TruSight Oncology Comprehensive

{0} # SUMMARY OF SAFETY AND EFFECTIVENESS DATA (SSED) ## I. GENERAL INFORMATION | Device Generic Name: | Next generation sequencing oncology panel, somatic or germline variant detection system | | --- | --- | | Device Trade Name: | TruSight™ Oncology Comprehensive | | Device Procode: | PQP | | Applicants' Name and Address: | Illumina Inc. 5200 Illumina Way, San Diego, CA 92122 | | Date(s) of Panel Recommendation: | None | | Premarket Approval Application (PMA) Number: | P230011/S006 | | Date of FDA Notice of Approval: | August 14, 2026 | The original PMA (P230011) for TruSight Oncology Comprehensive (TSO Comp) was approved on August 21, 2024, for the detection of genetic alterations in patients who may benefit from an FDA-approved therapy (larotrectinib) for solid tumors and an FDA-approved therapy (selpercatinib) for non-small cell lung cancer (NSCLC). The test also was approved to provide tumor profiling information for use by qualified health care professionals in accordance with professional guidelines in oncology for patients with solid malignant neoplasms. The SSED to support these indications is available on the CDRH website and is incorporated by reference here. The current supplement was submitted to expand the indications for TSO Comp to include companion diagnostic (CDx) indications for the detection of RET fusions in thyroid cancer (TC) and RET small DNA variant mutations in medullary thyroid cancer (MTC) patients who may benefit from treatment with RETEVMO (selpercatinib). ## II. INDICATIONS FOR USE TruSight™ Oncology Comprehensive is a qualitative in vitro diagnostic test that uses targeted next-generation sequencing to detect variants in 517 genes using nucleic acids extracted from formalin-fixed, paraffin embedded (FFPE) tumor tissue samples from cancer patients with solid malignant neoplasms using the Illumina® NextSeq™ 550Dx instrument. The test can be used to detect single nucleotide variants, multi-nucleotide variants, insertions, and deletions from DNA, and fusions in 24 genes and splice variants in one gene from RNA. The test also reports a Tumor Mutational Burden (TMB) score and Microsatellite Instability (MSI) status. The test is intended to be used as a companion diagnostic to identify cancer patients who may benefit from treatment with the targeted therapies listed in Table 1, in accordance with the approved therapeutic product labeling. PMA P230011/S006: FDA Summary of Safety and Effectiveness Data Page 1 of 63 {1} In addition, the test is intended to provide tumor profiling information for use by qualified health care professionals in accordance with professional guidelines in oncology for patients with solid malignant neoplasms. Genomic findings other than those listed in Table 1 of the intended use statement are not conclusive or prescriptive for labeled use of any specific therapeutic product. Table 1: Companion Diagnostic Indications | Tumor Type | Biomarker(s) Detected | Therapy | | --- | --- | --- | | Solid Tumors | NTRK1/2/3 fusions | VITRAKVI® (larotrectinib) | | Non-Small Cell Lung Cancer (NSCLC) or Thyroid Cancer (TC) | RET fusions | RETEVMO® (selpercatinib) | | Medullary Thyroid Cancer (MTC) | RET SNVs, MNVs, insertions and deletions | RETEVMO® (selpercatinib) | ### III. CONTRAINDICATIONS There are no known contraindications. ### IV. WARNINGS AND PRECAUTIONS The warnings and precautions can be found in the TruSight Oncology Comprehensive labeling. ### V. DEVICE DESCRIPTION TruSight Oncology (TSO) Comprehensive is a distributed in vitro diagnostic (IVD) test. The assay is for use as part of a test system with the NextSeq 550Dx instrument and sequencing reagents. The TSO Comprehensive assay contains reagents, software, and procedures for testing DNA and RNA extracted from formalin-fixed, paraffin-embedded (FFPE) tumor samples. The assay employs qualitative next-generation sequencing (NGS) comprehensive genomic profiling (CGP) that assesses genomic variants in a large panel of cancer-related genes. It is an enrichment based targeted NGS test comprised of library preparation and enrichment reagents to enable DNA and/or RNA sequencing from FFPE tissue from solid tumors encompassing multiple cancer types. DNA and/or RNA extracted from FFPE tissue is used to prepare libraries, which are then enriched for cancer-related genes and sequenced on the NextSeq 550Dx instrument. TSO Comprehensive user-facing software allows sequencing run set up, secondary analysis, and annotation of detected variants from the sequencing results generated on the NextSeq 550Dx instrument. TSO Comprehensive can be run with DNA and RNA, DNA only, or RNA only samples. The reported results will be reflective of the input nucleic acid. The test detects single nucleotide variants (SNVs), multi-nucleotide variants (MNVs) and insertions and deletions from DNA, and gene fusions and a splice variant from RNA. The assay detects small DNA variants in 517 genes, RNA fusions in 24 genes and RNA splice variants in one gene. The TSO Comprehensive assay also reports a Tumor Mutational Burden (TMB) score and Microsatellite Instability (MSI) status. PMA P230011/S006: FDA Summary of Safety and Effectiveness Data Page 2 of 63 {2} ## Test Output The output of the test includes results from the following levels: - Level 1: Companion Diagnostic (CDx) Claims noted in Table 1 of the Intended Use - Level 2: Cancer Mutations with Evidence of Clinical Significance - Level 3: Cancer Mutations with Evidence of Potential Clinical Significance Genomic findings other than those listed in Table 1 of the Companion diagnostic indications table of the intended use statement (i.e., Levels 2 and 3) are not prescriptive or conclusive for labeled use of any specific therapeutic product. ## Test Kit Contents The TSO Comprehensive assay is for use as part of a test system with the NextSeq 550Dx instrument and associated sequencing reagents. Components of the TSO Comprehensive assay are listed in Table 2. Illumina provides reagent kits and software (Local Run Manager TSO Comprehensive (US) analysis module), which supports both tumor profiling and companion diagnostic (CDx) claims (please refer to the “Software” section below for additional details). The Knowledge Base (updated monthly) is also required to be used with the assay for tumor profiling and is available for download on the Illumina Lighthouse Portal. The assay contains reagents with sufficient volume to generate 24 DNA and 24 RNA libraries inclusive of patient samples and controls (sold separately). Materials required but not provided are described in the text below Table 2. A detailed list of required instruments, software, reagents, consumables, and storage conditions is further described in the product labeling (TSO Comprehensive Package Insert). Table 2. Reagent Components of the TSO Comprehensive Assay | Storage Temp | Component Name | Volume (μL) | Quantity | | --- | --- | --- | --- | | TSO Comprehensive RNA Library Prep | | | | | -25 to -15 | First Strand Synthesis Mix (FSM) | 260 | 1 | | -25 to -15 | Second Strand Mix (SSM) | 720 | 1 | | -25 to -15 | Elution Primer Frag Mix (EPH3) | 250 | 1 | | -25 to -15 | Reverse Transcriptase (RVT) | 70 | 1 | | TSO Comprehensive Library Prep | | | | | -25 to -15 | End Repair A-tailing A (ERA1-A) | 85 | 2 | | -25 to -15 | End Repair A-tailing B (ERA1-B) | 210 | 2 | | -25 to -15 | Adapter Ligation Buffer 1 (ALB1) | 1730 | 2 | | -25 to -15 | DNA Ligase 3 (LIG3) | 190 | 2 | | -25 to -15 | Short Universal Adapters 1 (SUA1) | 290 | 1 | | -25 to -15 | UMI Adapters v1 (UMI) | 290 | 1 | | -25 to -15 | Stop Ligation Buffer (STL) | 480 | 2 | | -25 to -15 | Enhanced PCR Mix (EPM) | 550 | 2 | | 2 to 8 | Resuspension Buffer (RSB) | 12400 | 1 | | 2 to 8 | Sample Purification Beads (SPB) | 6110 | 2 | | 2 to 8 | TE Buffer (TEB) | 10000 | 1 | | TSO Comprehensive Index Primers | | | | PMA P230011/S006: FDA Summary of Safety and Effectiveness Data Page 3 of 63 {3} | Storage Temp | Component Name | Volume (μL) | Quantity | | --- | --- | --- | --- | | -25 to -15 | UP Index Primers | 24 | 16 | | -25 to -15 | CP Index Primers | 20 | 16 | | TSO Comprehensive Enrichment | | | | | 2 to 8 | Target Capture Buffer 1 (TCB1) | 870 | 2 | | 2 to 8 | Streptavidin Mag Beads (SMB) | 7780 | 2 | | 2 to 8 | 2N NaOH (HP3) | 400 | 2 | | 2 to 8 | Elute Target Buffer 2 (ET2) | 290 | 2 | | 2 to 8 | Library Normalization Beads 1 (LNB1) | 1040 | 1 | | 2 to 8 | Library Normalization Wash 1 (LNW1) | 4800 | 2 | | 2 to 8 | Library Normalization Storage Buffer 1 (LNS1) | 3500 | 2 | | 2 to 8 | Resuspension Buffer (RSB) | 12400 | 1 | | 2 to 8 | Sample Purification Beads (SPB) | 6110 | 2 | | -25 to -15 | Target Capture Additives 1 (TCA1) | 521 | 2 | | -25 to -15 | Enhanced Enrichment Wash (EEW) | 50400 | 1 | | -25 to -15 | Enrichment Elution 2 (EE2) | 1650 | 3 | | -25 to -15 | Enhanced PCR Mix (EPM) | 550 | 2 | | -25 to -15 | PCR Primer Cocktail 3 (PPC3) | 150 | 2 | | -25 to -15 | Library Normalization Additives 1 (LNA1) | 4600 | 1 | | -25 to -15 | PhiX Internal Control (PX3 or PhiX) | 10 | 1 | | TSO Comprehensive Content Set | | | | | -25 to -15 | Oncology RNA Probe Pool (OPR1) | 290 | 1 | | -25 to -15 | Oncology DNA Probe Pool 2 (OPD2) | 290 | 1 | ### Reagents/Consumables Required but not Provided For a detailed list of required, but not provided reagents and consumables, please refer to the product labeling (TSO Comprehensive Package Insert). - DNA/RNA Extraction and Purification Reagents - DNA/RNA Quantification Reagents - TruSight Oncology DNA Control v2 - TruSight Oncology RNA Control v2 - Ethanol (EtOH) 100% (200 proof), molecular biology grade - RNase/DNase-free water - NextSeq 550Dx High-Output Reagent Kit v2.5 (300 cycles) ### Required Equipment/Materials, Not Provided For