Who is the manufacturer of PGDx elio plasma focus Dx?

Personal Genome Diagnostics, Inc. filed the De Novo classification request for PGDx elio plasma focus Dx (DEN230046).

What is the FDA product code for PGDx elio plasma focus Dx?

SBY. It is classified under 21 CFR 866.6085 as a Class 2 device in the Pathology panel.

What is the regulatory pathway for PGDx elio plasma focus Dx?

De Novo classification (submission type: Direct).

Who can help prepare an FDA De Novo submission like PGDx elio plasma focus Dx?

Innolitics — a medical-device software consultancy — provides regulatory and engineering support for FDA De Novo submissions. See https://innolitics.com/services/regulatory/ or contact https://innolitics.com/contact.

PGDx elio plasma focus Dx

DEN230046 · Personal Genome Diagnostics, Inc. · SBY · Aug 1, 2024 · Pathology

Device Facts

Record ID	DEN230046
Device Name	PGDx elio plasma focus Dx
Applicant	Personal Genome Diagnostics, Inc.
Product Code	SBY · Pathology
Decision Date	Aug 1, 2024
Decision	DENG
Submission Type	Direct
Regulation	21 CFR 866.6085
Device Class	Class 2
Attributes	PCCP

Intended Use

PGDx elio plasma focus Dx is a qualitative next generation sequencing-based in vitro diagnostic device that uses targeted high throughput hybridization-based capture technology for detection of single nucleotide variants (SNVs), insertions and deletions (indels), copy number amplifications (CNAs), and translocations in human genomic circulating cell-free DNA (cfDNA) on the Illumina NextSeq 550Dx instrument. PGDx elio plasma focus Dx utilizes cfDNA from plasma of peripheral whole blood collected in Streek Cell-Free DNA blood collection tubes (BCTs). PGDx elio plasma focus Dx is a tumor mutation profiling test intended to provide information on mutations to be used by qualified health care professionals in accordance with professional guidelines in oncology for cancer patients with solid malignant neoplasms. The test is for use with patients previously diagnosed with cancer and in conjunction with other laboratory and clinical findings. A negative result from a plasma specimen does not assure that the patient's tumor is negative for genomic findings. Genomic findings are not prescriptive or conclusive for use of any specific therapeutic product.

Device Story

Qualitative NGS-based IVD; uses targeted hybridization-based capture to detect SNVs, indels, CNAs, and translocations in cfDNA from peripheral blood plasma. Operates on Illumina NextSeq 550Dx instrument. Used by qualified healthcare professionals in clinical settings to aid management of previously diagnosed cancer patients. Provides genomic mutation data to supplement clinical/laboratory findings; results are not prescriptive or conclusive for specific therapies. Includes authorized Pre-defined Change Control Plan (PCCP) for variant promotions and unmasking.

Clinical Evidence

Bench-only study. Analytical accuracy evaluated using 931 clinical plasma specimens and 48 contrived samples across 35 solid tumor types. Concordance assessed against six orthogonal methods. PPA for SNVs/indels with clinical significance in plasma ranged from 63.8% to 100% (97.6%-100% for variants ≥ LoD of orthogonal methods). NPA was 99.6%-99.9%. Precision/reproducibility assessed across three sites (504 replicates) with APA >95% for SNVs, deletions, and amplifications. No clinical prospective/retrospective study data provided.

Technological Characteristics

Targeted high-throughput hybridization-based capture NGS; utilizes Illumina NextSeq 550Dx sequencer. Analyzes cfDNA from peripheral blood plasma collected in Streck Cell-Free DNA BCTs. Detects SNVs, indels, CNAs, and translocations. Includes software with authorized Pre-defined Change Control Plan (PCCP).

Indications for Use

Indicated for patients previously diagnosed with cancer and solid malignant neoplasms to provide tumor mutation profiling information to qualified healthcare professionals.

Regulatory Classification

Identification

A high throughput sequencing based tumor profiling test of circulating cell-free nucleic acids is a qualitative in vitro diagnostic test intended for next generation sequencing analysis of circulating cell-free nucleic acids from plasma samples collected from peripheral whole blood to detect mutations in a panel of targeted genes to aid in the management of previously diagnosed cancer patients by qualified health care professionals. The results of the test are not prescriptive or conclusive for use of any specific therapeutic product.

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Submission Summary (Full Text)

