(398 days)
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Yes
The document explicitly mentions "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change control Plan for Artificial Intelligence/Machine Learning (AI/ML) - Enabled Device Software Functions; Draft Guidance for Industry and Food and Drug Administration Staff (2023)". This reference strongly suggests that the device or its software incorporates AI/ML technology, as the guidance is specifically for such devices.
No
Explanation: The device is an in vitro diagnostic (IVD) device used for tumor mutation profiling to provide information on mutations. It is intended to aid qualified healthcare professionals in oncology but does not directly treat or prevent a disease, which is characteristic of a therapeutic device. The device's output is not prescriptive or conclusive for the use of any specific therapeutic product.
Yes
The "Intended Use / Indications for Use" section explicitly states that "PGDx elio plasma focus Dx is a qualitative next generation sequencing-based in vitro diagnostic device." It also describes its purpose as 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," which directly indicates its role in diagnosing or aiding in the diagnosis of a medical condition.
No
The device description explicitly states that the assay consists of "library preparation and sample indexing reagents," "PGDx elio platform software," and a "server inclusive of all essential data analysis software." It also requires "blood collection and DNA extraction materials" and an "Illumina NextSeq 550Dx instrument." While software is a component, it is part of a larger in vitro diagnostic system that includes hardware and reagents.
Yes, this device is an IVD (In Vitro Diagnostic).
Here's why:
- Explicit Statement in Intended Use: The very first sentence of the "Intended Use / Indications for Use" section explicitly states: "PGDx elio plasma focus Dx is a qualitative next generation sequencing-based in vitro diagnostic device..."
- Explicit Statement in Device Description: The "Device Description" section also states: "The PGDx elio plasma focus Dx assay is a hybrid-capture, next generation sequencing (NGS)based in vitro diagnostic assay (IVD)..."
- Nature of the Test: The device analyzes human biological specimens (cfDNA from plasma) to provide information about mutations in the context of cancer diagnosis and management. This is the core function of an in vitro diagnostic device.
Yes
The clearance letter explicitly states, "The PGDx elio plasma focus Dx includes a predetermined change control plan (PCCP) approved by the US Food and Drug Administration (FDA)." This phrase directly confirms that the FDA has reviewed and authorized the PCCP for this device.
Intended Use / Indications 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.
Product codes (comma separated list FDA assigned to the subject device)
SBY
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.
Mentions image processing
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Mentions AI, DNN, or ML
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Input Imaging Modality
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Anatomical Site
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Indicated Patient Age Range
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Intended User / Care Setting
qualified health care professionals in accordance with professional. guidelines in oncology
Description of the training set, sample size, data source, and annotation protocol
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Description of the test set, sample size, data source, and annotation protocol
Accuracy Study:
- Sample Size: A total of 931 clinical plasma specimens across 35 solid tumor types and 48 contrived samples were assessed. The contrived cohort consisted of 34 spiked cell lines, 13 spiked cell line blends, and 1 clinical plasma blend.
- Data Source: Clinical plasma specimens from patients with various solid tumor types and contrived samples (spiked cell lines/blends).
- Annotation Protocol: Reference status was assigned to each variant based on comparison to six orthogonal methods.
Limit of Blank (LoB) Study:
- Sample Size: Cohort 1: 29 normal plasma specimens (tested in duplicate). Cohort 2: 38 normal plasma specimens.
- Data Source: Plasma samples from healthy donors without a cancer diagnosis.
- Annotation Protocol: Pre-screened for mutation negativity based on sequencing with an externally validated orthogonal method. Cohort 2 also included matched buffy coat sequencing to identify non-tumor somatic alterations (CHIP) and germline variants, which were excluded.
Limit of Detection (LoD) Study:
- Sample Size: Established LoD used 11 cell line blends in a dilution series (10 replicates at five dilution levels). Confirmed LoD used 20 unique cfDNA clinical sample pools (10 replicates across each of two reagent kit lots).
- Data Source: Cell line blends spiked into plasma and clinical plasma blends.
- Annotation Protocol: Variants were established using either probit or empirical approaches. LoD was defined as the lowest average VAF/FRF/fold at which ≥ 95% of replicates are detected.
Interlaboratory Reproducibility Study:
- Sample Size: Seven clinical blends and seven cell line blends. 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.
- Data Source: CfDNA extracted from clinical blends (mix of intended use clinical specimens from more than one donor) and cell line blends (mix of more than one cell line spiked into human plasma and extracted).
- Annotation Protocol: Precision analysis performed for known mutations and all additional variants. Assessed by positive call rate, average positive agreement (APA) and average negative agreement (ANA), and positive or negative modal analysis.