a detailed list of required, but not provided equipment, please refer to the product labeling (TSO Comprehensive Package Insert). - NextSeq 550Dx Instrument - Ultrasonicator - Thermal cycler - Vortexer - Microsample incubators PMA P230011/S006: FDA Summary of Safety and Effectiveness Data Page 4 of 63 {4} - Microcentrifuge - Plate Centrifuge - Plate shaker - Sealing wedge or roller - Magnetic Stand - Precision pipettes - Pipette-aid - Ice or cold block - 10 mL serological pipettes - Adhesive seals for 96-well plates - Microcentrifuge tubes (1.7mL and 2mL), nuclease-free - Nuclease-free reagent reservoirs - 15mL and 50mL conical tubes - Aerosol-resistant pipette tips - 96-well storage plates, 0.8 mL - 96-well PCR plates compatible with thermal cycler, 0.2 ml (polypropylene wells) - Dry heat block ## Software The Local Run Manager TSO Comprehensive (US) analysis module resides on the NextSeq 550Dx instrument as part of the Local Run Manager software to facilitate TSO Comprehensive assay run setup and to perform the secondary analysis of sequencing results. It contains the following components: - TSO Comprehensive Claims Packages - TSO Comprehensive Software Suite - TSO Comprehensive USB Kit Knowledge Base: Updated regularly and available for download on the Illumina Lighthouse Portal. ## Instruments TSO Comprehensive assay is validated for use on the Illumina NextSeq 550Dx instrument as part of a test system. An Illumina service representative installs the appropriate version of the TSO Comprehensive (US) analysis module on the Local Run Manager NextSeq 550Dx instrument, including the Knowledge Base, prior to use of the TSO Comprehensive assay and conducts training for the end user. Other required equipment and the specifications for the specific equipment for use with the TSO Comprehensive assay are described in the “Required Equipment/Materials, Not Provided” section. ## Principles of Operation The TSO Comprehensive assay involves the processes described below. ## Specimen Requirements, Collection, and Preparation The TSO Comprehensive assay requires nucleic acid (DNA and/or RNA) isolated from FFPE tissue specimens. Before performing the TSO Comprehensive assay, tissue samples should be examined by a pathologist to ensure that it is appropriate for this test. Tissue cannot be decalcified and should be fixed using formalin fixative suitable for molecular analyses (for example, 10% neutral-buffered PMA P230011/S006: FDA Summary of Safety and Effectiveness Data Page 5 of 63 {5} formalin). Recommended tissue volume is ≥1.0 mm³, which is equivalent to a cumulative viable tissue area (the sum of the viable tissue area in all sections submitted for extraction) of ≥200 mm² using 5 μm thick sections, or ≥100 mm² using 10 μm thick sections. Tumor content for RNA variants depends on the extent of expression. A minimum of 20% tumor content (by area) is required to detect somatic driver mutations. A minimum of 40% tumor content is necessary for robust reporting of MSI status. An outcome of MS-Stable may not be reliable if tumor content is less than 40%. A high amount of necrotic tissue (≥25%) can interfere with the TSO Comprehensive assay's ability to detect RNA fusions. If sample sections contain more than 25% necrosis in total tissue area, then the necrotic tissue must be macrodissected. ## Nucleic Acid Extraction The TSO Comprehensive assay requires nucleic acid (DNA and/or RNA) isolated from FFPE tissue using appropriate extraction methods. The required input for the TSO Comprehensive assay is 40 ng RNA and/or 40 ng DNA. For DNA, extraction kits should be able to yield this input amount at a minimum concentration of 3.33 ng/uL. Shearing requires a final volume of 52 μl (0.77 ng/μl) with a minimum of 40 μl TEB (provided) used as the diluent. For RNA, extraction kits should be able to yield this input amount in a final volume of 8.5 uL (minimum concentration of 4.7 ng/uL). Proteinase K or equivalent enzyme should not be increased during extraction from the standard concentration provided (interference was tested using 0.04 mg/mL, as well as 2.6 mg/mL and 5.2 mg/mL, 2 times and 4 times the excess from a concentration of 1.3 mg/mL per assay reaction, respectively). This assay has been validated with extracted DNA stored at -25°C to -15°C for up to 28 days, and extracted RNA stored at -85°C to -65°C for up to 28 days. ## Library Preparation Library preparation is performed using the TSO Comprehensive Library Prep Kit (PN 20031118, PN 20031119) for DNA and the RNA Library Prep Kit (PN 20031127) for RNA. For RNA, 40 ng total is converted to double-stranded complementary DNA (cDNA). For genomic DNA (gDNA), 40 ng of gDNA is sheared into small fragments. Universal adapters for sequencing are ligated onto the cDNA and gDNA fragments. P5 and P7 adapter sequences are incorporated into each library to enable the capture of library fragments onto the surface of the flow cell during sequencing. The adapters include i5 and i7 index sequences to identify each individual sample and, in the case of libraries from gDNA samples, individual molecules with the use of Unique Molecular Identifiers (UMIs). ## Enrichment Resultant libraries are enriched for specific genes of interest using a capture-based method. Biotinylated probe sequences that span gene regions of interest targeted by the assay are hybridized to the libraries. The probes and hybridized targeted libraries are isolated from non-targeted libraries by capture with streptavidin magnetic particles. The targeted enriched libraries are washed and amplified. The quantity of each enriched library is normalized using a bead-based method to ensure equal representation in the pooled libraries for sequencing. ## Sequencing Normalized, enriched libraries are pooled and clustered onto a flow cell and then sequenced using sequencing by synthesis (SBS) chemistry on the NextSeq 550Dx instrument. SBS chemistry uses a reversible terminator method to detect single, fluorescently labeled deoxynucleotide triphosphate (dNTP) bases as they are incorporated into growing DNA strands. During each sequencing cycle, a PMA P230011/S006: FDA Summary of Safety and Effectiveness Data Page 6 of 63 {6} single dNTP is added to the nucleic acid chain. The dNTP label serves as a terminator for polymerization. After each dNTP incorporation, the fluorescent dye is imaged to identify the base and then cleaved to allow incorporation of the next nucleotide. Four reversible terminator-bound dNTPs (A, G, T, and C) are present as single, separate molecules. As a result, natural competition minimizes incorporation bias. ## Data Analysis ### Primary Analysis During the primary analysis, base calls are made directly from signal intensity measurements during each sequencing cycle, resulting in base-by-base sequencing. A quality score is assigned to each base call, and the sequencing data is stored in binary base call (BCL) format. ### Secondary Analysis Secondary analysis includes the following sequential steps: - Validation of run processing and quality control Sequencing run quality metrics are evaluated to determine if they are within an acceptable range. Please refer to Table 3 for additional information about run-level quality metrics. - FASTQ File Generation Sequencing data stored in BCL format is demultiplexed using index sequences unique to each sample added during the library preparation step to assign clusters to the library from which they originated. Each cluster contains two indexes (i5 and i7 sequences, one at each end of the library fragment). The combination of those index sequences is used to demultiplex the pooled libraries. After demultiplexing, FASTQ files are generated, which are files that contain the sequencing reads for each individual sample library and the associated quality scores for each base call, excluding reads from any clusters that did not pass filter. - Alignment and Error Correction Sequencing reads derived from DNA sample libraries are aligned to a reference genome using the Burrows-Wheeler Aligner (BWA-MEM) to align DNA sequences to the hg19 reference genome, generating BAM files (*.bam), and BAM index files (*.bam.bai). These files are further processed to remove errors (including errors introduced during PCR amplification or sequencing). Reads derived from the same unique DNA molecule are collapsed into a single representative sequence using their unique molecular identifier (UMI). Indel realignment is performed to recover signal for insertions and deletions that may have been lost during initial alignment. Simultaneously, overlapping read pairs are bioinformatically combined into a single consensus read. All reads are then output as a final set of BAM files with corresponding BAM index files. Sequencing reads derived from RNA sample libraries are downsampled to approximately 30 million reads per RNA sample library by randomly selecting reads from the input FASTQ files following a probability distribution. The ends of the RNA sequences are then trimmed to a maximum length of 76 base pairs and are aligned to the hg19 reference genome. Candidate splice junctions are identified, generating BAM files and BAM index files for aligned reads, PMA P230011/S006: FDA Summary of Safety and Effectiveness Data Page 7 of 63 {7} and a tab-delimited text file for candidate splice junctions, using STAR aligner. Duplicate reads are then marked in the BAM files so they can be excluded from downstream steps. - Variant Calling Small variant calling is performed for DNA sample libraries to detect small variants [single-nucleotide variants (SNVs), multi-nucleotide variants (MNVs) up to 3 base pairs (bp) in length, insertions, and deletions. TSO Comprehensive performance has been demonstrated for insertions up to 24 base pairs and