{0}------------------------------------------------ Image /page/0/Picture/0 description: The image shows the logo of the U.S. Food and Drug Administration (FDA). The logo consists of two parts: the Department of Health and Human Services seal on the left and the FDA acronym with the full name of the agency on the right. The FDA part of the logo is in blue, with the acronym in a square and the full name written out to the right of the square. The full name of the agency is "U.S. Food & Drug Administration". # EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR PGDx elio plasma focus Dx DECISION SUMMARY #### I Background Information: ## A De Novo Number DEN230046 # B Applicant Personal Genome Diagnostics, Inc. ## C Proprietary and Established Names PGDx elio™ plasma focus Dx ## D Regulatory Information | Product Code(s) | Classification | Regulation Section | Panel | |--------------------|----------------|----------------------------------------------------------------------------------------------------------------------------|-----------| | SBY | Class II | 21 CFR 866.6085 - High throughput sequencing based tumor profiling test of circulating cell-free nucleic acids | Pathology | #### II Submission/Device Overview: ### A Purpose for Submission: De Novo request for evaluation of automatic class III designation for the PGDx elio plasma focus Dx # B Measurand: Single nucleotide variants, insertions, deletions, and translocations in genes in human genomic cell-free DNA (cfDNA) obtained from plasma. Refer to Table 1 in the Device Description for the complete list of genes and alterations reported by the test. # C Type of Test: Next generation sequencing based tumor profiling assay {1}------------------------------------------------ #### III Indications for Use: ## A Indication(s) for Use: PGDx elio plasma focus Dx is a qualitative next generation sequencing-based in vitro diagnostic device that uses targeted high throughput hybridization-based capture technology for detection of single nucleotide variants (SNVs), insertions and deletions (indels), copy number amplifications (CNAs), and translocations in human genomic circulating cell-free DNA (cfDNA) on the Illumina NextSeq 550Dx instrument. PGDx elio plasma focus Dx utilizes cfDNA from plasma of peripheral whole blood collected in Streek Cell-Free DNA blood collection tubes (BCTs). PGDx elio plasma focus Dx is a tumor mutation profiling test intended to provide information on mutations to be used by qualified health care professionals in accordance with professional. guidelines in oncology for cancer patients with solid malignant neoplasms. The test is for use with patients previously diagnosed with cancer and in conjunction with other laboratory and clinical findings. A negative result from a plasma specimen does not assure that the patient's tumor is negative for genomic findings. Genomic findings are not prescriptive or conclusive for use of any specific therapeutic product. ### B Special Conditions for Use Statement(s): For Prescription Use Only For in vitro diagnostic use ### C Special Instrument Requirements: Illumina® NextSeq™ 550Dx (qualified by PGDx) #### IV Device/System Characteristics: ### A Device Description: The PGDx elio plasma focus Dx assay is a hybrid-capture, next generation sequencing (NGS)based in vitro diagnostic assay (IVD) for the qualitative reporting of sequence mutations (SNVs and indels) in 33 genes, translocations in 3 genes, and amplifications in 5 genes. The assay consists of library preparation and sample indexing reagents. PGDx elio platform software, and a server inclusive of all essential data analysis software. The input of the test is cfDNA extracted from blood collected in the Streck Cell-Free DNA BCT using the OIAGEN OIAamp DSP Circulating NA Kit. The blood collection and DNA extraction materials are required but not supplied with the PGDx elio plasma focus Dx assay. Extracted cfDNA is used to prepare an indexed, targeted DNA library suitable for NGS on an Illumina NextSeq 550Dx instrument qualified by PGDx. Data analysis occurs on a dedicated server running the PGDx elio plasma focus Dx software that performs demultiplexing, alignment, variant calling, and filtering to generate reports containing detected and reportable alterations. Table 1 contains the 33 genes on the PGDx elio plasma focus Dx panel. SNVs and indels are reportable in all 33 genes, amplifications are reportable in 5 genes, and translocations are reportable in 3 genes. {2}------------------------------------------------ | Table 1. PGDx ello plasma focus Dx gene list | | | | | |----------------------------------------------|--------|--------|--------|------| | AKT1 | BRCA2 | ERBB23 | MET3 | POLE | | ALK2 | BRIP1 | EZH2 | MYC | RAF1 | | APC | CCND13 | FGFR1 | NRAS | RET2 | | ARID1A | CD2743 | FGFR23 | NTRK12 | ROS1 | | ATM | CDH1 | HRAS | PDGFR | TP53 | | BRAF | CSF1R | KIT | PIK3CA | - | | BRCA1 | EGFR | KRAS | POLD1 | - | #### Table 1 PCDx elio plasma focus Dy gene listl SNV and indels are reported for all genes 2 Translocations reported 3 Amplifications reported PGDx elio plasma focus Dx assay is capable to reporting ctDNA variants classified as FDA Levels 2, 3, or 4 variants. Variants classified as FDA Level 2 are classified in the report as ctDNA Variants with Evidence of Clinical Significance in Plasma. Variants classified as FDA Level 3 are classified in the report as ctDNA Variants with Evidence of Clinical Significance in Tissue. Additionally, the PGDx elio plasma focus Dx reports FDA Level 4 cancer mutations as Variants with Potential Clinical Significance (Table 2). ### Table 2. Category Definitions | FDA Levels of Evidence | Definition | PGDx elio plasma focus Dx Reporting Category | |------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------| | Level 2 | Clinical evidence from FDA-approved liquid biopsy companion diagnostic biomarkers for the specific tumor type at the biomarker or variant level. Analytical validity supported for each biomarker from accuracy, limit of blank (LoB), limit of detection (LoD), and precision/reproducibility, at the biomarker or variant level. | ctDNA Variants with Evidence of Clinical Significance in Plasma | | Level 3 | Clinical evidence from FDA-approved tissue-based companion diagnostic biomarkers, and/or professional guidelines for liquid or tissue Analytical validity supported by a representative approach for SNVs and indels from accuracy, LoB, LoD, and precision/reproducibility studies. Analytical validity supported for each translocation or copy number alteration from accuracy, LoB, LoD, and precision/reproducibility studies, at the gene level. | ctDNA Variants with Evidence of Clinical Significance in Tissue | | Level 4 | Biomarkers not categorized into Levels 2 or 3 can be included under Level 4 for informational purposes or to be used to direct patients toward clinical trials for which they may be eligible. Such claims can be supported by clinical rationale for inclusion in the panel. Such rationale could also include peer-reviewed publications for genes/variants in tissue, variant information from well curated public databases, or in vitro pre-clinical models. Analytical validity supported by a representative approach for SNVs and indels from accuracy, LoB, LoD, and precision/reproducibility studies. Analytical validity supported for each translocation or copy number alteration from accuracy, LoB, LoD, and precision/reproducibility studies, at the gene level. | ctDNA Variants with Potential Clinical Significance | # Reagents The PGDx elio plasma focus Dx product consists of assay kit components, PGDx elio platform software, and a server inclusive of all essential data analysis software. {3}------------------------------------------------ The PGDx elio plasma focus Dx kit provides material to accommodate 14 clinical samples across a maximum of three sequencing batches. PGDx elio plasma focus Dx accommodates flexible batch sizes. For details on the minimum and maximum batch sizes, refer to the product labeling (PGDx elio plasma focus Dx User Manual). PGDx elio plasma focus Dx kit reagent