End-to-End Precision Study:
- Sample Size: 14 clinical plasma specimens, each assessed in duplicate at both plasma isolation and cfDNA extraction, with each extracted cfDNA replicate run in duplicate through the assay (112 total observations). A supplemental study conducted with three amplifications and one translocation.
- Data Source: Clinical plasma specimens.
- Annotation Protocol: Assessment of variant calling from whole blood collection through sequencing and reporting.
Lot-to-Lot Precision Study:
- Sample Size: Three contrived samples, each tested in quadruplicate using three unique reagent lots (12 observations per lot).
- Data Source: Contrived samples (cell line DNA spiked into plasma).
- Annotation Protocol: Assessment of variant concordance.
Interfering Substances Study:
- Sample Size: Seven contrived samples for each of 11 potential interferents, tested in triplicate.
- Data Source: Contrived samples (cell line DNA spiked into plasma).
- Annotation Protocol: Assessed overall success rate, DEC coverage fold change, and percentage of exons/bases covered. Concordance of variant calls assessed between test and reference conditions.
DNA Input Robustness Study:
- Sample Size: Five contrived samples and three clinical plasma samples, run at five replicates per condition for varying DNA input concentrations.
- Data Source: Contrived samples (cell line DNA spiked into plasma) and clinical plasma samples.
- Annotation Protocol: Assessed overall success rate, DEC coverage fold change, and percentage of exons/bases covered. Concordance was assessed for observed variants using the modal status of each variant in the reference condition as truth.
Assay Guard Banding Study:
- Sample Size: Three contrived samples run in triplicate, varying critical steps in the assay protocol.
- Data Source: Contrived samples (cell line DNA blend spiked into plasma).
- Annotation Protocol: Assessed overall success rate and concordance of variants detected by calculating PPA and NPA against a reference condition.
Whole Blood Stability Study:
- Sample Size: Whole blood drawn from 15 cancer patients into four BCTs.
- Data Source: Whole blood from cancer patients.
- Annotation Protocol: Plasma isolated at different time points and temperatures. Assessed overall success rate, average plasma volume/cfDNA concentration/yield, relative exon-level sequencing coverage, and concordance.
Plasma Stability Study:
- Sample Size: 42 cancer patients.
- Data Source: Plasma isolated from peripheral whole blood of cancer patients.
- Annotation Protocol: Plasma stored at defined temperature and storage conditions. Assessed overall success rate, DEC coverage fold change, percentage of exons/bases covered, and concordance.
cfDNA Stability Study:
- Sample Size: Seven clinical samples (84 replicates) from the interlaboratory reproducibility study and clinical plasma samples from analytical performance studies.
- Data Source: CfDNA stored at -20°C.
- Annotation Protocol: Compared variant calls to prequalification results and analyzed overall pass rates and concordance by cfDNA age.
Sample Carryover and Cross-Contamination Study:
- Sample Size: 15 cases (14 mutation-positive, 1 mutation-negative reference cell line).
- Data Source: High-positive variants (including germline) and mutation-negative reference cell line.
- Annotation Protocol: Processed in checkerboard configuration, alternating positive and negative samples, to assess contamination within and between sequencing runs.
Lot Interchangeability Study:
- Sample Size: Three contrived samples, each tested in duplicate using two different lots each of library preparation and hybrid capture reagents.
- Data Source: Contrived samples (cell line DNA spiked into plasma).
- Annotation Protocol: Assessed concordance between reference and reagent lot exchange configurations.
Lane Combination Study:
- Sample Size: Seven variant-positive clinical plasma samples.
- Data Source: Variant-positive clinical plasma samples from full sequencing batches.
- Annotation Protocol: Compared results with and without PGDx elio diversiPhi as filler at six different ratios. Assessed concordance using PPA and NPA.
Incomplete Mixing and Underfilled Tube Study:
- Sample Size: Whole blood from 15 cancer patients collected into five tubes.
- Data Source: Whole blood from cancer patients.
- Annotation Protocol: Varied mixing and blood draw volume. Assessed overall success rate, plasma volume/cfDNA concentration/yield, DEC coverage fold change, relative exon-level sequencing coverage, and concordance.
BCT Lot-to-Lot Reproducibility Study:
- Sample Size: Whole blood from 60 cancer patients into four BCTs per patient.
- Data Source: Whole blood from cancer patients using different BCT lots.
- Annotation Protocol: Assessed overall success rate, cfDNA yield, and APA/ANA between and within BCT lots.
BCT Concordance Study:
- Sample Size: Whole blood from 60 advanced cancer patients in EDTA and Streck cfDNA BCTs.
- Data Source: Whole blood from advanced cancer patients.
- Annotation Protocol: Assessed overall success rates and concordance between the two tube types.
Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)
Analytical Performance:
-
Accuracy Concordance to Orthogonal Method(s):
- Study type: Concordance study.