deletions up to 25 base pairs. The error-corrected BAM files (collapsed and insertions and deletions realigned) are used as input by an initial variant calling algorithm to detect small variants. This creates an unfiltered genome Variant Call Format (gVCF) file, which contain reference or variant calls for each targeted locus. Candidate variants are then filtered for assay-specific and sample processing artifacts by using adjusted quality scores calculated by comparing the observed variant frequency against a baseline noise distribution for the same site (assay-specific artifacts) and by comparing the evidence for the candidate variant against the baseline level of noise associated with that base change in the sample (sample-specific artifacts). This results in filtered gVCF files, which then uses a phased variant caller to identify MNVs, indels, and deletions in the EGFR and RET genes that are candidates for phasing. Overlapping reads are clustered in the neighborhood into a minimal set of clusters that contain the same variants. Variants are detected by examining the Concise Idiosyncratic Gapped Alignment Report (CIGAR) strings in the BAM file and comparing read sequences to the reference genome sequence. Finally, MNVs, indels, and deletions detected by the phased variant caller are merged into the filtered gVCF files resulting in merged gVCF files. Fusion calling and splice variant calling are performed for RNA sample libraries. For fusion calling, candidate fusions are identified from supplemental alignments and anomalous read pairs (reads aligning to different chromosomes or in unexpected orientations) in the BAM files for the fusion genes targeted by the TSO Comprehensive assay. These reads are assembled into candidate fusion contigs, which are then aligned back to the reference genome (hg19). These candidate fusion contigs are then evaluated against various filters before being reported as detected (please refer to 'RNARNA Fusion Calling' in the Local Run Manager TruSight Oncology Comprehensive (US) analysis module Workflow Guide for additional information). For splice variant calling, candidate splice variants (junctions) from the alignment stage are compared against a database of known transcripts and a splice variant baseline of non-tumor junctions. Any splice variants that match the database or baseline are filtered out unless they are in a set of junctions with known oncological function. If there is sufficient read support, the candidate splice variant is kept. ### Tertiary Analysis Tertiary analysis, performed by the Local Run Manager TSO Comprehensive (US) analysis module, consists of annotation of the different variant types followed by the calling/score calculation specific to the intended variants. - Annotation Detected small DNA variants are annotated using the Nirvana annotation engine with information from the RefSeq database and various population databases (COSMIC, ClinVar, dbSNP, 1000 Genomes, and gnomAD). For TMB, using the gVCF from the small variant PMA P230011/S006: FDA Summary of Safety and Effectiveness Data Page 8 of 63 {8} phasing step as input, Nirvana annotates filtered small variant calls with static (not updatable) annotation databases for use by the downstream TMB calculation. For tumor profiling variants, Nirvana annotates filtered small variant calls with an updatable RefSeq database (included as part of the KB and may be updated periodically). RNA fusions identified during RNA fusion calling are merged with fusions from proximal genes identified during RNA splice variant calling. The merged fusions are then annotated with gene symbols or names corresponding to a static database of transcripts (GENCODE Release 19). Detected RNA splice variants are annotated using the Nirvana annotation engine with information from the RefSeq database. Nirvana annotates detected RNA splice variant calls with a static (not updatable) RefSeq database for use by downstream Companion Diagnostic calling. Splice variants are annotated with transcript-level changes (affected exons in the gene transcript) with respect to RefSeq, the same as the static RefSeq database used by the small variant annotation process. For tumor profiling variants, Nirvana annotates detected RNA splice variant calls with an updatable RefSeq database (included as part of the KB and may be updated periodically). Splice variants are annotated with transcript-level changes (affected exons in the gene transcript) with respect to RefSeq. - TMB Score Calculation Calculation of the TMB score [which includes SNVs, insertion, and deletion variants and is derived from the count of non-driver somatic variants per megabase (evaluable region)] is generated from the gVCF file generated by the Small Variant Filter step and the annotations generated during small variant Annotation by calculating the number of somatic non-hotspot variants with variant allele frequency (VAF) 2 5% divided by the evaluable region size. Driver mutations are identified and filtered based on COSMIC count. Variants are flagged as likely germline for calculating the TMB score, applying a combination of population database and post-database filtering strategies. After database filtering, the proximity filter labels variants as germline if they are surrounded by database-labeled germline variants, and variants identified as likely germline are excluded from the TMB score calculation. The evaluable region is the dynamically adjusted per sample based on sequencing depth, and genomic regions with a high background noise level are excluded from the TMB calculation. - MSI Score Calculation To determine the MSI status of a sample, a total of 130 predefined MSI homopolymer sites are evaluated. Sites with sufficient coverage are considered usable, and TSO Comp needs at least 40 sites to be usable for an MSI score to be reported. For each site, the repeat length distribution is compared against the baseline database samples to determine if the repeat distribution is significantly shifted. The final MSI score is calculated as the number of unstable microsatellite sites divided by the total number of usable microsatellite sites assessed. A sample is considered MSI-High if its MSI score is ≥ 20.00%. - Companion Diagnostic (CDx) Calling For each installed companion diagnostic (CDx) intended use, the TSO Comprehensive (US) PMA P230011/S006: FDA Summary of Safety and Effectiveness Data Page 9 of 63 {9} analysis module determines the applicability of the CDx intended use for each patient sample based on the tumor type of the patient sample. If the tumor type of the patient is an exact match or a descendant of the tumor type for a CDx intended use, it is considered applicable to that CDx intended use. If the tumor type of the patient is not applicable to a CDx intended use, then the CDx intended use is not evaluated for that sample. If a required nucleic acid type for a CDx intended use is not sequenced or fails QC, or required controls in the run fail, then the patient sample is not evaluated for that CDx intended use. If a variant type or biomarker required for a CDx intended use fails QC, then the patient sample is not evaluated for that CDx intended use. When it is determined that a CDx intended use is applicable for a patient sample, the required libraries are sequenced, required QC measures pass, and required controls pass, the companion diagnostic intended use is evaluated for the patient sample. Detected variants and/or biomarkers in the patient sample are evaluated to determine the result for the CDx intended use. The evaluation is done through an algorithm specific to the CDx intended use, which assesses the presence and/or absence of variants/biomarkers that match the CDx intended use. # - • Tumor Profiling Variant Calling After companion diagnostic results are determined, all passing, detected variants in a patient sample are matched against the installed Knowledge Base to determine the cancer mutations that have evidence of clinical significance (Level 2) or have potential clinical significance (Level 3). A genomic finding is either a single variant with evidence of clinical significance or potential clinical significance, or a grouping of variants that, when detected together, have evidence of clinical significance or potential clinical significance. - When multiple variants are listed together as a genomic finding, it means that there is evidence for clinical significance or potential clinical significance for those variants together, in at least one of the sources listed in the Informatics Details of the report. If there are multiple genomic findings, and a variant is included in more than one of these findings, then that variant may be listed more than one time on a report. A genomic finding with a single variant will only be listed at the highest level, including the “Companion Diagnostics Results” section, where it meets criteria for reporting. Refer to “Positive CDx Results” section in the Local Run Manager TruSight Oncology Comprehensive (US) analysis module Workflow Guide for additional details. ### ***Results Report Generation*** The interpreted variant results, the TMB score, and the MSI Status are summarized in the TSO Comprehensive assay results report generated for the testing laboratory, which consists of a “Companion Diagnostics Results” section, an “Other Alterations and Biomarkers Identified” section for reporting “Cancer Mutations with Evidence of Clinical Significance” (Level 2) or “Cancer Mutations with Potential Clinical Significance” (Level 3), a “Companion Diagnostics QC” section, and a “Companion Diagnostics Intended Use Evaluated” section. Please refer to the LRM TSO Comp Workflow Guide for additional information about the test reports. ### **Controls** PMA