components are listed in Table 3. Materials required but not provided are described in the text below Table 3. A description of required equipment, software, reagents, and storage conditions are described in the product labeling (PGDx elio plasma focus Dx Manual). | Storage Temp. (°C) | Component Name | Volume (µL) | Cap Label | |-------------------------------------|-----------------------------------------------------------------------------|-------------|-----------------| | Library Preparation Kit, Box 1 of 2 | | | | | -25 to -15 | ER/AT Enzyme | 83 | ER/AT Enzyme | | -25 to -15 | ER/AT Buffer | 185 | ER/AT Buffer | | -25 to -15 | DNA Ligase | 275 | DNA Ligase | | -25 to -15 | Ligation Buffer | 825 | Ligation Buffer | | -25 to -15 | 10x Library Amplification Primer Mix | 132 | Primer Mix | | -25 to -15 | HotStart PCR Mix (2x) | 660 | PCR Mix | | Adapter Kit Box A, B, or C | | | | | -25 to -15 | Kit A (Adapters 1-16), Kit B (Adapters 17-32), or Kit C (Adapters 33-48) | 13 | RSB_XX (01-48) | | Library Preparation Kit, Box 2 of 2 | | | | | 2 to 8 | Pre-PCR Beads | 2790 | Pre-PCR | | 2 to 8 | Nuclease-Free Water | 2500 | NF Water | | Capture Kit, Box 1 of 4 | | | | | -25 to -15 | Hybridization Buffer | 400 | Hyb Buffer | | -25 to -15 | RNase Block | 14 | RNase Block | | -25 to -15 | Hybridization Blocker Mix | 132 | Hyb Block | | -25 to -15 | 10x Library Amplification Primer Mix | 132 | Primer Mix | | -25 to -15 | HotStart PCR Mix (2x) | 660 | PCR Mix | | Capture Kit, Box 2 of 4 | | | | | RT | Binding Buffer | 20000 | Binding Buffer | | RT | Wash Buffer 1 | 4840 | WB 1 | | RT | Wash Buffer 2 | 29000 | WB 2 | | Capture Kit, Box 3 of 4 | | | | | 2 to 8 | Capture Beads | 1320 | Capture Beads | | 2 to 8 | Post-PCR Beads | 2800 | Post-PCR | | 2 to 8 | Nuclease-Free Water | 2500 | NF Water | | Capture Kit, Box 4 of 4 | | | | | -85 to -65 | Capture Baits | 55 | Capture Baits | | External Control | | | | | Storage Temp. (°C) | Component Name | Volume (µL) | Cap Label | | 2 to 8 | External Control | 10 | Ex Control | Table 3: Reagent Components PGDx elio™ plasma focus Dx Kit {4}------------------------------------------------ # Materials Required but Not Provided Additional components required to run the assay but not provided by PGDx include blood collection tubes, cfDNA extraction materials, sequencing instrument and reagents, and molecular laboratory equipment. For a detailed list of required but not provided reagent and consumables, refer to the product labeling (PGDx elio plasma focus Dx User Manual). # PGDx elio Server and Software The proprietary PGDx elio™ server contains analysis and reporting software necessary for the PGDx elio plasma focus Dx assay (software versions are displayed within the PGDx elio platform user interface and on reports). The software is only compatible with NextSeq 550Dx instruments that have been qualified by PGDx with the PGDx elio local run manager (LRM) module installed. The PGDx elio server saves reports only and does not provide storage or backup of raw sequencing data. ### Sample Preparation PGDx elio plasma focus Dx requires cfDNA isolated from whole blood specimens collected in Streck Cell-Free DNA BCTs and extracted with OIAGEN® OIAamp DSP Circulating NA Kit. Extracted cfDNA should be quantified using a fluorometer. The cfDNA input for the assay is 25 ng of cfDNA. ## Library Preparation Sequence libraries are prepared through cfDNA end-repair, phosphorylation, and adenylation. Indexed adapters are then ligated to the adenvlated DNA molecules. Unincorporated adapters and reagents are removed by magnetic bead purification. Ligated libraries may be left overnight at -20 ℃. Adapter-ligated DNA is enriched by PCR amplification. Amplified libraries may be stored overnight at 4 °C in thermal cycler. Primer dimers and residual reagents are removed by magnetic bead purification. Purified libraries may be stored for up to 4 weeks at -20 ℃. Library quality is assessed using a fluorometer and a DNA fragment analyzer prior to hybrid capture. Purified, ligated, and amplified samples should have a concentration of > 25 ng/uL by fluorometer and an average peak size ≥ 230 bp between 180-1000 bp region by DNA fragment analyzer to ensure adequate hybridization for capture. # Hybrid Capture The adapter-ligated library is normalized to 300 ng. The normalized library is then hybridized with biotinylated RNA capture baits, and targeted regions are enriched using magnetic streptavidin coated beads. Captured libraries are purified to remove unincorporated baits and incompletely hybridized DNA fragments. Captured libraries are enriched by PCR amplification. Captured libraries may be stored overnight at 4 ℃ in thermal cycler. Primer dimers and residual reagents are removed by magnetic bead purification. Final library quality is assessed using a DNA fragment analyzer prior to sequencing. Purified capture library samples should have a concentration ≥ 5 nM and average peak size ≥ 230 bp between 180-1000 bp region by DNA fragment analyzer to ensure adequate sequencing. {5}------------------------------------------------ # Sequencing Captured libraries are normalized into a sequencing pool of up to 7 samples and the external control. Using the fragment analyzer data of the captured libraries. 2-5 uL of each sample is pooled to 10 nM. Pooled capture libraries (sequencing pool) may be stored for up to 4 weeks at -20 °C. The sequencing pool is fluorometrically quantified. Then 14 uL of the 10 nM pool is diluted to 4 nM then 5 uL of the 4 nM pool is denatured and further diluted to 20 pM with Illumina HT1 buffer. From the 20 pM pool, 117 uL is diluted with Illumina HT1 buffer to make 1300 uL of 1.8 pM, which is loaded in the sequencing cartridge and sequencing using a NextSeq 550Dx instrument that has been pre-qualified by PGDx with the PGDx elio LRM module installed. ## Data Analysis Sequence data is processed using the PGDx elio platform contains a user interface that tracks sample status from sequencing through analysis and reporting. Users configure sequencing runs, and the automated software performs bioinformatic analysis to identify and report genomic alterations. After processing, the software generates FASTQ files containing sequences and quality scores for each sample. The FASTO files are then aligned to a reference genome to generate BAM files, which are processed for variant calling of different alteration types (SNVs, indels, amplifications, and translocations). ### Report Generation The PGDx elio plasma focus Dx platform generates reports for each sample and the respective sequencing run processed. There are 3 report outputs, which are all available for download from the PGDx elio platform user interface. - . The Complete Run Record (CRR). A CSV report containing a summary of the sequencing metrics per run. This file is used to assess the quality of the sequencing run and is typically used for troubleshooting. Further details are provided in this guide. - . The Complete Case Record (CCR). A report containing quality control (OC) data for the run, sample, and external control, as well as a list of variant calls per sample. - The Case Report. A report organizing variants by association with clinical evidence per . FDA reporting Levels 2, 3, and 4. The PGDx elio plasma focus Dx product uses a positive reporting scheme only, meaning only detected variants passing the software's filters will be reported. Positive variants are reported in 1 of 3 categories: ctDNA Variants with Evidence of Clinical Significance in Plasma (FDA Level 2), ctDNA Variants with Evidence of Clinical Significance in Tissue (FDA Level 3), and ctDNA Variants of with Potential Clinical Significance (FDA Level 4). These reporting levels are also consistent with AMP/ASCO/CAP guidelines and OncoKB levels of evidence. Quality metrics are summarized, and results definitions are provided. # Controls An external control derived from cell-line material containing (five) 5 variants (i.e., AKTI E17K, EGFR M766 A767insASV, EGFR E746 A750del. KRAS G13D, and PIK3CA E545K) is used as a positive control in every run and is prepared