- Sample size: 931 clinical plasma specimens, 48 contrived samples (34 spiked cell lines, 13 spiked cell line blends, 1 clinical plasma blend). 785 samples passed QC.
- Key results:
- SNVs/indels with Evidence of Clinical Significance in Plasma (clinical plasma): PPA 63.8% to 100%. PPA at or above LoD: 97.6% to 100%. NPA 99.1% to 100%.
- SNVs/indels with Evidence of Clinical Significance in Tissue (clinical plasma): PPA 60.5%. PPA at or above LoD: 95.7%. NPA 99.99%.
- SNVs/indels with Potential Clinical Significance (clinical plasma): PPA 53.4%. PPA at or above LoD: 94.3%. NPA 99.99%.
- Amplifications (clinical plasma): PPA 62.5% to 100%. PPA at or above LoD: 64.3% to 100%. NPA 99.7% to 99.8%.
- Translocations (clinical plasma): PPA 50% to 77.8%. PPA at or above LoD for ALK 77.8%, NTRK1 50%, RET 75%. NPA 100% for all.
- Overall pass rate: >95% (892/931).
-
Contrived Sample Functional Characterization (CSFC):
- Study type: Comparability study using dilution series.
- Sample size: Not explicitly stated, but cell line blends and clinical plasma used.
- Key results: Similar performance between clinical plasma specimens and contrived samples for SNVs, translocations, and amplifications (p ≥ 0.05).
-
Analytical Sensitivity - Limit of Blank (LoB):
- Study type: LoB determination.
- Sample size: Cohort 1: 29 normal plasma specimens; Cohort 2: 38 normal plasma specimens.
- Key results: Per sample FPR for clinically significant SNVs/indels, amplifications, and translocations was 0% across both cohorts. Per variant FPR across all variant types was 95% for SNVs, deletions, amplifications; >94% for insertions and translocations.
- Interlaboratory ANA: 99.4% to 99.9%.
-
Precision/Reproducibility - End-to-end Precision:
- Study type: Precision study.
- Sample size: 14 clinical plasma specimens (112 observations).
- Key results: Aggregated APA ≥ 96.2%; Aggregated ANA ≥ 99.9%. APA at or above LoD was 100% for all structural variants.
-
Precision/Reproducibility - Lot-to-lot Precision:
- Study type: Precision study.
- Sample size: Three contrived samples tested in quadruplicate using three unique reagent lots.
- Key results: SNVs (APA 90.5%, ANA 99.9%); Deletions (APA 86.6%, ANA 99.9%); Amplification (APA 100%, ANA 100%); Translocation (APA 100%, ANA 100%).
-
Analytical Specificity/Interference - Interfering Substances:
- Study type: Interference study.
- Sample size: Seven contrived samples for each of 11 potential interferents (tested in triplicate).
- Key results: Overall success rate >85%. Concordance (PPA) ≥92.6% and NPA ≥99.9% across all variant types compared to reference.
-
Analytical Specificity/Interference - Hybrid Capture Probe Specificity:
- Study type: In silico sequence homology assessment.
- Sample size: 9,093 target probes.
- Key results: 97.1% uniquely mapped. Minimal risk of false positives due to off-target sequences.
-
Robustness Studies - DNA Input:
- Study type: Robustness study.
- Sample size: Five contrived samples, three clinical plasma samples, at five replicates per condition.
- Key results: Overall success rate >85%. PPAs >96% and NPAs ≥99.9% for clinical plasma samples across input levels.
-
Robustness Studies - Assay Guard Banding:
- Study type: Robustness study.
- Sample size: Three contrived samples run in triplicate.
- Key results: Overall success rate 100%. PPA >94% and NPA >99.9% for all test conditions, except -25% ligation master mix (88.6% PPA).
-
Sample Stability - Whole Blood Stability:
- Study type: Stability study.
- Sample size: Whole blood from 15 cancer patients.
- Key results: Overall success rate >86.7%. PPAs 100% and NPA ≥99% compared to reference condition. Whole blood stable for up to 7 days.
-
Sample Stability - Plasma Stability:
- Study type: Stability study.
- Sample size: Plasma from 42 cancer patients.
- Key results: Overall success rate >92%. PPA 100% for 25-hour and 46-day timepoints, 95.0% for 1-year timepoint. NPA ≥99%. Plasma stable at 2-8°C for 24 hours or -80°C for 1 year.
-
Sample Stability - cfDNA Stability:
- Study type: Stability assessment.
- Sample size: Seven clinical samples from interlaboratory reproducibility study, clinical plasma samples from analytical performance studies.