P230011/S006: FDA Summary of Safety and Effectiveness Data Page 10 of 63 {10} # Positive Controls TruSight Oncology DNA Control v2 and TruSight Oncology RNA Control v2 are required for use as positive controls. These controls consist of a multiplexed blend of biosynthetic DNA/RNA with multiple verified sequence mutations in a background of genomic DNA/RNA. The controls should be included for each DNA and RNA sequencing run (including combined runs) within a given library preparation event. The controls will be checked for quality of library preparation after sequencing. Failure of the control to meet the pre-defined quality metrics will result in all test samples on the run related to that specific control (DNA Control for DNA samples, RNA Control for RNA samples) to be invalidated. # No Template Control (NTC) A no template control (NTC) consists of Tris-EDTA Buffer solution (provided in TSO Comprehensive kit as reagent “TEB”) for DNA samples, and DNase/RNase-free water (not included in the TSO Comprehensive kit) for RNA samples to determine if the library preparation is free of contamination by evaluating for the presence of sequencing reads (median exon coverage for DNA, number of genes with median deduplicated coverage for RNA) that map to the genes targeted by the assay. The NTC should be included for each DNA and RNA sequencing run (including combined runs) within a given library preparation event. If a NTC control fails, the assay instructions for use will require that the entire library preparation event is repeated for all samples. # Quality Metrics Quality metrics (Table 3) are assessed across the following categories: - Run-level: Metrics that are quantified per sequencing run; if external control fails these criteria, no results are reported for the entire batch of samples. - Sample-level: Metrics that are quantified per sample; no device results are generated for samples failing these metrics. - Analyte-level: Metrics that are quantified for individual alteration types and positions. Variants passing analyte-level metrics are reported. Table 3. Summary of TSO Comprehensive post-sequencing library quality control metrics | Variant Type | Metric | Run/ Sample/ Analyte | Required Value | Description | Impact of Failure | | --- | --- | --- | --- | --- | --- | | All variants | PCT_RF_READ S (%) | Run-Level | ≥ 80.0 | Percentage of reads passing filter (PF). | Sequencing run invalidated. No results reported for any sample in the run. | | | PCT_Q30_R1 (%) | Run-Level | ≥ 80.0 | Average percent of base calls with quality score of Q30 or higher for Read 1. | | | | PCT_Q30_R2 (%) | Run-Level | ≥ 80.0 | Average percent of base calls with quality score of Q30 or higher for Read 2. | | PMA P230011/S006: FDA Summary of Safety and Effectiveness Data Page 11 of 63 {11} | Variant Type | Metric | Run/ Sample/ Analyte | Required Value | Description | Impact of Failure | | --- | --- | --- | --- | --- | --- | | DNA variants | CONTAMINATION_SCORE | Sample-Level | ≤ 3106 OR (> 3106 and P VALUE ≤ 0.049) | A metric assessing the likelihood of contamination using the VAF of common variants. The contamination score is based on VAF distribution of SNPs. The contamination P value is used to assess highly rearranged genomes. It is only applicable when contamination score is above Upper Spec Limit. | No TMB or small variant results called | | | MEDIAN_INSERT_SIZE (bp) | Sample-Level | ≥ 70 | The median fragment length in the sample. | | | | MEDIAN_EXON_COVERAGE | Sample-Level | ≥ 150 | Median exon fragment coverage across all exon bases. | | | | PCT_EXON_50X (%) | Sample-Level | ≥ 90.0 | Percent exon bases with 50X fragment coverage. | | | | Small DNA Variants^{6} | Analyte-Level | AQ ≥ 20 for Frequent COSMIC mutations^{1} AQ ≥ 60 for other mutations^{1} | Metric to determine whether a small DNA variant is called | Small DNA variant not called | | | Small DNA Variants^{6} | Analyte-Level | LQ ≥ 20 for Frequent COSMIC mutations^{2} LQ ≥ 60 for other mutations^{2} | Metric to determine whether a small DNA variant is called | | | | TMB | Analyte-Level | N/A^{3} | N/A | N/A | | | MSI | Analyte Level | ≥40 evaluable sites | Minimum number of sites evaluable for MSI score calculation | MSI status not determined | PMA P230011/S006: FDA Summary of Safety and Effectiveness Data Page 12 of 63 {12} | Variant Type | Metric | Run/ Sample/ Analyte | Required Value | Description | Impact of Failure | | --- | --- | --- | --- | --- | --- | | RNA variants | Median_CV_Gene_500X | Sample-Level | ≤ 0.93 | For each gene with at least 500x coverage, the coefficient of variation in coverage across the gene body is computed. This metric is the median of these values. A high value indicates a high level of variation and indicates a problem in library preparation such as low sample input and/or probe pulldown issues. This metric is computed using all reads (including reads marked as duplicates). | No fusions or splice variant results called. | | | Total_On_Target_Reads | Sample-Level | ≥ 9,000,000 | The total number of reads that map to the target regions. This metric is computed using all reads (including reads marked as duplicates). | | | | MEDIAN_INSERT_SIZE (bp) | Sample-Level | ≥ 80 | The median fragment length in the sample. | | | | RNA Fusions | Analyte-Level | Fusion score ≥ 0.45^{4} Deduped supporting reads ≥ 5 | Metric to determine whether a RNA fusion gene is called | RNA fusion not called | | | RNA Splice Variants | Analyte-Level | Splice score = 1^{5} | Metric to determine whether a RNA splice variant is called | RNA splice variant not called | $^{1}$Variant quality score (AQ) is calculated as -10*log$_{10}$(p-value), where p-value is the binomial test by comparing the observed VAF and depth to a baseline of normal FFPE samples. $^{2}$Likelihood ratio (LQ) is calculated as -10log$_{10}$(Likelihood (observed variant is an error 1 DP error rate/Likelihood (observed variant is a mutation 1 DP, VAF) $^{3}$Evaluation of TMB does not have a reporting threshold, as the value is quantitative (score), not qualitative (classification) $^{4}$Fusion score is a weighted sum that takes into account the number of unique supporting reads, fusion contig length, breakpoint homology, and other associated metrics. PMA P230011/S006: FDA Summary of Safety and Effectiveness Data Page 13 of 63 {13} ⁵Splice score increments by 0.1 for each supporting reads for the splice junction and is capped at 1. ⁶Accuracy of small DNA tumor profiling variants below 5% variant allele frequency (VAF) has not been established. Table 4. TruSight Oncology Control Metrics | Output Type | Metric | Specification | Impact of Specification Failure | | --- | --- | --- | --- | | Positive Control | DNA External Control | 51 of 54 specified variants detected | No small DNA variant, TMB, or MSI results reported. | | | | MSI-High (MSI score ≥ 20% unstable sites) and Usable MSI Sites ≥ 30 | No MSI results reported. | | | RNA External Control | 20 of 22 specified variants detected | No fusions or splice variant results reported. | | Non-Template Control (NTC) | DNA Median Exon Coverage | ≤ 8 | No small DNA variant, TMB, or MSI results reported | | | RNA Gene above Median Cutoff | ≤ 1 | No fusions or splice variant results reported. | | DNA or RNA Positive Control | CDx Control Requirements | CDx control validation requires detection of at least one representative variant for a biomarker | No CDx results reported for a biomarker. | ## VI. ALTERNATIVE PRACTICES AND PROCEDURES There are FDA approved companion diagnostic (CDx) alternatives for the detection of genetic alterations using FFPE tumor specimens as listed in Table 1 of the TSO Comprehensive assay intended use statement (Section II). The approved CDx tests are listed in Table 5 below. Table 5. Alternative FDA-approved CDx assays for CDx biomarkers identified by the TSO Comprehensive Assay | Indication | Gene | Device (PMA) | Company | Technology | Therapy | | --- | --- | --- | --- | --- | --- | | Solid Tumors | NTRK1/2/3 Fusions | FoundationOne® CDx (P170019/S017) | Foundation Medicine, Inc. | NGS | VITRAKVI® (larotrectinib) | PMA P230011/S006: FDA Summary of Safety and Effectiveness Data Page 14 of 63 {14} | Indication | Gene | Device (PMA) | Company | Technology | Therapy | | --- | --- | --- | --- | --- | --- | | Non-Small Cell Lung Cancer (NSCLC) | RET Fusions | OncomineDx Target Test (P160045/S019 and P160045/S031) | Life Technologies Corporation | NGS | RETEVMO® (selpercatinib) | | Solid Tumors | RET Fusions | FoundationOne® CDx (P170019/S043) | Foundation Medicine, Inc. | NGS | RETEVMO® (selpercatinib) | | Medullary Thyroid Cancer (MTC) | RET mutations (SNVs, MNVs, and deletions) | OncomineDx Target Test (P160045/S031) | Life Technologies Corporation | NGS | RETEVMO® (selpercatinib) | | Thyroid Cancer (TC) | RET Fusions | OncomineDx Target Test (P160045/S031) | Life Technologies Corporation | NGS | RETEVMO® (selpercatinib) | For additional details see the FDA List of Cleared or Approved Companion Diagnostic Devices at: https://www.fda.gov/medical-devices/vitro-diagnostics/list-cleared-or-approved-companion-diagnostic-devices-vitro-and-imaging-tools ### VII. MARKETING HISTORY The TSO Comprehensive Premarket Approval (P230011) was originally approved on August 21, 2024, by FDA and has been commercially available in the U.S. since November 2024. ### VIII. POTENTIAL ADVERSE EFFECTS OF THE DEVICE ON HEALTH Failure of the device to perform as expected or failure to correctly interpret test results may lead to incorrect TSO Comprehensive assay results and subsequently improper patient management decisions. Patients with false positive results may undergo treatment with the therapy listed in the intended use statement without clinical benefit and may experience adverse