in the same manner as a clinical sample through library preparation, capture, and sequencing. If the external control does not meet pre-defined library preparation quality criteria, all libraries in the batch should fail. The pre-capture and final library yields must be ≥ 25 ng/uL and ≥ 5nM, respectively, with an average size of ≥ 2230bp assessed within the 180-1000bp region. The analysis pipeline will fail all samples in a {6}------------------------------------------------ sequencing run if the five (5) expected variants are not detected or sufficient coverage is not achieved (i.e., ≥ 950x de-duplicated error-corrected [DEC] coverage). The no template control (NTC) is used in the workflow as a negative control and is processed through hybrid capture. The NTC undergoes a quality check post-library prep and post-capture prior to sample loading onto the sequencer. If the NTC fails to meet the pre-defined library preparation quality criteria, then the laboratory process is contaminated and should be repeated. # Quality Metrics The PGDx elio plasma focus Dx assay cutoffs include laboratory, sequencing, and analytical quality control metrics. Metrics and cutoffs were established and optimized using combinations of noncancerous donors, reference cell lines with known mutations, and clinical cfDNA specimens. Laboratory quality metrics include cutoffs at library preparation and hybrid capture. Sequencing quality metrics include sequence coverage depth, uniformity of coverage, and minimum base quality scores (Table 4). PGDx elio plasma focus Dx supports repeating the samples one-time through the workflow if key laboratory and sequencing quality metrics are not met. | Quality Metric | Level of Qualification | Passing Criteria | |-------------------------------------------------------------------------------------------|-------------------------|-------------------------------------------------------------------------------------------------------------------------------------| | Q30 Score | Run Level Sequencing QC | Reads 1-4 ≥ 75% | | Cluster Density | Run Level Sequencing QC | ≥ 130 K/mm2 | | Bases Covered (%) | Sample | ≥ 75% bases with ≥ 300x de- duplicated error-corrected (DEC) coverage | | Bases Mapped to ROI (%) | Sample | ≥ 20% bases mapped to ROI | | Contamination | Sample | No contamination detected | | Reads Identified per Sample (%) | Sample | ≥ 8% AND ≤ 25% | | Distinct Coverage | External Control | ≥ 950x DEC coverage | | Variants Expected | External Control | Absence of any of the following 5 variants: AKTI E17K, EGFR E746 A750del, EGFR M766 A767insASV, PIK3CA E545K, KRAS G13D | | Sequence Mutations for select ctDNA Variants with Evidence of Clinical Significance | Per-Variant Coverage | ≥ 100x DEC coverage for detected variants | | Sequence Mutations for select ctDNA Variants with Evidence of Clinical Significance | Per-Variant Coverage | Based on de-duplicated error- corrected (DE) variant observation threshold when no variant is detected | | ALK, RET, and NTRKI translocations | Per-Variant Coverage | ≥ 475x de-duplicated coverage across all reportable regions when no variant is detected | | MET and ERBB2 amplification genes | Per-Variant Coverage | ≥ 98% bases ≥ 100x de-duplicated coverage | | CCND1, CDK274, and FGFR2 amplification genes | Per-Variant Coverage | ≥ 98% bases ≥ 100x de-duplicated coverage | ### Table 4. Sequencing quality metrics Analytical quality metrics include variant calling thresholds, such as minimum sequence read coverage and minimum number of variant observations for a positive call. The SNV and indel {7}------------------------------------------------ reporting thresholds are defined in terms of number of variant observations and variant allele frequency (VAF). Variants with evidence of clinical significance require > 3 de-duplicated errorcorrected (DE) variant observations and a VAF > 0.1%. Hotspot mutations require > 3 DE variant observations and VAF ≥ 0.3% to be reported. Non-hotspot mutations require ≥ 6 DE variant observations and VAF ≥ 0.5%. Amplification status is determined by evaluating fold change and allelic imbalance. Fold change is calculated by comparing the normalized sample coverage to a collection of non-cancerous samples. Allelic imbalance is evaluated using a set of heterozygous single nucleotide polymorphisms (SNPs) when detected. A fold change > 1.2 or 1.33, depending on the gene, is reported as amplified. The pipeline identifies inter-chromosomal translocations and select intra-chromosomal events (i.e., inversions, deletions, or duplications). Regions evaluated for translocations within chromosomes must be greater than 6 kbp apart to be reported. These alterations are identified in genomic alignments of select gene regions using discordantly mapped read pairs, based on the analysis methodology of personalized analysis of rearranged ends (PARE). Translocation reporting thresholds are defined in terms of variant observations (i.e., number of fusion reads), and directly relates to the fusion read fraction (FRF) which is defined as the number of variant observations (fusion reads) divided by the coverage at that position. Reported hotspot translocations require ≥ 3 variant observations. Non-hotspot translocations require ≥ 7 variant observations. Metrics were first established and optimized using well-characterized cell lines with known variants. Once initial thresholds were established, clinical samples were run through the assay to measure device performance prior to assay lock and the commencement of analytical performance testing. ## B Predetermined Changed Control Plan The PGDx elio plasma focus Dx includes a predetermined change control plan (PCCP) approved by the US Food and Drug Administration (FDA). The PCCP describes a set of specific test methods for clinical and analytical validation, which include sample size determination, analysis methods, and acceptance criteria, for promoting and unmasking validated variants in the report without further regulatory review. PGDx will perform testing of the to be promoted or unmasked variants according to the specified protocols, and if the validation data meet the specified acceptance criteria, they may promote or unmask those variants in the report without additional premarket review. Variant promotion within the scope of the PCCP are variants promoted from FDA Levels 4 or 3 to Level 2, or Level 4 to 3. Variant unmasking within the scope of the PCCP are variants not previously reported under any FDA Level being unmasked to be reported under Levels 2, 3, and/or 4. The PCCP is limited to the promotion and unmasking of specific single nucleotide variants, insertions, deletions, copy number amplifications, and translocations in the 33 genes on the PGDx elio plasma focus Dx panel. The plan describes the specific clinical validation criteria that must be met to demonstrate the variant is eligible for promotion or unmasking into a specific reporting level. Specific analytical validation protocols and acceptance criteria are also detailed in the plan to ensure that device maintains the following performance characteristics for each promoted or unmasked variant: - · < 5% per sample false positive rate (FPR) AND < 0.000025% per variant FPR {8}------------------------------------------------ - . Limit of Detection (LoD) is established with clinical or contrived samples. If contrived samples are used to establish LoD due to low prevalence of the biomarker, LoD is confirmed with clinical samples as part of the precision evaluation. - . Accuracy point estimates of ≥ 90% positive percent agreement (PPA) and ≥ 95% negative percent agreement (NPA) much be achieved for SNVs and indels when considering variants at or above LoD of the PGDx elio plasma focus Dx only. Accuracy point estimates of ≥ 60% PPA and ≥ 95% NPA for all SNVs and indels must be achieved for all variants that will reported by PGDx elio plasma focus Dx irrespective of variant level. - Accuracy point estimates of ≥ 80% PPA and ≥ 95% NPA for copy number amplifications . and translocations when considering variants at or above LoD of the PGDx elio