- Key results: Modal PPA ≥ 93% and modal NPA ≥ 99% for all variant types. Overall pass rates similar for different cfDNA ages (96.7% to 100%). PPAs for different cfDNA age bins: 93.1%, 97.2%, 97.1%.
-
Sample Carryover and Cross-Contamination:
- Study type: Carryover/cross-contamination study.
- Sample size: 15 cases (14 positive, 1 negative).
- Key results: No evidence of contamination in 100% of mutation-negative samples within and between sequencing runs.
-
Lot Interchangeability:
- Study type: Interchangeability study.
- Sample size: Three contrived samples (tested in duplicate) using different reagent lots.
- Key results: PPA > 91% and NPA ≥ 99.9% across all aggregated variant types.
-
Lane Combination:
- Study type: Impact of partial sequencing batches.
- Sample size: Seven variant-positive clinical plasma samples.
- Key results: 100% PPA and ≥ 99.99% NPA for all variants at all lane ratio test conditions.
-
Blood Collection Tube Characterization and Concordance Studies - Incomplete Mixing and Underfilled Tube:
- Study type: BCT impact study.
- Sample size: Whole blood from 15 cancer patients (into five tubes).
- Key results: Overall success rate >93% (except 5mL underfilled at 73.3%). PPAs 100% and NPA >99% for all variants.
-
Blood Collection Tube Characterization and Concordance Studies - BCT Lot-to-Lot Reproducibility:
- Study type: Reproducibility study.
- Sample size: Whole blood from 60 cancer patients (into four BCTs per patient).
- Key results: Overall success rate 99.2%. APA >93% and ANA ≥99% between and within BCT lots.
-
Blood Collection Tube Characterization and Concordance Studies - Concordance:
- Study type: BCT type concordance.
- Sample size: Whole blood from 60 advanced cancer patients (in EDTA and Streck cfDNA BCTs).
- Key results: Overall success rates ≥95% for both tube types. PPA 95.8% and NPA 99.9% for aggregated variant types.
Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)
Accuracy:
- Positive Percent Agreement (PPA):
- SNVs/indels with Evidence of Clinical Significance in Plasma (clinical plasma): 63.8% to 100% (overall); 97.6% to 100% (≥ LoD).
- SNVs/indels with Evidence of Clinical Significance in Tissue (clinical plasma): 60.5% (overall); 95.7% (≥ LoD).
- SNVs/indels with Potential Clinical Significance (clinical plasma): 53.4% (overall); 94.3% (≥ LoD).
- Amplifications (clinical plasma): 62.5% to 100% (overall); 64.3% to 100% (≥ LoD).
- Translocations (clinical plasma): 50% to 77.8% (overall).
- Negative Percent Agreement (NPA):
- SNVs/indels with Evidence of Clinical Significance in Plasma (clinical plasma): 99.1% to 100%.
- SNVs/indels with Evidence of Clinical Significance in Tissue (clinical plasma): 99.99%.
- SNVs/indels with Potential Clinical Significance (clinical plasma): 99.99%.
- Amplifications (clinical plasma): 99.7% to 99.8%.
- Translocations (clinical plasma): 100%.
Limit of Blank (LoB):
- Per Sample False Positive Rate (FPR): 0% for clinically significant SNVs/indels, amplifications, and translocations. 5.17% for SNVs/indels with Potential Clinical Significance (for Cohort 1).
- Per Variant FPR: 95% for SNVs, deletions, amplifications; >94% for insertions and translocations.
- End-to-end aggregated APA: ≥ 96.2%.
- Lot-to-lot APA: 86.6% to 100%.
Reproducibility (Average Negative Agreement - ANA):
- Interlaboratory ANA: 99.4% to 99.9%.
- End-to-end aggregated ANA: ≥ 99.9%.
- Lot-to-lot ANA: 99.9% to 100%.
Predicate Device(s): If the device was cleared using the 510(k) pathway, identify the Predicate Device(s) K/DEN number used to claim substantial equivalence and list them here in a comma separated list exactly as they appear in the text. List the primary predicate first in the list.
Not Found
Reference Device(s): Identify the Reference Device(s) K/DEN number and list them here in a comma separated list exactly as they appear in the text.
Not Found
Predetermined Change Control Plan (PCCP) - All Relevant Information for the subject device only (e.g. presence / absence, what scope was granted / cleared under the PCCP, any restrictions, etc).
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:
- 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.
N/A
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
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:
- · 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
-
- Instrument Name:
Illumina NextSeq 550Dx (qualified by PGDx)
- Instrument Name:
-
- Specimen Identification:
9
Plasma obtained from peripheral whole blood
-
- Specimen Sampling and Handling:
Refer to Section IV.A., Sample Preparation
- Specimen Sampling and Handling:
-
- Calibration:
Not applicable
- Calibration:
-
- Quality Control:
Refer to Section IV.A., Quality Metrics
- Quality Control:
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.