reactions associated with the therapy. Patients with false negative results may not be considered for treatment with the indicated therapy. There is also a risk of delayed results, which may lead to delay of treatment with the appropriate indicated therapy. No adverse events were reported in connection with the clinical studies used to support this PMA as the studies were performed retrospectively using banked samples. For the specific adverse events related to the approved therapeutics, please see approved FDA therapeutic product labeling. ### IX. SUMMARY OF NONCLINICAL STUDIES #### A. Laboratory Studies A summary of the evidence in support of the performance of the TSO Comprehensive assay in PMA P230011/S006: FDA Summary of Safety and Effectiveness Data Page 15 of 63 {15} detecting RET fusions in thyroid cancer (TC) and RET small DNA variant mutations in medullary thyroid cancer (MTC) is provided below. Studies evaluating analytical accuracy/concordance, precision, reproducibility, and limit of detection (LoD) were conducted to support the indication for RET fusions in TC and RET mutations in MTC. # 1. Analytical Accuracy/Concordance # RET Fusion Detection in Thyroid Cancer (TC) An accuracy study was performed to evaluate the concordance between TSO Comprehensive assay and an externally validated NGS assay (comparator method) for detection of RET fusions in TC. The accuracy study was conducted with TC and MTC samples obtained from the LIBRETTO-001 trial and commercial sources. Across both sets of samples, 272 samples were tested using both the TSO Comprehensive assay and the comparator method. Of the 272 samples, 86 samples returned invalid results with the comparator method (out of these, 35 were also invalid by TSO Comp) and 47 samples returned no calls with comparator (out of these, 1 was invalid by TSO Comp); while 18 samples had invalid results by TSO Comprehensive assay only (invalid rate of 6.6%). Thus, 121 of the 272 samples had valid results by both methods. The agreement analysis is shown in Table 6. Based on valid test results, the PPA and NPA between the TSO Comprehensive assay and comparator method were 81.8% (18/22; 95% CI: 59.7%, 94.8%), and 100% (99/99; 95% CI: 96.3%, 100%), respectively. There were four samples that were positive by the comparator method but negative by the TSO Comprehensive assay. Three of these samples had KIF5B-RET fusions called by the comparator method with 117, 4472, and 6913 supporting reads. These were all supplemental TC samples that were negative by the TSO Comprehensive assay and the representative local test used in the RET clinical performance bridging study. KIF5B-RET fusions are the most prevalent RET fusion species seen in lung adenocarcinoma and are not observed in TC. The other sample had a CUX1-RET fusion called by the comparator method with number of supporting reads close to its LoD. This was a LIBRETTO-001 sample from an MTC patient who was RET mutation positive by the local test used for LIBRETTO-001 enrollment but whose RET fusion status is unknown. The most common oncogenic drivers of MTC are RET DNA mutations, while MTC tumors harboring RET fusions are extremely rare. Table 6. Concordance Between the TSO Comprehensive Assay and Comparator Method for Detection of RET Fusions in Thyroid Cancer | | Comparator Method Result | | | | | --- | --- | --- | --- | --- | | | | RET Fusion Positive | RET Fusion Negative | Total | | TSO Comprehensive Assay Result | RET Fusion Positive | 18 | 0 | 18 | | | RET Fusion Negative | 4^{2} | 99 | 103 | | | Invalid | 0 | 18 | 18 | | | Total | 22 | 117 | 139 | PMA P230011/S006: FDA Summary of Safety and Effectiveness Data Page 16 of 63 {16} | | Comparator Method Result | | | | | --- | --- | --- | --- | --- | | | | RET Fusion Positive | RET Fusion Negative | Total | | Agreement Statistics | PPA% (n/N; 95% CI^{1}) | 81.8% (18/22; 59.7% -94.8%) | | | | | NPA% (n/N; 95% CI^{1}) | 100.0% (99/99; 96.3% - 100%) | | | | Agreement Statistics Including Invalids | PPA% (n/N; 95% CI^{1}) | 81.8% (18/22; 59.7% -94.8%) | | | | | NPA% (n/N; 95% CI^{1}) | 84.6% (99/117; 76.8% - 90.6%) | | | $^{1}$CI based on Clopper-Pearson (exact) method ### RET Small DNA Variant Mutations Detection in Medullary Thyroid Cancer (MTC) An accuracy study was performed to evaluate the concordance between TSO Comprehensive assay and externally validated NGS assays (comparator methods) for detection of RET small DNA variant mutations in MTC. The accuracy study was conducted using MTC samples obtained from the LIBRETTO-001 trial, and supplemental MTC and TC samples obtained from commercial sources. Combining LIBRETTO-001 and supplemental samples, 283 samples were tested using the TSO Comprehensive assay and a comparator method. Of the 283 samples, 22 had invalid comparator method results, and 42 samples had invalid results by TSO Comprehensive assay only (invalid rate of 14.8%). Thus, 219 of the 283 samples had valid results by the TSO Comprehensive assay and a comparator method. There were 21 SNVs, 2 MNVs, and 4 deletions represented in the samples that were positive by TSO Comprehensive and the comparator method. The agreement analysis is shown in Table 7. For the primary analysis, concordance was assessed by analyzing the CDx RET variants that are detected by both the TSO Comprehensive assay and comparator method. Based on valid test results, the PPA and NPA between the TSO Comprehensive assay and comparator method were 81.8% (27/33;95% CI: 64.5, 93.0) and 99.5% (185/186; 97.0, >99.9), respectively. Table 7. Concordance Between the TSO Comprehensive Assay and Comparator Method for Detection of RET Small DNA Variants Mutations in Medullary Thyroid Cancer | | Comparator Method Result | | | | | --- | --- | --- | --- | --- | | | | RET Mutation Positive | RET Mutation Negative | Total | | TSO Comprehensive Assay Result | RET Mutation Positive | 27 | 1 | 28 | | | RET Mutation Negative | 6^{2} | 185 | 191 | | | Invalid | 6 | 36 | 42 | | | Total | 39 | 222 | 261 | | Agreement Statistics | PPA% (n/N; 95% CI^{1}) | 81.8% (27/33; 64.5% -93.0%) | | | | | NPA% (n/N; 95% CI^{1}) | 99.5% (185/186; 97.0% - >99.9%) | | | PMA P230011/S006: FDA Summary of Safety and Effectiveness Data Page 17 of 63 {17} | Agreement Statistics Including Invalids | PPA% (n/N; 95% CI^{1}) | 69.2% (27/39; 52.4% -83.0%) | | --- | --- | --- | | | NPA% (n/N; 95% CI^{1}) | 83.3% (185/222; 77.8% - 88.0%) | $^{1}$CI based on Clopper-Pearson (exact) method. There were six samples that were comparator method positive (all with a RET Val804Met mutation with VAFs ranging from 0.02 to 0.09) but negative by the TSO Comprehensive assay. Five of these 6 samples had VAFs below the TSO Comprehensive assay's average LoD of 0.049 for SNVs. All six samples were supplemental TC samples (classified as Papillary Thyroid Cancer, PTC) that were also RET mutation negative by the representative local test used in the RET clinical performance bridging study. The RET Val804Met variant is most frequently of germline origin and is found in MTC, not PTC, which has not been reported to harbor activating RET DNA mutations. The VAFs reported by the comparator method are characteristic of somatic variants and are inconsistent with the typical range for germline variants. The range of VAF values reported in multiple samples is consistent with previously reported deamination-related artifacts for the comparator method. There was one sample that was comparator method negative but positive by the TSO Comprehensive assay. The TSO Comprehensive called a RET Met918Thr variant with a VAF of 0.35. The RET Met918Thr variant is the most common RET mutation in acquired MTC. The local test used in the LIBRETTO-001 study also called this mutation a RET Met918Thr variant. ## 2. Precision and Reproducibility ### a. Precision Within-laboratory precision was evaluated for RET fusions in 4 samples from 3 tumor types (non-small cell lung cancer, thyroid cancer, and atypical Spitz tumor from skin tissue specimen), and RET small DNA variants (SNV, MNV, deletion) from five FFPE samples of medullary thyroid cancer. One RET insertion and three RET delins (at the protein level, the variants are net deletions or an MNV at the DNA level) were tested in the FFPE-treated cell lines. Each sample was tested at two variant levels: ~1x LoD (low variant level) and ~2–3x LoD (high variant level) with the exception of the sample harboring CCDC6-RET, which was only tested at the low variant level. Each of the samples at each test level was run in duplicates in each library preparation event across three (3) operators. Each operator started library preparation on three (3) non-consecutive start days and sequenced on three (3) designated NextSeq 550Dx instruments. Three (3) reagents lots were tested, generating 54 observations per sample per level. Three variant/levels had fewer than 54 observations due to invalid libraries. Variant calling was evaluated separately for the two variant levels for a given variant from pooled observations across all variables (operators, reagent lots, instruments, days, and replicates). The PPC, PNC, and associated two-sided 95% confidence interval (Wilson score) are summarized in the following tables (Table 8 and Table 9). It should be noted that some levels had fewer than 54 observations; these were due to invalid libraries that did not meet the necessary QC metrics (4 out of 1096 libraries failed, 0.4% invalid rate). For RET small DNA variants at the high variant level (~2–3x LoD), the TSO Comprehensive PMA P230011/S006: FDA Summary of Safety and Effectiveness Data Page 18 of 63 {18} assay demonstrated 100% Percent positive calls (PPC) and percent negative calls (PNC) for each variant except one RET delins variant (E632_V637delinsAA, net nucleotide deletion) which could not be consistently detected by TSO Comprehensive assay (Table 8). Refer to Limit of Detection for CDx study results. For RET small DNA variants at the low variant level (~1x LoD), PPCs ranged from 83.3% to 100.0%. For variants with PPC < 95%, the mean VAFs (RET C634Y and RET D898_E901del) were below the respective limits of detection. At the low variant level, 100% PNC was achieved for all variants. Table 8. Summary Results for Targeted RET Small DNA Variants | Variant Level | Variant | Variant Type | Mean VAF | PPC (95% CI) | PNC (95% CI) | LoD Level | | --- | --- | --- | --- | --- | --- | --- | | Low (~1x LoD) | RET p.