plasma focus Dx only. Accuracy point estimates of > 60% PPA and > 95% NPA for all copy number amplifications and translocations must be achieved for all variants that will reported by PGDx elio plasma focus Dx irrespective of variant level. - . > 95% PPA for mutation positive samples with alteration at 1-1.5x LoD and > 95% NPA for mutation negative samples for precision evaluation Additionally, software verification and validation activities are detailed in the PCCP and all must be completed successfully to modify the report to promote or unmask variants. If a new release version of the software is available, due to implementation of modifications made in accordance with the PCCP, PGDx notifies all existing customers that the update is available so that customers can have the update installed. # C Principle of Operation The PGDx elio plasma focus Dx assay is an in vitro diagnostic assay that uses targeted next generation sequencing to detect tumor gene alterations in cfDNA from plasma of peripheral whole blood in a 33 gene panel. PGDx elio™ plasma focus Dx targets cancer-associated genes that are enriched from genomic libraries using a high throughput hybridization-based capture technology. Genomic libraries are prepared through a series of enzymatic steps including end repair, A-base addition, ligation of index adaptors, and PCR amplification. Adapter-ligated libraries are hybridized with biotinylated RNA capture baits, and targeted regions are enriched using magnetic streptavidin coated beads followed by PCR amplification. Sample libraries are pooled and sequenced on an Illumina NextSeq 550Dx. Sequence data is processed using the PGDx elio platform and reads are aligned to the reference human genome. Somatic alterations in the sequence data are identified by comparing the identity of bases from the cfDNA to the reference human genome. The PGDx elio plasma focus Dx platform generates reports for each sample, including a summary of the alterations found, and the respective sequencing run processed. # D Instrument Description Information - 1. Instrument Name: Illumina NextSeq 550Dx (qualified by PGDx) - 2. Specimen Identification: {9}------------------------------------------------ Plasma obtained from peripheral whole blood - 3. Specimen Sampling and Handling: Refer to Section IV.A., Sample Preparation - 4. Calibration: Not applicable - 5. Quality Control: Refer to Section IV.A., Quality Metrics #### V Standards/Guidance Documents Referenced: - CLSI EP07 Interference Testing in Clinical Chemistry (3rd ed) . - CLSI EP25-A Evaluation of Stability of In Vitro Diagnostic Reagents . - CLSI EP17-A2 Evaluation of Detection Capability for Clinical Laboratory Measurement . Procedures - . CLSI EP05-A3 Evaluation of Precision of Quantitative Measurement Procedures (3rd ed) - . CLSI EP06 Evaluation of the Linearity of Quantitative Measurement Procedures (2nd ed) - CLSI EP12-A2 User Protocol for Evaluation of Qualitative Test Performance (2nd ed0 . - . Guidance on Informed Consent for In Vitro Diagnostic Device Studies Using Leftover Human Specimens that are Not Individually Identifiable; Guidance for Sponsors, Institutional Review Boards, Clinical Investigators, and Food and Drug Administration Staff (2006) - Guidance for the Content of Premarket Submissions for Software Contained in Medical . Devices; Guidance for Industry and FDA Staff (2005) - Content of Premarket Submissions for Management of Cybersecurity in Medical . Devices; Guidance for Industry and Food and Drug Administration Staff (2018) - Medical Device Accessories Describing Accessories and Classification Pathways: . Guidance for Industry and FDA Staff (2017) - Information to Support a Claim of Electromagnetic Compatibility (EMC) of Electrically . Powered Medical Devices; Guidance for Industry and Food and Drug Administration Staff (2016) - De Novo Classification Process (Evaluation of Automatic Class III Designation); . Guidance for Industry and Food and Drug Administration Staff (2021) - Acceptance Review for De Novo Classification Requests; Guidance for Industry and . Food and Drug Administration Staff (2021) - . Off-The-Shelf Software Use in Medical Devices; Guidance for Industry and Food and Drug Administration Staff (2019) - 9 Cybersecurity for Networked Medical Devices Containing Off-the-Shelf (OTS) Software; Guidance for Industry (2005) - . Content of Premarket Submissions for Management of Cybersecurity in Medical Devices; Guidance for Industry and Food and Drug Administration Staff (2014) {10}------------------------------------------------ - Postmarket Management of Cybersecurity in Medical Devices: Guidance for Industry and . Food and Drug Administration Staff (2016) - . Cybersecurity in Medical Devices: Quality System Considerations and Content of Premarket Submissions; Draft Guidance for Industry and Food and Drug Administration Staff (2022) - Cybersecurity in Medical Devices: Refuse to Accept Policy for Cyber Devices and . Related Systems Under Section 524B of the FD&C Act; Guidance for Industry and Food Food Food and Drug Administration Staff (2023) - . Marketing Submission Recommendations for a Predetermined change control Plan for Artificial Intelligence/Machine Learning (AI/ML) - Enabled Device Software Functions; Draft Guidance for Industry and Food and Drug Administration Staff (2023) - . Deciding When to Submit a 510(k) for a Change to an Existing Device: Guidance for Industry and Food and Drug Administration Staff" (2017) - Leveraging Existing Clinical Data for Extrapolation to Pediatric Uses of Medical . Devices; Guidance for Industry and Food and Drug Administration Staff (2016) #### VI Performance Characteristics: # A Analytical Performance: The PGDx elio plasma focus Dx is a targeted NGS panel with 33 genes. The targeted regions of interest in the panel are designed to detect SNVs as well as insertions and deletions (indels) in the coding exons of the targeted genes. The test also detects and reports CCND1, CD274, ERBB2, FGFR2, and MET amplifications, ALK, NTRK1, and RET translocations, For SNVs and indels with evidence of clinical significance in plasma, analytical performance at the biomarker or variant level across key studies (i.e., accuracy, limit of blank, limit of detection, and reproducibility/precision) was evaluated. For SNVs and indels with evidence of clinical significance in tissue and potential clinical significance, a representative approach to validation of the targeted genes in the panel was submitted with data representing variant types for SNVs and indels. For amplifications and translocations, analytical performance was determined at the gene level across key studies (i.e., accuracy, limit of blank, limit of detection, and reproducibility/precision). In addition, the assay was evaluated for performance regarding the panel wide quality metrics. - 1. Accuracy Concordance to Orthogonal Method(s): The detection of all reportable variant types by PGDx elio plasma focus Dx was compared to results across six orthogonal methods. A total of 931 clinical plasma specimens across 35 solid tumor types were assessed, along with 48 contrived samples. The contrived cohort of samples consisted of 34 spiked cell lines (i.e., DNA from a single cell line spiked into human plasma and extracted), 13 spiked cell line blends (i.e., DNA from a mix of at least two cell lines spiked into human plasma and extracted), and 1 clinical plasma blend (i.e., DNA from a mix of at least two donors of the intended use population) (Table 5). A total of 785 samples passed QC of PGDx elio plasma focus Dx and at least one orthogonal method. | Cancer Type | Clinical Plasma | Contrived¹ | Total | |-------------|-----------------|------------|-------| | NSCLC | 200 | 11 | 235 | | Colorectal | 170 | 6 | 176 | ### Table 5 Samples enrolled in accuracy study by tumor type {11}------------------------------------------------ | Cancer Type | Clinical Plasma | Contrived1 | Total | |-----------------------------------|-----------------|------------|-------| | Breast | 131 | 2 | 141 | | Colon | 86 | 0 | 86 | | Gastric | 46 | 2 | 49 | | Pancreas | 32 | 3 | 35 | | Ovarian | 31 | 0 | 31 | | Unknown Primary2 | 29 | 0 | 29 | | Endometrium | 28 | 0 | 28 | | Rectum | 24 | 0 | 24 | | Head and Neck | 23 | 0 | 23 | | Bladder | 17 | 3 | 20 | | Esophagus | 16 | 0 | 17 | | Prostate | 16 | 0 | 16 | | Thyroid | 16 | 0 | 16 | | Melanoma | 13 | 1 | 14 | | Liver | 12 | 1 | 13 | | Kidney | 9 | 0 | 9 | | Cholangiocarcinoma | 5 | 0 | 5 | | Glioblastoma | 5 | 0 | 5 | | Brain | 4 | 0 | 4 | | Appendix | 3 | 0 | 3 | | Neuroendocrine | 3 | 1 | 4 | | Cervix | 2 | 0 | 2 | | Sarcoma | 2 | 0 | 2 | | Small Intestine | 2 | 0 | 2 | | Squamous Cell Carcinoma | 1 | 0 | 1 | | Gall bladder | 1 | 0 | 1 | | Gastrointestinal Stromal Tumor | 1 | 0 | 1 | | Larynx | 1 | 0 | 1 | | Not Applicable3 | 0 | 14 | 20 | | Peritoneum | 1 | 0 | 1 | | Small Cell Lung | 1 | 2 | 3 | | Leukemia4 | 0 | 1 | 1 | | Lymphoma4 | 0 | 1 | 1 | | Total | 931 | 48 | 979 | 1 Includes 1 clinical plasma blend, 13 spiked cell line blends, 34 spiked cell lines. 