 (10/10) (69.2, 100.0) | 100 (30/30) (88.6, 100.0) |

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| Test Condition | PCR (%) (n/N) (95% CI) | NCR (%) (n/N) (95% CI) |
| --- | --- | --- |
| Hemoglobin | 100 (10/10) (69.2, 100.0) | 100 (35/35) (90.1, 100.0) |

**Table 25. Interfering Substances Concordance by Test Condition for RNA Fusions (ALK, RET, and ROS1)**

| Test Condition | PCR (%) (n/N) (95% CI) | NCR (%) (n/N) (95% CI) |
| --- | --- | --- |
| Ethanol | 100 (15/15) (78.2, 100) | 100 (30/30) (88.4, 100) |
| Adaptor | 100 (15/15) (78.2, 100) | 100 (30/30) (88.4, 100) |
| Proteinase K | 100 (15/15) (78.2, 100) | 100 (29/29) (88.1, 100) |
| Wash Buffer | 100 (15/15) (78.2,…