(M918T) | SNV | 0.042 | 96.2 (51/53) (87.2, 99.0) | 100 (483/483) (99.2, 100) | 0.9x | | | RET p.(D898_E901del) | Deletion | 0.048 | 87.0 (47/54) (75.6, 93.6) | 100 (482/482) (99.2, 100) | 0.9x | | | RET p.(D631_L633delins E*) | Deletion | 0.056 | 98.1 (53/54) (90.2, 99.7) | 100 (482/482) (99.2, 100) | 1.0x | | | RET p.(C634Y) | MNV | 0.028 | 83.3 (45/54) (71.3, 91.0) | 100 (482/482) (99.2, 100) | 0.6x | | | RET p.(C618R) | SNV | 0.046 | 94.4 (51/54) (84.9, 98.1) | 100 (482/482) (99.2, 100) | 0.9x | | | RET p.(C634_R635delins WG) | MNV | 0.014 | 98.1 (53/54) (90.2, 99.7) | 100 (482/482) (99.2, 100) | 0.5x | | | RET p.(C634_T636dup) | Insertion | 0.034 | 98.1 (51/52) (89.9, 99.7) | 100 (484/484) (99.2, 100) | 0.5x | | | RET p.(L629_I638delinsC D) | Deletion | 0.021 | 100 (54/54) (93.4, 100) | 100 (482/482) (99.2, 100) | 1.0x | | | RET p.(E632_V637delins AA) | Deletion | N/A | Not tested | 100 (536/536) (99.3, 100) | N/A | | High (~3x LoD) | RET p.(M918T) | SNV | 0.078 | 100 (52/52) (93.1, 100) | 100 (464/464) (99.2, 100) | 1.7x | PMA P230011/S006: FDA Summary of Safety and Effectiveness Data Page 19 of 63 {19} | Variant Level | Variant | Variant Type | Mean VAF | PPC (95% CI) | PNC (95% CI) | LoD Level | | --- | --- | --- | --- | --- | --- | --- | | | RET p.(D898_E901del) | Deletion | 0.088 | 100 (54/54) (93.4, 100) | 100 (462/462) (99.2, 100) | 1.6x | | | RET p.(D631_L633delins E*) | Deletion | 0.161 | 100 (32/32) (89.3, 100) | 100 (484/484) (99.2, 100) | 2.9x | | | RET p.(C634Y) | MNV | 0.095 | 100 (54/54) (93.4, 100) | 100 (462/462) (99.2, 100) | 2.1x | | | RET p.(C618R) | SNV | 0.146 | 100 (54/54) (93.4, 100) | 100 (462/462) (99.2, 100) | 2.8x | | | RET p.(C634_R635delins WG) | MNV | 0.030 | 100 (54/54) (93.4, 100) | 100 (462/462) (99.2, 100) | 1.0x | | | RET p.(C634_T636dup) | Insertion | 0.071 | 100 (54/54) (93.4, 100) | 100 (462/462) (99.2, 100) | 1.0x | | | RET p.(E632_V637delins AA) | Deletion | 0.127 | 77.8 (42/54) (65.1, 86.8) | 100 (462/462) (99.2, 100) | N/A | | | RET p.(L629_I638delinsC D) | Deletion | 0.055 | 100 (54/54) (93.4, 100) | 100 (462/462) (99.2, 100) | 2.5x | For RET fusions, at the higher variant level (~2 – 3x LoD), the TSO Comprehensive assay demonstrated 100% PPC for all fusions (Table 9). It should be noted that KIF5B-RET at the low level had more than 54 observations; this was due to the preparation of two dilution mixtures targeting the same variant level. Therefore, the observations were combined. At the low variant level (~1x LoD), the PPC for RET fusions ranged from 90.7% to 98.1%. For variants with PPC < 95% (NCOA4-RET), the supporting reads were below the respective Limits of Detection (15.8 supporting reads for NCOA4-RET). 100% PNC was achieved for all variants at both levels. Table 9. Summary Results for RET Fusions | Variant Level | Targeted Fusions | Mean Supporting Reads | PPC (n/N) (95% CI) | PNC (n/N) (95% CI) | | --- | --- | --- | --- | --- | | ~1x LoD | NCOA4-RET^{1} | 13.3 | 90.7 (49/54) (80.1, 96.0) | 100.0 (537/537) (99.3, 100.0) | PMA P230011/S006: FDA Summary of Safety and Effectiveness Data Page 20 of 63 {20} | Variant Level | Targeted Fusions | Mean Supporting Reads | PPC (n/N) (95% CI) | PNC (n/N) (95% CI) | | --- | --- | --- | --- | --- | | | CCDC6-RET^{2} | 18.7 | 98.1 (53/54) (90.2, 99.7) | 100.0 (591/591) (99.4, 100.0) | | | KIF5B-RET (sample 1)^{3} | 17.3 | 95.4 (103/108) (89.6, 98.0) | 100.0 (430/430) (99.1, 100.0) | | | KIF5B-RET (sample 2)^{3} | 17.3 | 96.2 (51/53) (87.2, 99.0) | | | ~3x LoD | NCOA4-RET^{1} | 24.8 | 100.0 (54/54) (93.4, 100.0) | 100.0 (481/481) (99.2, 100.0) | | | CCDC6-RET^{2} | N/A | Not tested^{4} | 100.0 (589/589) (99.4, 100.0) | | | KIF5B-RET (sample 1)^{3} | 43.8 | 100.0 (54/54) (93.4, 100.0) | 100.0 (428/428) (99.1, 100.0) | | | KIF5B-RET (sample 2)^{3} | 44.6 | 100.0 (53/53) (93.2, 100.0) | | $^{1}$Thyroid tissue $^{2}$Atypical Spitz tumor $^{3}$Lung Tissue from NSCLC $^{4}$CCDC6-RET was tested at one level only (i.e., ~1x LoD due to insufficient materials. Table 10. Results for RET Fusions by Operator | Variant Level | Targeted Fusions | Operator^{2} | N | PPC (n/N) | 95% CI^{1} | | --- | --- | --- | --- | --- | --- | | ~1x LoD | NCOA4-RET | 1 | 18 | 94.4 (17/18) | (74.2, 99.0) | | | | 2 | 18 | 83.3 (15/18) | (60.8, 94.2) | | | | 3 | 18 | 94.4 (17/18) | (74.2, 99.0) | | | CCDC6-RET^{3} | 1 | 18 | 100.0 (18/18) | (82.4, 100.0) | | | | 2 | 18 | 100.0 (18/18) | (82.4, 100.0) | | | | 3 | 18 | 94.4 (17/18) | (74.2, 99.0) | | | KIF5B-RET^{4} (Sample ID 10755) | 1 | 36 | 91.7 (33/36) | (78.2, 97.1) | | | | 2 | 16 | 93.8 (15/16) | (71.7, 98.9) | | | | 3 | 36 | 97.2 (35/36) | (85.8, 99.5) | | | | 4 | 20 | 100.0 (20/20) | (83.9, 100.0) | | | KIF5B-RET (Sample ID 10756) | 1 | 18 | 94.4 (17/18) | (74.2, 99.0) | | | | 2 | 7 | 100.0 (7/7) | (64.6, 100.0) | | | | 3 | 18 | 94.4 (17/18) | (74.2, 99.0) | | | | 4 | 10 | 100.0 (10/10) | (72.2, 100.0) | | Aggregate RET fusions (low level) | | | 269 | 95.2 (256/269) | (91.9, 97.2) | | ~3x LoD | | 1 | 18 | 100.0 (18/18) | (82.4, 100.0) | PMA P230011/S006: FDA Summary of Safety and Effectiveness Data Page 21 of 63 {21} | | NCOA4-RET | 2 | 18 | 100.0 (18/18) | (82.4, 100.0) | | --- | --- | --- | --- | --- | --- | | | | 3 | 18 | 100.0 (18/18) | (82.4, 100.0) | | | KIF5B-RET (Sample ID 10755) | 1 | 18 | 100.0 (18/18) | (82.4, 100.0) | | | | 2 | 8 | 100.0 (8/8) | (67.6, 100.0) | | | | 3 | 18 | 100.0 (18/18) | (82.4, 100.0) | | | | 4 | 10 | 100.0 (10/10) | (72.2, 100.0) | | | KIF5B-RET (Sample ID 10756) | 1 | 18 | 100.0 (18/18) | (82.4, 100.0) | | | | 2 | 7 | 100.0 (7/7) | (64.6, 100.0) | | | | 3 | 18 | 100.0 (18/18) | (82.4, 100.0) | | | | 4 | 10 | 100.0 (10/10) | (72.2, 100.0) | | Aggregate RET Fusions (high level) | | | 161 | 100.0 (161/161) | (97.7, 100.0) | ¹ 2-sided 95% CI with Wilson Score test. ² For the RNA fusions KIF5B-RET (Sample ID 10755) and KIF5B-RET (Sample ID 10756) operator 4 replaced operator 2 with 10 of the 18 replicates and 20 of the 36 replicates for high and low levels, respectively for testing with lots IUO6 and IUO6.5. ³ CCDC6-RET was tested at one level only (i.e., ~1x LoD due to insufficient materials. ⁴ Two dilution mixtures for KIF5B-RET (sample 10755) “low” level and LMNA-NTRK1 (sample 19219) “high” level were tested. The two dilution mixtures having slightly different dilution-fold in the normal diluent were intended to target to the same variant level. Since the observed mean supporting reads for both mixtures were comparable, the observations from both mixtures were combined for analysis. Table 11. Results for RET Fusions by Lot | Variant Level | Targeted Fusions | Lot² | N | PPC (n/N) | 95% CI¹ | | --- | --- | --- | --- | --- | --- | | ~1x LoD | NCOA4-RET | M | 18 | 83.3 (15/18) | (60.8, 94.2) | | | | PQ2 | 18 | 94.4 (17/18) | (74.2, 99.0) | | | | PQ3 | 18 | 94.4 (17/18) | (74.2, 99.0) | | | CCDC6-RET³ | M | 18 | 94.4 (17/18) | (74.2, 99.0) | | | | PQ2 | 18 | 100.0 (18/18) | (82.4, 100.0) | | | | PQ3 | 18 | 100.0 (18/18) | (82.4, 100.0) | | | KIF5B-RET⁴ (Sample ID 10755) | IUO5 | 36 | 91.7 (33/36) | (78.2, 97.1) | | | | IUO6 | 36 | 97.2 (35/36) | (85.8, 99.5) | | | | IUO6.5 | 36 | 97.2 (35/36) | (85.8, 99.5) | | | KIF5B-RET (Sample ID 10756) | IUO5 | 17 | 94.1 (16/17) | (73.0, 99.0) | | | | IUO6 | 18 | 94.4 (17/18) | (74.2, 99.0) | | | | IUO6.5 | 18 | 100.0 (18/18) | (82.4, 100.0) | | Aggregate RET fusions (low level) | | | 269 | 95.2 (256/269) | (91.9, 97.2) | | ~3x LoD | NCOA4-RET | M | 18 | 100.0 (18/18) | (82.4, 100.0) | | | | PQ2 | 18 | 100.0 (18/18) | (82.4, 100.0) | | | | PQ3 | 18 | 100.0 (18/18) | (82.4, 100.0) | | | | IUO5 | 18 | 100.0 (18/18) | (82.4, 100.0) | PMA P230011/S006: FDA Summary of Safety and Effectiveness Data Page 22 of 63 {22} | | KIF5B-RET (Sample ID 10755) | IUO6 | 18 | 100.0 (18/18) | (82.4, 100.0) | | --- | --- | --- | --- | --- | --- | | | | IUO6.5 | 18 | 100.0 (18/18) | (82.4, 100.0) | | | KIF5B-RET (Sample ID 10756) | IUO5 | 17 | 100.0 (17/17) | (81.6, 100.0) | | | | IUO6 | 18 | 100.0 (18/18) | (82.4, 100.0) | | | | IUO6.5 | 18 | 100.0 (18/18) | (82.4, 100.0) | | Aggregate RET fusions (low level) | | | 161 | 100.0 (161/161) | (97.7, 100.0) | ¹ 2-sided 95% CI with Wilson Score test ² 3 waves of testing took place over course of study. 3 sets of unique lots were tested per wave (i.e. Lots M, PQ2, and PQ3 for wave 1, Lots PQ1, PQ2 and PQ3 for wave 2 and Lots IUO5, IUO6 and IUO 6.5 for wave 3) ³ CCDC6-RET was tested at one level only (i.e., ~1x LoD) due to insufficient materials. ⁴ Two dilution mixtures for KIF5B-RET (sample 10755) “low” level and LMNA-NTRK1 (sample 19219) “high” level were tested. The two dilution mixtures having slightly different dilution-fold in the normal diluent were intended to target to the same variant level. Since the observed mean supporting reads for both mixtures were comparable, the observations from both mixtures were combined for analysis. Restricted maximum likelihood (REML) variance components analysis was performed to evaluate total variation of the underlying continuous variable (supporting reads for RNA fusions) and estimate the components of precision [standard deviation (SD), coefficient of variation (CV)] for each source of variation [operators, instruments, days, reagent lots, residual and total]. The results are presented in Table 12 for RET small DNA variants, and Table 13 for RET fusions. The residual component was the largest contributor to total variance for both small DNA variants and RNA fusions at both levels, supporting the conclusion that detection of these variants by TSO Comprehensive assay is robust to operators, lots, instruments, and days. Table 12. Variance Components Analysis of VAF for Targeted RET Small DNA Variants | VAF Level | Variant | Variant Type | N Valid Attempts | Mean VAF | Operator SD (%CV) | Instrument SD (%CV) | Lot SD (%CV) | Day SD (%CV) | Residual SD (%CV) | Total SD (%CV) | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | Low (~1x LoD) | RET p.