2Includes unknown carcinoma, unknown adenocarcinoma, and unknown primary. 3Includes 1 clinical plasma blend and 13 cell line blends spiked into plasma. 4Cell lines spiked into plasma. Samples were enrolled into the study by selection with one of the orthogonal methods, an independent assay, or were fully uncharacterized. Reference status was assigned to each variant based on the orthogonal method. Concordance was assessed considering 1) all variants detected by either PGDx elio plasma focus Dx and/or an orthogonal method and 2) variants detected at VAF levels at or above LoDs of the orthogonal method(s) The LoDs of the orthogonal method(s) were comparable to that of the PGDx elio plasma focus Dx. A summary of PPA and NPA with 95% confidence intervals (Cls) across variant types, stratified by analysis (primary v. secondary) and sample type (clinical plasma specimens v. all sample types) is provided in Table 6. {12}------------------------------------------------ | | | All Variants | | | | Variants ≥ LoD of Orthogonal Method | | | | |-----------------------------------------------------------------------------|-----------------------------------|---------------------------------------------------------|---------------------------------------------------------------|---------------------------------------------------------|---------------------------------------------------------------|---------------------------------------------------------|--------------------------------------------------------------|---------------------------------------------------------|---------------------------------------------------------------| | Variant Type | Gene/Variant | Clinical Plasma | | All Samples | | Clinical Plasma | | All Samples | | | | | PPA n/N (95%CI) | NPA n/N (95%CI) | PPA n/N (95%CI) | NPA n/N (95%CI) | PPA n/N (95%CI) | NPA n/N (95%CI) | PPA n/N (95%CI) | NPA n/N (95%CI) | | | ATM | 69.2% 9/13 (42.4, 87.3) | 99.6% 1224/1229 (99.1, 99.8) | 77.8% 14/18 (54.8, 91.0) | 99.8% 4410/4417 (99.7, 99.9) | 100% 7/7 (64.6, 100) | 99.9% 4135/4140 (99.7, 99.9) | 100% 12/12 (75.8, 100) | 99.8% 4416/4423 (99.7, 99.9) | | SNVs/indels with Evidence of Clinical Significance in Plasma | EGFR exon 19 deletions | 100% 14/14 (78.5, 100) | 100% 403/403 (99.1, 100) | 90.6% 29/32 (75.8, 96.8) | 99.45% 546/549 (98.0, 100) | 100% 12/12 (75.8, 100) | 100% 501/501 (99.2, 100) | 90.0% 27/30 (74.4, 96.5) | 98.0% 625/638 (96.5, 98.8) | | | EGFR L858R | 87.5% 7/8 (52.9, 97.8) | 100% 668/668 (99.4, 100) | 94.7% 18/19 (75.4, 99.1) | 100% 801/801 (99.5, 99.9) | 100% 7/7 (64.6, 100) | 100% 765/765 (99.5, 100) | 100% 18/18 (82.4, 100) | 100% 802/802 (99.6, 100) | | | KRAS G12C | 87.9% 29/33 (72.7, 95.2) | 99.8% 597/598 (99.1, 99.9) | 89.7% 35/39 (76.4, 95.9) | 99.87% 780/781 (99.2, 99.9) | 100% 25/25 (86.7, 100) | 100% 747/747 (99.5, 100) | 100% 29/29 (88.3, 100) | 100% 791/791 (99.5, 100) | | | PIK3CA | 63.8% 51/80 (52.8, 73.4) | 99.9% 3974/3977 (99.8, 99.9) | 70.5% 74/105 (61.2, 78.4) | 99.88% 6067/6074 (99.7, 99.9) | 97.6% 41/42 (87.7, 99.6) | 99.97% 5744/5746 (99.9, 99.9) | 98.4% 63/64 (91.7, 99.7) | 99.9% 6103/6108 (99.8, 99.9) | | SNVs/indels with Evidence of Clinical Significance in Tissue | All | 60.5% 320/529 (56.3, 64.6) | 99.99% 221438/22 1453 (99.9, 99.9) | 70.0% 515/736 (66.6, 73.2) | 99.99% 235958/23 5982 (99.9, 99.9) | 95.7% 268/280 (92.7, 97.5) | 99.99% 221691/22 1702 (99.9, 99.9) | 96.6% 453/469 (94.5, 97.9) | 99.99% 236230/23 6249 (99.9, 99.9) | | SNVs/indels with Potential Clinical Significance | All | 53.4% 725/1359 (50.7, 56.0) | 99.99% 232782510 /23278257 2 (99.9, 99.9) | 64.7% 1199/1853 (62.5, 66.9) | 99.99% 145146092 /14514621 6 (99.9, 99.9) | 94.3% 593/629 (92.2, 95.8) | 99.99% 133865120 /13386513 6 (99.9, 99.9) | 96.2% 1026/1067 (94.8, 97.2) | 99.99% 145146954 /14514700 2 (99.9, 99.9) | | Insertions | All | 70.8% 17/24 (50.8, 85.1) | 100% 49454521/ 49454523 (99.9, 99.9) | 86.4% 51/59 (75.5, 93.0) | 99.99% 55089777/ 55089792 (99.9, 99.9) | 84.2% 16/19 (62.4, 94.5) | 99.99% 49454527/ 49454528 (99.9, 99.9) | 91.5% 43/47 (80.1, 96.6) | 99.99% 55089795/ 55089796 (99.9, 99.9) | | Deletions | All | 75.0% 54/72 (63.9, 83.6) | 99.99% 49454473/ 49454475 (99.9, 99.9) | 79.5% 89/112 (71.1, 85.9) | 99.99% 55089723/ 55089731 (99.9, 99.9) | 94.1% 48/51 (84.1, 98.0) | 99.99% 49454495/ 49454496 (99.9, 99.9) | 90.8% 79/87 (82.9, 95.3) | 99.99% 55089752/ 55089756 (99.9, 99.9) | | Amp- | CCNDI | 70.0% 14/20 (48.1, 85.5) | 99.8% 395/396 (98.6, 99.9) | 57.9% 22/38 (42.2, 72.1) | 99.8% 425/426 (98.7, 99.9) | 70.0% 14/20 (48.1, 85.5) | 99.7% 395/396 (98.6, 99.9) | 65.6% 21/32 (48.3, 79.6) | 99.8% 431/432 (98.7, 99.9) | | lifications | CD274 | 76.9% 10/13 (49.7, 91.8) | 99.7% 306/307 (98.2, 99.9) | 68.8% 11/16 (44.4, 85.8) | 99.7% 351/352 (98.4, 99.9) | 76.9% 10/13 (49.7, 91.8) | 99.7% 308/309 (98.2,99.9) | 91.7% 11/12 (64.6, 98.5) | 99.7% 361/362 (98.5, 99.9) | | | | All Variants | | Variants ≥ LoD of Orthogonal Method | | | | | | | Variant Type | Gene/Variant | Clinical Plasma | All Samples | Clinical Plasma | All Samples | | | | | | | ERBB2 | PPA n/N (95%CI) 79.4% 27/34 (63.2, 89.7) | NPA n/N (95%CI) 99.4% 476/479 (98.2, 99.8) | PPA n/N (95%CI) 62.7% 37/59 (50.0, 73.9) | NPA n/N (95%CI) 99.3% 528/532 (98.1, 99.7) | PPA n/N (95%CI) 79.4% 27/34 (63.2, 89.7) | NPA n/N (95%CI) 99.4% 476/479 (98.2, 99.8) | PPA n/N (95%CI) 67.3% 35/52 (53.8, 78.5) | NPA n/N (95%CI) 99.4% 512/515 (98.3, 99.8) | | | FGFR2 | PPA n/N (95%CI) 100% 6/6 (61.0, 100) | NPA n/N (95%CI) 99.5% 408/410 (98.2, 99.9) | PPA n/N (95%CI) 88.9% 8/9 (56.5, 98.0) | NPA n/N (95%CI) 99.6% 453/455 (98.4, 99.9) | PPA n/N (95%CI) 100% 5/5 (56.5, 100) | NPA n/N (95%CI) 99.5% 409/411 (98.2,99.9) | PPA n/N (95%CI) 85.7% 6/7 (48.7, 97.4) | NPA n/N (95%CI) 99.6% 461/463 (98.4, 99.9) | | | MET | PPA n/N (95%CI) 62.5% 10/16 (38.6, 81.5) | NPA n/N (95%CI) 99.8% 496/497 (98.9, 99.96) | PPA n/N (95%CI) 71.9% 23/32 (54.6, 84.4) | NPA n/N (95%CI) 99.6% 527/529 (98.6, 99.9) | PPA n/N (95%CI) 64.3% 9/14 (38.8, 83.7) | NPA n/N (95%CI) 99.8% 498/499 (98.9,99.9) | PPA n/N (95%CI) 70.8% 17/24 (50.8, 85.1) | NPA n/N (95%CI) 99.8% 542/543 (99.0, 99.9) | | Trans- locations | ALK 1 | PPA n/N (95%CI) 77.8% 7/9 (45.3, 93.7) | NPA n/N (95%CI) 100% 504/504 (99.2, 100) | PPA n/N (95%CI) 88.2% 15/17 (65.7, 96.7) | NPA n/N (95%CI) 99.63% 542/544 (98.6, 99.9) | PPA n/N (95%CI) 77.8% 7/9 (45.3, 93.7) | NPA n/N (95%CI) 100% 504/504 (99.2, 100) | PPA n/N (95%CI) 88.2% 15/17 (65.7, 96.7) | NPA n/N (95%CI) 99.63% 542/544 (98.6, 99.9) | | | NTRK1 2 | PPA n/N (95%CI) 50.0% 1/2 (9.5, 90.5) | NPA n/N (95%CI) 100% 511/511 (99.3, 100) | PPA n/N (95%CI) 75.0% 3/4 (30.1, 95.4) | NPA n/N (95%CI) 100% 557/557 (99.32, 100) | PPA n/N (95%CI) 50.0% 1/2 (9.5, 90.5) | NPA n/N (95%CI) 100% 511/511 (99.3, 100) | PPA n/N (95%CI) 75.0% 3/4 (30.1, 95.4) | NPA n/N (95%CI) 100% 557/557 (99.3, 100) | | | RET 3 | PPA n/N (95%CI) 75.0% 3/4 (30.1, 95.4) | NPA n/N (95%CI) 100% 509/509 (99.25, 100) | PPA n/N (95%CI) 92.9% 13/14 (68.5, 98.7) | NPA n/N (95%CI) 99.82% 546/547 (98.9, 99.9) | PPA n/N (95%CI) 75.0% 3/4 (30.1, 95.4) | NPA n/N (95%CI) 100% 509/509 (99.25, 100) | PPA n/N (95%CI) 92.3% 12/13 (66.7, 98.6) | NPA n/N (95%CI) 99.8% 548/549 (99.0, 99.97) | # Table 6. Summary of accuracy results - primary and secondary concordance assessments by variant and sample type {13}------------------------------------------------ ITwo ALK translocations were reported in orthogonal tests that were not reported by PGDx elio plasma focus Dx. For one of them, the orthogonal detected 6 fusion reads, which is considered a low level positive. 