-
- 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.
- Accuracy Concordance to Orthogonal Method(s):
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, |
-
| 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 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 I1417Lfs2 identified in a cell line blend and APC M1431Cfs42 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 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 | 6 | 35 | 1 | 0 | 0 |
| Cell Line
Blend | PIK3CA H1047R;
PIK3CA E545K | 3 | 33 | 2 | ALK; RET; | 0 |
| Cell Line
Blend | ATM Q2210*;
PIK3CA E542K;
EGFR L858R | 2 | 15 | 2 | 0 | 0 |
| Cell Line
Blend | PIK3CA E545K | 3 | 24 | 3 | ALK | 0 |
| Cell Line
Blend | EGFR L858R;
PIK3CA H1047R | 2 | 20 | 0 | 0 | ERBB2 |
| Cell Line
Blend | ATM Q2210*;
PIK3CA E542K | 2 | 22 | 0 | ALK | 0 |
| Cell Line
Blend | 0 | 0 | 11 | 0 | 0 | CD274 |
The precision analysis was performed for the known mutations (as listed in Table 12), and for all additional variants identified in the samples, but were not the basis for sample selection. The reproducibility of the assay was assessed in three ways; 1) agreement for each positive variant detected across replicates is reported (positive call rate), 2) average positive agreement (APA) and average negative agreement (ANA), and 3) positive or negative modal analysis (i.e., majority call across replicates).
The positive call rate and modal negative percent agreement (NPA) stratified by variant type, median LoD level assessed, and sample type is summarized in Table 13. The positive call rate of aggregate variants (inclusive of all variant types) assessed at or above the LoD was 97.0% (4995/5148). The positive call rate of variants with evidence of clinical significance in plasma assessed at or above the LoD was 98.5% (532/540). In general, variants assessed at levels below the LoD had, expectedly, positive call rates 3x | 100% (36/36) | n/a | 100% (108/108) | n/a |
| | All
Observed
Variants | 73.9% (2768/3744) | 83.44% (781/936) | 73.6% (6305/8568) | 83.5% (1654/1980) |
| | Below LoD | 61.2% (1431/2340) | 82.8% (715/864) | 57.9% (3002/5184) | 83.3% (1619/1944) |
| SNVs | 1-1.5x | 99.7% (287/288) | n/a | 95.1% (1506/1584) | 97.2% (35/36) |
| | 1.5-2x | 100% (324/324) | n/a | 99.6% (825/828) | n/a |
| | 2-3x | 100% (216/216) | n/a | 100% (576/576) | n/a |
| | >3x | 88.5% (510/576)1 | 91.7% (66/72)1 | 100% (396/396) | n/a |
| | All
Observed
Variants | 70.4% (76/108) | 80.6% (29/36) | 97.2% (35/36) | n/a |
| | Below LoD | 70.4% (76/108) | 80.6% (29/36) | 97.2% (35/36) | n/a |
| Insertions | 1-1.5x | n/a | n/a | n/a | n/a |
| | 1.5-2x | n/a | n/a | n/a | n/a |
| | 2-3x | n/a | n/a | n/a | n/a |
| | >3x | n/a | n/a | n/a | n/a |
| | All
Observed
Variants | 84.0% (272/324) | 94.4% (34/36) | 72.0% (285/396) | 97.2% (70/72) |
| | Below LoD | 79.4% (200/252) | 94.4% (34/36) | 67.0% (217/324) | 97.2% (70/72) |
| Deletions | 1-1.5x | 100% (36/36) | n/a | 88.9% (32/36) | n/a |
| | 1.5-2x | 100% (36/36) | n/a | 100% (36/36) | n/a |
| | 2-3x | n/a | n/a | n/a | n/a |
| | >3x | n/a | n/a | n/a | n/a |
| | All
Observed
Variants | 100% (144/144) | n/a | 94.0% (203/216) | n/a |
| | Below LoD | n/a | n/a | 86.1% (62/72) | n/a |
| Amplifications | 1-1.5x | 100% (36/36) | n/a | n/a | n/a |
| | 1.5-2x | 100% (72/72) | n/a | 97.2% (35/36) | n/a |
| | 2-3x | 100% (36/36) | n/a | n/a | n/a |
| | >3x | n/a | n/a | n/a | n/a |
| Translocations | All
Observed
Variants | 100% (36/36) | n/a | 82.4% (178/216) | 55.6% (20/36) |
| | Below LoD | n/a | n/a | 82.4% (178/216) | 55.6% (20/36) |
| | 1-1.5x | n/a | n/a | n/a | n/a |
| | 1.5-2x | 100% (36/36) | n/a | n/a | n/a |
| | 2-3x | n/a | n/a | n/a | n/a |
| | >3x | n/a | n/a | n/a | n/a |
19
IOf the 16 SNVs >3x LoD, 2 variants were borderline to the germline filter, RAF' 1201T and ALK P157S at 38.8% VAF and 39.3% VAF, respectively. The call rate for the RAFI 201T SNV was 1/36 and the call rate for the ALK P157S SNV was 5/36, which contribute to both the positive call rate and modal NPA.