(M918T) | SNV | 53 | 0.042 | 0.000 (0.0) | 0.001 (3.0) | 0.000 (0.0) | 0.000 (0.0) | 0.011 (25.6) | 0.011 (25.7) | | | RET p.(D898_E 901del) | Deletion | 54 | 0.048 | 0.000 (0.0) | 0.000 (0.0) | 0.000 (0.0) | 0.004 (8.7) | 0.014 (30.0) | 0.015 (31.2) | | | RET p.(D631_L 633delinsE) | Deletion | 54 | 0.056 | 0.000 (0.0) | 0.002 (3.0) | 0.006 (11.6) | 0.000 (0.0) | 0.010 (18.5) | 0.012 (22.0) | | | RET p.(C634Y) | MNV | 54 | 0.028 | 0.000 (0.0) | 0.000 (0.0) | 0.001 (3.3) | 0.000 (0.0) | 0.009 (30.7) | 0.009 (30.9) | | | RET p.(C618R) | SNV | 54 | 0.046 | 0.000 (0.0) | 0.000 (0.0) | 0.000 (0.0) | 0.000 (0.0) | 0.014 (31.3) | 0.014 (31.3) | PMA P230011/S006: FDA Summary of Safety and Effectiveness Data Page 23 of 63 {23} | VAF Level | Variant | Variant Type | N Valid Attempts | Mean VAF | Operator SD (%CV) | Instrument SD (%CV) | Lot SD (%CV) | Day SD (%CV) | Residual SD (%CV) | Total SD (%CV) | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | RET p.(C634_R 635delins WG) | MNV | 54 | 0.014 | 0.000 (0.0) | 0.000 (0.0) | 0.000 (0.0) | 0.000 (0.0) | 0.014 (29.9) | 0.014 (29.9) | | | RET p.(C634_T 636dup) | Insertion | 52 | 0.034 | 0.000 (0.0) | 0.000 (1.4) | 0.002 (7.0) | 0.000 (0.0) | 0.007 (20.6) | 0.007 (21.8) | | | RET p.(L629_I6 38delinsCD) | Deletion | 54 | 0.021 | 0.000 (0.0) | 0.000 (0.0) | 0.000 (0.0) | 0.000 (0.0) | 0.007 (32.6) | 0.007 (32.6) | | High (~3x LoD) | RET p.(M918T) | SNV | 52 | 0.078 | 0.002 (3.1) | 0.000 (0.0) | 0.000 (0.0) | 0.007 (9.1) | 0.018 (23.1) | 0.020 (25.0) | | | RET p.(D898_E 901del) | Deletion | 54 | 0.088 | 0.000 (0.0) | 0.000 (0.0) | 0.001 (1.4) | 0.006 (7.0) | 0.017 (19.2) | 0.018 (20.5) | | | RET p.(D631_L 633delinsE) | Deletion | 52* | 0.164 | 0.000 (0.0) | 0.000 (0.0) | 0.005 (3.0) | 0.000 (0.0) | 0.020 (12.1) | 0.020 (12.4) | | | RET p.(C634Y) | MNV | 54 | 0.095 | 0.000 (0.0) | 0.002 (2.5) | 0.002 (2.1) | 0.000 (0.0) | 0.014 (15.0) | 0.015 (15.3) | | | RET p.(C618R) | SNV | 54 | 0.146 | 0.003 (1.7) | 0.000 (0.0) | 0.020 (13.7) | 0.002 (1.1) | 0.018 (12.6) | 0.027 (18.7) | | | RET p.(C634_R 635delins WG) | MNV | 54 | 0.030 | 0.001 (4.8) | 0.000 (0.0) | 0.000 (0.0) | 0.001 (3.4) | 0.006 (19.5) | 0.006 (20.4) | | | RET p.(C634_T 636dup) | Insertion | 54 | 0.071 | 0.001 (2.1) | 0.000 (0.0) | 0.000 (0.0) | 0.000 (0.0) | 0.009 (13.4) | 0.010 (13.6) | | | RET p.(E632_V 637delinsA A) | Deletion | 54 | 0.127 | 0.000 (0.0) | 0.000 (0.0) | 0.000 (0.0) | 0.000 (0.0) | 0.072 (56.2) | 0.072 (56.2) | | | RET p.(L629_I6 38delinsCD) | Deletion | 54 | 0.055 | 0.000 (0.0) | 0.000 (0.0) | 0.000 (0.0) | 0.001 (2.2) | 0.013 (23.8) | 0.013 (23.9) | PMA P230011/S006: FDA Summary of Safety and Effectiveness Data Page 24 of 63 {24} * Includes 20 observations that failed QC for Median Exon Coverage but have coverage at the RET variant position well above the low depth cutoff of 100 (Mean Depth = 374). Table 13. Variance Components Analysis of Supporting Reads for Targeted RET RNA Fusions | Supporting Reads Level | Fusion | N Valid Attempts | Mean Supporting | Operator SD (%CV) | Instrument SD (%CV) | Lot SD (%CV) | Day SD (%CV) | Residual SD (%CV) | Total SD (%CV) | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | ~1x LoD | NCOA4-RET | 54 | 13.3 | 1.67 (13) | 0.00 (0) | 0.00 (0) | 1.67 (13) | 5.09 (38) | 5.61 (42) | | | CCDC6-RET | 54 | 18.7 | 0.00 (0) | 1.14 (6) | 5.44 (29) | 0.00 (0) | 6.17 (33) | 8.30 (44) | | | KIF5B-RET (Sample 1) | 108 | 17.3 | 2.11 (12) | 2.50 (14) | 2.89 (17) | 3.52 (20) | 7.09 (41) | 9.04 (52) | | | KIF5B-RET (Sample 2) | 53 | 17.3 | 2.05 (12) | 3.72 (22) | 3.65 (21) | 2.41 (14) | 5.95 (34) | 8.52 (49) | | ~2-3x LoD | NCOA4-RET^{1} | 54 | 24.8 | 3.05 (12) | 0.00 (0) | 5.92 (24) | 0.00 (0) | 6.78 (27) | 9.50 (38) | | | KIF5B-RET (Sample 1) | 54 | 43.8 | 4.15 (9) | 0.96 (2) | 12.57 (29) | 6.52 (15) | 15.23 (35) | 21.23 (48) | | | KIF5B-RET (Sample 2) | 53 | 44.6 | 5.37 (12) | 4.97 (11) | 13.73 (31) | 0.00 (0) | 12.41 (28) | 19.90 (45) | %CV: Percent coefficient of variation. SD: Standard deviation. ### b. Reproducibility Reproducibility of the TSO Comprehensive assay across 3 testing sites (1 internal, 2 external). At each site, each operator (2) tested the panel members in duplicate over the course of 3 non-consecutive testing days, generating 6 observations per target per panel member. In total, 36 observations were generated per panel member (3 sites/instruments × 2 operators × 3 start days × 2 within-run replicates). Testing was conducted with a DNA panel that included nine panel members from FFPE samples (from medullary thyroid cancer tissue), and three from a DNA cell line, that contained RET small DNA variants. There were four panel members from FFPE samples (from papillary thyroid, unclassified thyroid, and lung tissue) that contained RET fusions. PMA P230011/S006: FDA Summary of Safety and Effectiveness Data Page 25 of 63 {25} Panel members were tested at both low (~1x LoD) and high (2-3x LoD) variant levels. Percent positive calls (PPC) and percent negative calls (PNC) for targeted small DNA variants and RNA fusion variants, at both the high and low levels, were determined. Two-sided 95% confidence intervals (CIs) associated were also calculated using the Wilson score method. Analyses were performed to estimate PPC and PNC (with associated 95% CIs) in the targeted high-level and low-level panel members by combining TSO Comprehensive assay observations for a given target in a group of panel members representing the applicable variant type (for example, small DNA variants and RNA fusions) across sites/instruments, operators, and runs. For each targeted variant, TSO Comprehensive assay observations in other panel members at the high level targeted for the same variant type but not containing the same variant as determined by the majority rule, were combined to calculate PNC. It should be noted that some levels had fewer than 36 observations; these were due to invalid libraries that did not meet the necessary QC metrics (4 out of 576 libraries failed, invalid rate of 0.7%). For the high-level small DNA variant panel members, the overall PPC was 100% (207/207; 95% CI: 98.2%, 100%) (Table 14). The overall PNC for the high-level small DNA variant panel members was 100% (1035/1035; 95% CI: 99.6%, 100%) (Table 15). For low-level targeted small DNA variant panel members, the overall PPC for the low-level targeted small DNA variant panel members was 99.1% (210/212; 95% CI: 96.6%, 99.7%) (Table 14), and the overall PNC was 100% (1026/1026; 95% CI: 99.6%, 100%) (Table 15). Table 14. PPC of Targeted RET Small DNA Variants | Variant Level | Variant Type | Targeted Variant (Nucleotide) | Targeted Variant (Amino Acid) | N | Mean VAF^{1} | Percent Positive Calls (%) | 95% CI^{2} | | --- | --- | --- | --- | --- | --- | --- | --- | | High | SNV | chr10_4361741 6_T_C | RET p.(M918T) | 34 | 0.156 | 100 (34/34) | (89.8, 100) | | | SNV | chr10_4360994 9_G_C | RET p.(C634S) | 36 | 0.140 | 100 (36/36) | (90.4, 100) | | | SNV | chr10_4361499 6_G_A | RET p.(V804M) | 33 | 0.116 | 100 (33/33) | (89.6, 100) | | | MNV | chr10_4360994 9_GC_AT | RET p.(C634Y) | 35 | 0.195 | 100 (35/35) | (90.1, 100) | | | Deletion | chr10_4361561 1_GAGATGTT TATGA_G | RET p.(D898_E901d el) | 33 | 0.199 | 100 (33/33) | (89.6, 100) | | | Insertion | chr10_4360994 6_T_TGTGCC GCAC | RET p.(C634_T636d up) | 36 | 0.095 | 100 (36/36) | (90.4, 100) | | | All small DNA | All small DNA variants high | All small DNA variants high | 207 | N/A^{1} | 100 (207/207) | (98.2, 100) | PMA P230011/S006: FDA Summary of Safety and Effectiveness Data Page 26 of 63 {26} | Variant Level | Variant Type | Targeted Variant (Nucleotide) | Targeted Variant (Amino Acid) | N | Mean VAF^{1} | Percent Positive Calls (%) | 95% CI^{2} | | --- | --- | --- | --- | --- | --- | --- | --- | | | variants high | | | | | | | | Low | SNV | chr10_43617416_T_C | RET p.(M918T) | 35 | 0.042 | 100 (35/35) | (90.1, 100) | | | SNV | chr10_43601830_G_A | RET p.(V292M) | 35 | 0.033 | 94.3 (33/35) | (81.4, 98.4) | | | SNV | chr10_43613840_G_C | RET p.(E768D) | 36 | 0.044 | 100 (36/36) | (90.4, 100) | | | MNV | chr10_43609949_GC_AT | RET p.(C634Y) | 36 | 0.071 | 100 (36/36) | (90.4, 100) | | | Deletion | chr10_43615611_GAGATGTTTATGA_G | RET p.(D898_E901del) | 34 | 0.065 | 100 (34/34) | (89.8, 100) | | | Insertion | chr10_43609946_T_TGTGCCGCAC | RET p.(C634_T636dup) | 36 | 0.037 | 100 (36/36) | (90.4, 100) | | | All small DNA variants low | All small DNA variants low | All small DNA variants low | 212 | N/A^{1} | 99.1 (210/212) | (96.6, 99.7) | N/A, not applicable. $^{1}$ VAF, variant allele frequency. $^{2}$ 95% two-sided confidence interval calculated via the Wilson score method. Table 15 PNC of Targeted RET Small DNA Variants | Variant Level | Variant Type | Targeted Variant (Nucleotide) | Targeted Variant (Amino Acid) | N^{1} | Percent Negative Calls (%) | 95% CI^{2} | | --- | --- | --- | --- | --- | --- | --- | | High | SNV | chr10_43617416_T_C | RET p.