2One NTRK I translocation was reported by an orthogonal test that was not reported by PGDx elio plasma focus Dx. The test detected the translocation at 0.095% variant allele frequency, which is considered a low level positive. This case was also tested with another orthogonal method, and the translocation was not detected. 3 One RET translocation was reported by an orthogonal test that was not reported by PGDx elio plasma focus Dx. The test detected the translocation at 6 fusion reads, which is considered a low level positive. The positive agreement rates for SNVs/indels with Evidence of Clinical Significance in Plasma assessed in clinical specimens ranged from 63.8% to 100% PPA. The positive agreement rates for these variants were 97.6% to 100% PPA when considering only variants with levels at or above the LoDs of the orthogonal methods. The discordances observed when considering all variants identified in the accuracy study are due to PGDx elio plasma focus Dx and orthogonal methods having high imprecision at low VAF levels. Please refer to the interlaboratory reproducibility study data in Section VI.A.4.a. A limitation is included in the device labeling to address the risk of false negative results by indicating that a negative result does not rule out the presence of an alteration in the patient's tumor and should be followed by additional confirmatory testing. The positive agreement rates for SNVs and indels with Evidence of Clinical Significance in Tissue and SNVs and indels with Potential Clinical Significance assessed in clinical specimens were 60.5% PPA and 53.4% PPA, respectively. When considering only variants with levels at or above the LoDs of the orthogonal methods, positive agreement rates were 95.7% and 94.3% PPA, respectively. {14}------------------------------------------------ The positive agreement rates for amplifications assessed in clinical specimens ranged from 62.5% to 100% PPA when considering all variants identified in the study, and 64.3% to 100% PPA when considering only variants with levels at or above the LoDs of the orthogonal methods. Concordance was also assessed for amplifications detected at >1.8-fold, which had an aggregate PPA of 90.7% (80.1%. 96.0%) relative to the aggregate PPA of 75.3% (65.4%, 83.1%) when considering all amplifications detected in the study. A limitation is included in the device labeling to address the risk of false negative results for amplifications with fold change < 1.8-fold. The positive agreement rates for translocations assessed in clinical specimens ranged from 50% to 77.8% PPA. and 75% to 92.3% PPA when considering all sample types. The PPA for NTRK I translocations was 75% (3/4), with one specimen harboring a NTRK I translocation as reported by an orthogonal method but not reported by PGDx elio plasma focus Dx. This specimen was also tested with a second orthogonal method, and the translocation was not detected. A summary of the PGDx elio plasma focus Dx overall pass rates of clinical specimens enrolled in the accuracy study is shown in Table 7. Across the ≥ 30 tumor types evaluated in the study, the overall pass rate was >95%. The lower overall success rate for esophagus cancers was due to two specimens failing the contamination OC metric. The lower overall success rates for head and neck and kidney cancers are likely due to their low shedding status. (i.e., head and neck and kidney cancers are known to shed less ctDNA into the blood relative to other tumor types). In general, these data demonstrate that pre-analytical and biological factors that can affect device performance are not significant across different tumor types supporting a pan tumor mutation profiling claim. | Cancer Type | Passing Samples | Total Samples | Overall Success Rate | |--------------------|-----------------|---------------|----------------------| | Bladder | 17 | 17 | 100% | | Cholangiocarcinoma | 5 | 5 | 100% | | Colon | 82 | 86 | 95% | | Colorectal | 161 | 170 | 95% | | Esophagus | 13 | 16 | 81% | | Gastric | 44 | 46 | 96% | | Melanoma | 13 | 13 | 100% | | NSCLC | 196 | 200 | 98% | | Ovarian | 31 | 31 | 100% | | Rectum | 23 | 24 | 96% | | Endometrium | 26 | 28 | 93% | | Liver | 12 | 12 | 100% | | Unknown Primary1 | 29 | 29 | 100% | | Breast | 123 | 131 | 94% | | Brain | 4 | 4 | 100% | | Glioblastoma | 5 | 5 | 100% | | Head and Neck | 19 | 23 | 83% | | Kidney | 8 | 9 | 89% | | Pancreas | 32 | 32 | 100% | | Prostate | 16 | 16 | 100% | | Thyroid | 16 | 16 | 100% | | Other2 | 17 | 18 | 94% | | Total | 892 | 931 | 96% | Table 7. Overall pass rates for clinical specimen enrolled in accuracy study 11ncludes Unknown Adenocarcinoma, Unknown Carcinoma, Unknown Primary cases {15}------------------------------------------------ 20ther (n ≤ 3 cases per tumor type): Appendix, Cervix, Gall Bladder, GIST, Larynx, Neuroendocrine, Peritoneum, Sarcoma, Small Cell Lung, Small Intestine, Squamous Cell Carcinoma, small intestine. ## 2. Contrived Sample Functional Characterization (CSFC) Comparable performance between clinical plasma specimens and contrived samples (i.e., cell line DNA spiked into plasma) for SNVs, translocations, and amplifications was established by testing a dilution series consisting of five dilution levels with 20 replicates per level across two reagent kit lots. Each dilution series was designed to match variant levels (i.e., VAF, fold, and FRF) to determine similar performance between the sample types. Hit rates at each dilution level were calculated and comparability was assessed across all five dilution levels using the Mann-Whitney U Test (p ≥ 0.05) and at each level using Fisher's Exact Test (p ≥ 0.05). Similar performance between clinical specimens and cell line samples was confirmed for an SNV (KRAS G12V), translocation (RET), and amplification (ERBB2). The Fisher's Exact Test evaluation showed no statistically significant differences (p-values > 0.05) in hit rates between clinical and cell line samples at all dilution levels. Additionally, the Mann-Whitney U Test showed no significant differences across all dilution levels. Comparability of performance between clinical plasma specimens and contrived samples was also assessed for indels by demonstrating equivalent hit rates across comparable VAF levels between the two sample types. Two different APC deletions were compared. APC I1417Lfs*2 identified in a cell line blend and APC M1431Cfs*42 identified in a clinical blend. Four VAF levels were assessed with at least 10 replicates per level, which demonstrated similar hit rates between the clinical and contrived sample. The results of the evaluation demonstrated that use of contrived samples to assess the analytical performance of the test does not lead to overestimation of assay performance. # 3. Analytical Sensitivity - a) Limit of Blank (False positive rate) The limit of blank (LoB) was established by evaluating plasma samples from healthy donors without a cancer diagnosis across two cohorts at 25 ng DNA input (Table 8). Cohort 1 consisted of 29 normal plasma specimens that passed OC and confirmed to be mutation negative based on sequencing with an externally validated orthogonal method, tested in duplicate. Cohort 2 consisted of 38 normal plasma specimens that passed OC and confirmed to be mutation negative based on sequencing with an externally validated orthogonal method. Cohort 2 also included sequencing of matched buffy coat to identify non-tumor somatic alterations, such as clonal hematopoiesis of indeterminate potential (CHIP), and germline variants, which were excluded from the analysis. | Cohort | Unique Donors | Observations at 25 ng | Pre-Screened | Matched Buffy Coat Sequencing | |--------|---------------|-----------------------|--------------|-------------------------------| | 1 | 29 | 58 | Yes | No | | 2 | 38 | 38 | Yes | Yes | | | Table 8. LoB study design across cohorts | | |--|------------------------------------------|--| |--|------------------------------------------|--| The LoB study results across the two cohorts is summarized in Table 9. In Cohort 1, no clinically significant variants were detected and the per sample FPR was 5.17%. The per {16}------------------------------------------------ sample FPR for amplifications and translocations was zero across both cohorts. The per variant FPR was calculated based on the number of sequenced bases in the targeted panel. 