Table 14 summarizes the positive call rates stratified by variant type and variant allele frequency (VAF). The overall positive call rate was 73.9% across all samples and replicates (9741/13176) with increased positive call rate at higher VAFs for SNVs and indels. The interlaboratory reproducibility study data indicate that reliable and reproducible results can be expected for SNVs at ≥ 1.0% VAF and deletions at ≥ 1.5% VAF. While there were no insertions evaluated at ≥ 1.0% VAF, there is an increased
20
trend in positive agreement rates with increased VAF suggesting that reliable and reproducible results are expected at higher VAF levels. All translocations and amplifications evaluated at levels above LoD had positive call rates >95%. In general, the interlaboratory reproducibility study data indicate that reliable and reproducible results can be expected for translocations at ≥ 0.5% FRF and amplifications at > 1.8-fold. Please also refer to the LoD and accuracy study results presented in Sections VI.A.3.b and VI.A.1, respectively.
| Variant
Type | VAF
Threshold | Positive Call Rate
(n/N) | Variants
Observed | Median
VAF/FRF/
fold
Ranges | Mean DEC
Observation/
FRF/fold
Ranges | Mean DEC
Coverage
Ranges |
|---------------------------------------|------------------|-----------------------------|----------------------|--------------------------------------|------------------------------------------------|--------------------------------|
| SNVs and | ≥ 0% VAF | 82.67% (744/900) | 25 | 0.325 -
25.8 | 4 - 549.6 | 1224-4971 |
| indels with | ≥ 0.5% VAF | 91.41% (724/792) | 22 | 0.5 - 25.8 | 9.8 - 549.6 | 1489 - 4971 |
| Evidence of | ≥ 0.75% VAF | 93.95% (575/612) | 17 | 0.78 - 25.8 | 12.3 - 549.6 | 1489-4971 |
| Clinical
Significance
in Plasma | ≥ 1.0% VAF | 99.07% (428/432) | 12 | 1.035 -
25.8 | 17 - 549.6 | 1754-3791 |
| | ≥ 1.5% VAF | 100% (252/252) | 7 | 1.72 - 25.8 | 45.7 - 549.6 | 2096-3157 |
| | ≥ 0% VAF | 73.69% (9073/12312) | 342 | 0.31 - 39.3 | 3 - 1628.4 | 496 - 8138 |
| | ≥ 0.5% VAF | 75.53% (8919/11808) | 328 | 0.5 - 39.3 | 6 - 1628.4 | 496 - 8138 |
| All SNVs | ≥ 0.75% VAF | 91.56% (7416/8100) | 225 | 0.75 - 39.3 | 7.8 - 1628.4 | 496 - 8138 |
| | ≥ 1.0% VAF | 96.35% (5619/5832) | 162 | 1 - 39.3 | 8 - 1628.4 | 496 - 8138 |
| | ≥ 1.5% VAF | 97.45% (4280/4392) | 122 | 1.51 - 39.3 | 8 - 1628.4 | 496 - 8138 |
| | ≥ 0% VAF | 77.08% (111/144) | 4 | 0.5 - 0.9 | 16.0 - 34.8 | 2944 - 5131 |
| | ≥ 0.5% VAF | 77.08% (111/144) | 4 | 0.5 - 0.9 | 16.0 - 34.8 | 2944 - 5131 |
| All Insertions | ≥ 0.75% VAF | 91.67% (33/36) | 1 | 0.89-0.9 | 34.9 - 34.8 | 3770 - 3770 |
| | ≥ 1.0% VAF | n/a | n/a | n/a | n/a | n/a |
| | ≥ 1.5% VAF | n/a | n/a | n/a | n/a | n/a |
| | ≥ 0% VAF | 77.36% (557/720) | 20 | 0.5 - 2.3 | 7 - 73.3 | 1037 - 4403 |
| | ≥ 0.5% VAF | 77.36% (557/720) | 20 | 0.5 - 2.3 | 7 - 73.3 | 1037 - 4403 |
| All Deletions | ≥ 0.75% VAF | 90.67% (457/504) | 14 | 0.835 - 2.3 | 11.9 - 73.3 | 1037 - 4403 |
| | ≥ 1.0% VAF | 90.05% (389/432) | 12 | 1.065 - 2.3 | 12.1 - 73.3 | 1037 - 4403 |
| | ≥ 1.5% VAF | 100% (252/252) | 7 | 1.53 - 2.3 | 18 - 73.3 | 1151 - 3004 |
| ALK trans | n/a | 100% (36/36)1 | 1 | 0.96 | 0.4 - 2.4 | 1854 - 4194 |
| ALK trans | n/a | 100% (36/36)1 | 1 | 0.72 | 0.3 - 1.6 | 2160 - 5111 |
| ALK trans | n/a | 44.4% (16/36)1 | 1 | 0.19 | 0.1 - 0.3 | 2058 - 3983 |
| NTRK1 trans | n/a | 100% (36/36) | 1 | 0.62 | 0.4 - 1.8 | 2157 - 8953 |