(M918T) | 173 | 100 (173/173) | (97.8, 100) | | | SNV | chr10_43609949_G_C | RET p.(C634S) | 171 | 100 (171/171) | (97.8, 100) | | | SNV | chr10_43614996_G_A | RET p.(V804M) | 174 | 100 (174/174) | (97.8, 100) | PMA P230011/S006: FDA Summary of Safety and Effectiveness Data Page 27 of 63 {27} | Variant Level | Variant Type | Targeted Variant (Nucleotide) | Targeted Variant (Amino Acid) | N^{1} | Percent Negative Calls (%) | 95% CI^{2} | | --- | --- | --- | --- | --- | --- | --- | | | MNV | chr10_43609949_GC_AT | RET p.(C634Y) | 172 | 100 (172/172) | (97.8, 100) | | | Deletion | chr10_43615611_GA_GATGTTTATGAG | RET p.(D898_E901del) | 174 | 100 (174/174) | (97.8, 100) | | | Insertion | chr10_43609946_T_TGTGCCGCAC | RET p.(C634_T636dup) | 171 | 100 (171/171) | (97.8, 100) | | | All small DNA variants high | All small DNA variants high | All small DNA variants high | 1035 | 100 (1035/1035) | (99.6, 100) | | Low | SNV | chr10_43617416_T_C | RET p.(M918T) | 177 | 100 (177/177) | (97.9, 100) | | | SNV | chr10_43601830_G_A | RET p.(V292M) | 143 | 100 (143/143) | (97.4, 100) | | | SNV | chr10_43613840_G_C | RET p.(E768D) | 176 | 100 (176/176) | (97.9, 100) | | | MNV | chr10_43609949_GC_AT | RET p.(C634Y) | 176 | 100 (176/176) | (97.9, 100) | | | Deletion | chr10_43615611_GA_GATGTTTATGA_G | RET p.(D898_E901del) | 178 | 100 (178/178) | (97.9, 100) | | | Insertion | chr10_43609946_T_TGTGCCGCAC | RET p.(C634_T636dup) | 176 | 100 (176/176) | (97.9, 100) | | | All small DNA variants low | All small DNA variants low | All small DNA variants low | 1026 | 100 (1026/1026) | (99.6, 100) | $^{1}$ All observations pooled from panel member-variant combinations for which the majority call is negative (targeted variants harboring fusions with less than 50% calls positive). $^{2}$ 95% two-sided confidence interval calculated via the Wilson score method. Table 16 shows the variance components analysis of variant allele frequencies (VAFs) across the approximately 36 observations for each panel member. The standard deviation (SD) and percent coefficient of variation (%CV; total and for each source) were calculated and presented for each targeted RET small DNA variant. Table 16. Variance Components Analysis of VAF in Targeted Small DNA Variants Panel Members PMA P230011/S006: FDA Summary of Safety and Effectiveness Data Page 28 of 63 {28} | Variant Level | Variant Type | Targeted Variant (Nucleotide) | Targeted Variant (Amino Acid) | N | Mean VAF | Site SD (%CV) | Operator SD (%CV) | Day SD (%CV) | Replicate SD (%CV) | Total SD (%CV) | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | High | SNV | chr10_43617416_T_C | RET p.(M918T) | 34 | 0.156 | 0.011 (7.2) | 0.000 (0.0) | 0.000 (0.0) | 0.017 (10.8) | 0.020 (13.0) | | | SNV | chr10_43609949_G_C | RET p.(C634S) | 36 | 0.140 | 0.006 (4.6) | 0.000 (0.0) | 0.005 (3.7) | 0.014 (10.2) | 0.017 (11.8) | | | SNV | chr10_43614996_G_A | RET p.(V804M) | 33 | 0.116 | 0.005 (4.1) | 0.000 (0.0) | 0.002 (1.7) | 0.012 (10.7) | 0.013 (11.6) | | | MNV | chr10_43609949_GC_AT | RET p.(C634Y) | 35 | 0.195 | 0.000 (0.0) | 0.000 (0.0) | 0.009 (4.4) | 0.012 (6.0) | 0.015 (7.5) | | | Deletion | chr10_43615611_GAGATGTTTATGA_G | RET p.(D898_E901 del) | 33 | 0.199 | 0.000 (0.0) | 0.000 (0.0) | 0.011 (5.5) | 0.017 (8.6) | 0.020 (10.2) | | | Insertion | chr10_43609946_T_TGTGCCGCAC | RET p.(C634_T636 dup) | 36 | 0.095 | 0.003 (3.0) | 0.000 (0.0) | 0.000 (0.0) | 0.009 (9.6) | 0.010 (10.1) | | Low | SNV | chr10_43617416_T_C | RET p.(M918T) | 35 | 0.042 | 0.000 (0.0) | 0.000 (0.0) | 0.000 (0.0) | 0.009 (22.2) | 0.009 (22.2) | | | SNV | chr10_43601830_G_A | RET p.(V292M) | 35 | 0.033 | 0.000 (0.0) | 0.003 (9.8) | 0.002 (6.2) | 0.007 (21.7) | 0.008 (24.6) | | | SNV | chr10_43613840_G_C | RET p.(E768D) | 36 | 0.044 | 0.003 (6.0) | 0.000 (0.0) | 0.000 (0.0) | 0.008 (17.5) | 0.008 (18.5) | | | MNV | chr10_43609949_GC_AT | RET p.(C634Y) | 36 | 0.071 | 0.000 (0.0) | 0.008 (10.7) | 0.000 (0.0) | 0.011 (14.9) | 0.013 (18.4) | | | Deletion | chr10_43615611_GAGATGTTTATGA_G | RET p.(D898_E901 del) | 34 | 0.065 | 0.002 (2.5) | 0.006 (9.9) | 0.004 (6.4) | 0.010 (16.2) | 0.013 (20.2) | | | Insertion | chr10_43609946_T_TGTGCCGCAC | RET p.(C634_T636 dup) | 36 | 0.037 | 0.005 (13.8) | 0.000 (0.0) | 0.003 (9.1) | 0.006 (15.9) | 0.008 (22.9) | For the RET fusion panel members at 2-3x LoD, PPC and PNC values were 100.0%. For the low-level RET fusion panel members, PPC values were 97.2% and 97.1%, and PNC values were both 100.0% (Table 17 and Table 18). Further, none of the individual sites showed any additional discordance (Table 19). Table 17. PPC of TSO Comprehensive Assay for Detection of RET Fusions in High- and Low-Level Targeted Panel Members PMA P230011/S006: FDA Summary of Safety and Effectiveness Data Page 29 of 63 {29} | Variant Level | Targeted Fusion | n | Mean Supporting Reads | PPC (%) (n/N) | 95% CI^{1} | | --- | --- | --- | --- | --- | --- | | ~1x LoD | NCOA4-RET^{3} | 36 | 15.8 | 97.2 (35/36) | (85.8, 99.5) | | | KIF5B-RET^{2,4} | 34 | 16.6 | 97.1 (33/34) | (85.1, 99.5) | | ~2-3x LoD | NCOA4-RET^{3} | 36 | 36.7 | 100.0 (36/36) | (90.4, 100.0) | | | CCDC6-RET^{2,3} | 36 | 33.4 | 100.0 (36/36) | (90.4, 100.0) | $^{1}$95% 2-sided confidence interval (CI) calculated via the Wilson Score method. $^{2}$2 different fusions (KIF5B-RET and CCDC6-RET) were used for low- and high-level panel members due to sample insufficiency. $^{3}$Thyroid tissue $^{4}$Lung tissue from NSCLC Table 18. PNC of TSO Comprehensive Assay for Detection of RET Fusions in High- and Low-Level Targeted Panel Members | Variant Level | Targeted Fusions | n^{1} | PNC (%) (n/N) | 95% CI^{2} | | --- | --- | --- | --- | --- | | ~1x LoD | NCOA4-RET | 213 | 100.0% (213/213) | (98.2%, 100.0%) | | | KIF5B-RET | 251 | 100.0% (251/251) | (98.5%, 100.0%) | | ~2-3x LoD | NCOA4-RET | 215 | 100.0% (215/215) | (98.2%, 100.0%) | | | CCDC6-RET | 251 | 100.0% (251/251) | (98.5%, 100.0%) | $^{1}$ All observations pooled from panel member-variant combinations for which the majority call is negative, i.e., targeted variants harboring fusions with less than 50% calls positive. $^{2}$ 95% 2-sided confidence interval (CI) calculated via the Wilson Score method. Table 19. PPC of TSO Comprehensive Assay for Detection of RET Fusions in High- and Low-Level Targeted Panel Members by Site | Variant Level | Targeted Fusions | Site | n | PPC (%) (n/N) | 95% CI^{1} | | --- | --- | --- | --- | --- | --- | | ~1x LoD | NCOA4-RET | S1 | 12 | 100.0 (12/12) | (75.8, 100.0) | | | | S2 | 12 | 100.0 (12/12) | (75.8, 100.0) | | | | S3 | 12 | 91.7 (11/12) | (64.6, 98.5) | | | KIF5B-RET | S1 | 11 | 100.0 (11/11) | (74.1, 100.0) | | | | S2 | 11 | 90.9 (10/11) | (62.3, 98.4) | | | | S3 | 12 | 100.0 (12/12) | (75.8, 100.0) | | ~2-3x LoD | NCOA4-RET | S1 | 12 | 100.0 (12/12) | (75.8, 100.0) | | | | S2 | 12 | 100.0 (12/12) | (75.8, 100.0) | | | | S3 | 12 | 100.0 (12/12) | (75.8, 100.0) | PMA P230011/S006: FDA Summary of Safety and Effectiveness Data Page 30 of 63 {30} | | CCDC6-RET | S1 | 12 | 100.0 (12/12) | (75.8, 100.0) | | --- | --- | --- | --- | --- | --- | | | | S2 | 12 | 100.0 (12/12) | (75.8, 100.0) | | | | S3 | 12 | 100.0 (12/12) | (75.8, 100.0) | ¹ 95% 2-sided confidence interval (CI) calculated via the Wilson Score method. Table 20 (RET Fusions) shows the variance components analysis of supporting reads across the approximately 36 observations within each targeted fusion. The SD and %CV (total and for each source) were calculated and presented for each targeted fusion. Table 20. Variance Components Analysis of Supporting Reads in Targeted RET Fusion Panel Members | Variant Level | Fusion | n | Mean Supporting Reads | Site SD (%CV) | Operator SD (%CV) | Day SD (%CV) | Replicate SD (%CV) | Total SD (%CV) | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | ~1x LoD | NCOA4-RET | 36 | 15.8 | 2.08 (13) | 1.03 (7) | 0.00 (0) | 5.11 (32) | 5.61 (36) | | | KIF5B-RET | 34 | 16.6 | 2.07 (12) | 0.00 (0) | 1.58 (10) | 5.83 (35) | 6.39 (39) | | ~2-3x LoD | NCOA4-RET | 36 | 36.7 | 4.64 (13) | 4.09 (11) | 6.17 (17) | 5.20 (14) | 10.17 (28) | | | CCDC6-RET | 36 | 33.4 | 7.25 (22) | 2.56 (8) | 6.53 (20) | 5.51 (16) | 11.49 (34) | %CV: Percent coefficient of variation. SD: Standard deviation. ### Controls Reproducibility The reproducibility of TruSight Oncology DNA Control v2 and TruSight Oncology RNA Control v2 was evaluated with the TSO Comprehensive assay at three external sites. At each site, two operators/instruments using three reagent lots over three non-consecutive days, tested three lots in duplicates of the TruSight Oncology DNA Control v2 and three lots in duplicates of the TruSight Oncology RNA Control v2 with three lots of the TSO Comprehensive assay. A total of 102 valid observations for each of the DNA and RNA controls v2 were assessed for library validity based on the QC metric used by the TSO Comprehensive assay. For small DNA variants, 51 out of 54 variant calls need to be detected to pass. For fusion and splice variants, 20 out of 22 variants need to be detected to pass. In summary, the percentage of TruSight Oncology DNA control v2 libraries that passed was 98.0% (100/102; 95% CI: 93.1%, 99.5%) for small DNA variants. The percentage of TruSight Oncology RNA control v2 libraries that passed was 96.1% (98/102; 95% CI: 90.3%, 98.5%). ### 3. Analytical Sensitivity: Limit of Blank (LoB) Please refer to the Summary of Safety and Effectiveness Data (SSED) for P230011 for Analytical Sensitivity: Limit of Blank (LoB). PMA P230011/S006: FDA Summary of Safety and Effectiveness Data Page 31 of 63 {31} #### 4. Analytical Sensitivity: Limit of Detection (LoD) The Limit of Detection (LoD) of RET small DNA variants was evaluated using four medullary thyroid cancer FFPE tissue samples, which included RET SNVs, deletion, and MNV, as well as four FFPE-treated cell…