121.866 bp. The per variant FPR for all variant types across both cohorts was <0.0025%. | | Cohort 1 | | Cohort 2 | | |------------------------------------------------------------------------------|-----------------------|------------------|------------------|----------------| | Variant Type | Per Variant FPR | Per Sample FPR | Per Variant FPR | Per Sample FPR | | SNVs/indels with Evidence of Clinical Significance in Plasma or Tissue | 0% (0/662) | 0% (0/58) | 0% (0/662) | 0% (0/38) | | SNVs/indels with Potential Clinical Significance | 0.0025% (3/121866) | 5.17%1 (3/58) | 0% (0/121866) | 0% (0/38) | | Amplifications | 0% (n/a) | 0% (0/58) | 0% (n/a) | 0% (0/38) | | Translocations | 0% (n/a) | 0% (0/58) | 0% (n/a) | 0% (0/38) | | Table 9. Summary of LoB study results across cohorts | | | | |------------------------------------------------------|--|--|--| | | | | | 1 An NTRKI A293V at 0.59% VAF, ROSI D2203N at 0.95% VAF, and FGFRI A94T at 0.59% VAF were observed in one replicate across different donors. The LoB study results demonstrate a zero FPR for clinically significant SNVs. indels. amplifications, and translocations, and a near zero FPR across the panel. - b) Limit of Detection (LoD) The LoD is defined as the lowest average VAF for SNVs and indels, FRF for translocations, and fold for amplifications at which ≥ 95% of replicates are detected. The LoD of the PGDx elio plasma focus Dx for representative SNVs, insertions, and deletions, and all reportable amplifications and translocations, was established at the test required DNA input of 25 ng with a dilution series using cell line blends spiked into plasma. Variant LoDs were established using either probit or empirical approaches with 11 cell line blends in a dilution series of 10 replicates at five dilution levels above and below the LoD. The LoD was confirmed with clinical plasma blends diluted in normal plasma to target 1-1.5x the established LoD, using 20 unique cfDNA clinical sample pools of 10 replicates across each of two reagent kit lots. The LoD was established and/or confirmed for 8 SNVs/indels with evidence of clinical significance in plasma and all reportable translocations and amplifications (Table 10). Table 10. Summary of established and confirmed LoD for variants with evidence of clinical significance in plasma, translocations, and amplifications | Variant Type | Variant | Established LoD VAF, FRF, or Fold Change (n/N) | Confirmed LoD VAF, FRF, or Fold Change (n/N) | |--------------|--------------------------------|------------------------------------------------------|----------------------------------------------------| | SNV | ATM R3008H | ND | 1.1 (35/36)1.2 | | SNV | ATM splice donor variant | ND | 1.1 (20/20)2 | | SNV | ATM stop gained | ND | 0.94 (36/36)1,2 | | SNV | EGFR L858R | 0.43 (10/10) | 0.31 (19/20) | | SNV | KRAS G12C | 0.80 (10/10) | 0.69 (36/36)1 | | SNV | PIK3CA E545K | 0.55 (5/5) | 2.1 (36/36)1 | | SNV | PIK3CA H1047R | 1.0 (10/10) | 1.2 (19/19) | | Deletion | EGFR exon 19 in-frame deletion | 1.1 (10/10) | 0.71 (20/20) | {17}------------------------------------------------ | Variant Type | Variant | Established LoD VAF, FRF, or Fold Change (n/N) | Confirmed LoD VAF, FRF, or Fold Change (n/N) | |---------------|---------|------------------------------------------------------|----------------------------------------------------| | Translocation | ALK | 1.2 (10/10) | 0.49 (13/13)3.4 | | Translocation | NTRK1 | 0.21 (10/10) | 0.47 (19/20)5 | | Translocation | RET | 0.30 (10/10) | 0.41 (20/20)6 | | Amplification | ERBB2 | 1.4 (10/10) | 1.3 (20/20) | | Amplification | CD274 | ND | 1.6 (20/20) | | Amplification | MET | 1.3 (10/10) | 1.7 (20/20) | | Amplification | CCND1 | 1.4 (10/10) | 1.4 (19/19) | | Amplification | FGFR2 | 1.3 (10/10) | 1.4 (19/19) | 1LoD was confirmed from the Multi-site Reproducibility Study across 36 replicates. 2Observed VAF with >95% detection rate without direct prior LoD establishment data. 3LoD was confirmed from a single-site precision study across multiple operators, lots, and days. 4 Average Fusion Reads = 32.3; Average Breakpoint Coverage = 6550 5 Average Fusion Reads = 24.1; Average Breakpoint Coverage = 5289 6 Average Fusion Reads = 19.1; Average Breakpoint Coverage = 4604 ND = not determined In total, the LoD was established and/or confirmed for 156 SNVs, 6 insertions, and 12 deletions across the panel-wide median LoD VAFs and VAF ranges for SNVs and indels are summarized in Table 11. #### Table 11. Panel-wide LoD for SNVs and indels | Variant Type | Median VAF | VAF Range (Median) | |-----------------------------------------------|------------|--------------------| | SNVs with Evidence of Clinical Significance | 0.97% | 0.31-3.4% | | Indels with Evidence of Clinical Significance | 0.71% | 0.52-0.83% | | SNVs with Potential Clinical Significance | 1.7% | 0.76-3.1% | | Indels with Potential Clinical Significance | 1.4% | 0.52-5.6%1 | 1 An LoD was established at 5.8% VAF for a single long indel that was not targeted as part of the dilution series. ### 4. Precision/Reproducibility: #### a) Interlaboratory Reproducibility Interlaboratory reproducibility of the PGDx elio plasma focus Dx assay was assessed across three different sites (1 internal and 2 external sites), using cfDNA extracted from seven clinical blends (i.e., a mix of intended use clinical specimens from more than one donor) and seven cell line blends (i.e., a mix of more than one cell line spiked into human plasma and extracted). Each of the 14 samples were tested in duplicate, across three nonconsecutive days, with two different operators, and across each of the three laboratory sites for 36 replicates per sample and 504 total replicates. Each replicate began with the assay workflow post-DNA extraction. The samples used in the interlaboratory reproducibility study, along with their expected variants, are presented in Table 12. #### Table 12. Expected variants in samples used in interlaboratory reproducibility study | Sample Type | SNVs with Evidence of Clinical Significance in Plasma | SNVs with Evidence of Clinical Significance in Tissue | SNVs with Potential Clinical Significance | Indel(s) | Trans- location(s) | Amp- lification(s) | |----------------|-------------------------------------------------------------------|-------------------------------------------------------------------|----------------------------------------------------|----------|-----------------------|-----------------------| | Clinical Blend | ATM R447 *: ATM R3008H | 2 | 7 | 1 | 0 | 0 | {18}------------------------------------------------ | Sample Type | SNVs with Evidence of Clinical Significance in Plasma | SNVs with Evidence of Clinical Significance in Tissue | SNVs with Potential Clinical Significance | Indel(s) | Trans- location(s) | Amp- lification(s) | |--------------------|-------------------------------------------------------------------|-------------------------------------------------------------------|----------------------------------------------------|----------|-----------------------|-----------------------| | Clinical Blend | ATM splice variant; PIK3CA E545K | 1 | 15 | 2 | 0 | CCND1; FGFR2 | | Clinical Blend | 0 | 2 | 10 | 2 | 0 | MET | | Clinical Blend | PIK3CA H1047R | 1 | 18 | 2 | NTRK1 | 0 | | Clinical Blend | KRAS G12C | 0 | 12 | 1 | 0 | ERBB2 | | Clinical Blend | 0 | 0 | 5 | 1 | 0 | 0 | | Clinical Blend | 0 | 1 | 4 | 2 | 0 | 0 | | Cell Line Blend | PIK3CA H1047R…