| RET trans | n/a | 77.8% (28/36)1 | 1 | 0.26 | 0.1 - 1.2 | 1569 - 4195 |
| CCND1 amp | n/a | 100% (36/36) | 1 | 2.73 | 2.37 - 3.02 | n/a |
| CD274 amp | n/a | 77.78 (28/36)1 | 1 | 1.50 | 1.34 - 1.65 | n/a |
| ERBB2 amp | n/a | 100% (36/36) | 1 | 2.07 | 1.81 - 2.27 | n/a |
| ERBB2 amp | n/a | 97.22% (35/36) | 1 | 2.26 | 1.86 - 2.68 | n/a |
| FGFR2 amp | n/a | 100% (36/36) | 1 | 2.93 | 2.74 - 2.99 | n/a |
| MET amp | n/a | 100% (36/36) | 1 | 1.82 | 1.59 - 1.93 | n/a |
| MET amp | n/a | 94.44 (34/36)1 | 1 | 1.33 | 1.21 - 1.42 | n/a |
Table 14. Interlaboratory reproducibility positive call rates by variant type and VAF | ||||
---|---|---|---|---|
--------------------------------------------------------------------------------------- | -- | -- | -- | -- |
1 Median fusion read fraction (FRF)/fold across replicates was below the LoD
DEC= de-duplicated error corrected; trans = translocation; amp = amplification;
21
The positive and negative modal analysis (i.e., majority call rates) per specimens for variants observed in ≥ 1 replicate are summarized in Table 15. The positive and negative call rates were comparable between clinical and cell line samples. Ninety (90) of 93 discordant variants had a median VAF below the LoD. For all other locations, the negative call rates are 100%. Refer to Section VI.A.3.b for the LoD estimated for the PGDx elio plasma focus Dx assay.
| Specimen Number
(Type) | Total Unique
Mutations Detected
Across All Replicates | Modal Positive Call
Rate1 (n/N) | Modal Negative Call
Rate2 (n/N) |
|---------------------------|-------------------------------------------------------------|------------------------------------|------------------------------------|
| 1 (clinical) | 16 | 98.15% (424/432) | 87.5% (126/144) |
| 2 (clinical) | 29 | 95.83% (690/720) | 78.09% (253/324) |
| 3 (clinical) | 17 | 96.03% (484/504) | 90.74% (98/108) |
| 4 (clinical) | 26 | 93.78% (709/756) | 85.56% (154/180) |
| 5 (clinical) | 20 | 90% (486/540) | 82.22% (148/180) |
| 6 (clinical) | 6 | 72.92% (105/144) | 80.56% (58/72) |
| 7 (clinical) | 7 | 100% (216/216) | 69.44% (25/36) |
| 8 (cell line) | 57 | 89.86% (1294/1440) | 78.10% (478/612) |
| 9 (cell line) | 45 | 87.90% (1329/1512) | 88.89% (96/108) |
| 10 (cell line) | 27 | 92.59% (600/648) | 76.23% (247/324) |
| 11 (cell line) | 35 | 91.67% (1023/1116) | 76.39% (110/144) |
| 12 (cell line) | 33 | 95.33% (755/792) | 86.11% (341/396) |
| 13 (cell line) | 40 | 92.78% (1002/1080) | 74.17% (267/360) |
| 14 (cell line) | 20 | 96.99% (419/432) | 96.18% (277/288) |
Table 15. Interlaboratory reproducibility modal call rates per specimen
1Positive call rate was calculated based on variants with majority call detected as positive.
2Negative call rate was calculated based on variants detected at least once, but with majority or equal call as negative.
Additionally, precision was assessed between laboratory sites, operators, and nonconsecutive days (runs). APA and ANA were calculated in a pair-wise fashion for each variant type (Table 16). The APA between laboratory sites, operators, and nonconsecutive days (runs) was >95% across SNVs, deletions, and amplifications, and >94% for insertions and translocations. The majority of insertions and translocations were detected at levels below LoD, contributing to the lower APA observed for these variant types.
Variant Type | Source of Variance | % Variance Explained |
---|---|---|
SNVs | Library Preparation | 20% |
Sequencing | 30% | |
Analysis | 50% | |
CNVs | Library Preparation | 30% |
Sequencing | 40% | |
Analysis | 30% |
Variant Type | Metric (CI95) | Overall | Inter-Site | Inter-Operator | Inter-Day |
---|---|---|---|---|---|
SNVs | APA | 95.8% | |||
(95.7, 95.9) | 95.8% | ||||
(95.7, 95.9) | 95.9% | ||||
(95.7, 96.0) | 95.9% | ||||
(95.7, 96.1) | |||||
ANA | 99.9% | ||||
(99.9, 100.0) | 99.9% | ||||
(99.9, 100.0) | 99.9% | ||||
(99.9, 100.0) | 99.9% | ||||
(99.9, 100.0) | |||||
Insertions | APA | 94.4% | |||
(93.4, 95.3) | 94.4% | ||||
(93.2, 95.5) | 94.1% | ||||
(91.6, 96.3) | 94.1% | ||||
(91.0, 96.8) | |||||
ANA | 99.9% | ||||
(99.9, 100.0) | 99.9% | ||||
(99.9, 100.0) | 99.9% | ||||
(99.9, 100.0) | 99.9% | ||||
(99.9, 100.0) | |||||
Deletions | APA | 97.0% | |||
(96.8, 97.3) | 97.0% | ||||
(96.7, 97.3) | 97.0% | ||||
(96.4, 97.6) | 97.2% | ||||
(96.4, 97.9) | |||||
ANA | 99.9% | ||||
(99.9, 100.0) | 99.9% | ||||
(99.9, 100.0) | 99.9% | ||||
(99.9, 100.0) | 99.9% | ||||
(99.9, 100.0) | |||||
Amplifications | APA | 96.1% | |||
(95.7, 96.5) | 95.4% | ||||
(94.9, 96.0) | 97.5% | ||||
(96.7, 98.3) | 97.5% | ||||
(96.5, 98.5) | |||||
ANA | 99.6% | 99.6% | 99.7% | 99.7% |
22
| Variant Type | Metric
(CI95) | Overall | Inter-Site | Inter-Operator | Inter-Day |
|----------------|------------------|-----------------------|-----------------------|-----------------------|-----------------------|
| | | (99.6, 99.7) | (99.5, 99.6) | (99.7, 99.8) | (99.6, 99.8) |
| Translocations | APA | 95.3%
(94.7, 95.9) | 95.3%
(94.5, 96.0) | 94.9%
(93.2, 96.3) | 95.6%
(93.8, 97.3) |
| | ANA | 99.4%
(99.3, 99.4) | 99.3%
(99.2, 99.4) | 99.6%
(99.4, 99.7) | 99.6%
(99.5, 99.7) |
b) End-to-end Precision
The precision of the assay for variant calling from whole blood collection through sequencing and reporting was assessed using 14 clinical plasma specimens. Each specimen was assessed in duplicate at both plasma isolation and cfDNA extraction, with each extracted cfDNA replicate run in duplicate through the assay for a total of 112 observations in the study. The inter-operator and inter-replicate performance for SNVs and indels identified at or above LoD is summarized in Table 17. The APA and ANA for aggregated variants was ≥ 96.2% and ≥ 99.9%, respectively.
A supplemental study was conducted to assess the end precision of the assay for structural variant (i.e., amplification and translocation) calling, which enrolled three amplifications and one translocation across multiple BCTs and operators. The APA at or above LoD was 100% for all structural variants (amplifications and translocations).
Table 17. End-to-end precision by variant type and sources of variance for SNVs and indels | |||
---|---|---|---|
(> 1x LoD) | |||
Comparison | SNVs (95%CI) | Indels (95%CI) | Aggregate variants |
(95%CI) | |||
Between Extraction | |||
Replicates APA (%) | 97.4% (95.4, 99.0) | 100.0% (94.3, 100.0) | 97.8% (96.2, 99.2) |
Between Extraction | |||
Replicates ANA (%) | 99.9% (99.9, 100.0) | 100.0% (99.99, 100.0) | 99.9% (99.9, 100.0) |
Between Extraction | |||
Operators APA (%) | 95.4% (93.7, 97.0) | 100.0% (97.1, 100.0) | 96.2% (94.8, 97.5) |
Between Extraction | |||
Operators ANA (%) | 99.9% (99.9, 100.0) | 100.0% (99.99, 100.0) | 99.9% (99.9, 100.0) |
Between Plasma Isolation | |||
Operators APA (%) | 95.4% (93.7, 97.0) | 100.0% (97.1, 100.0) | 96.2% (94.8, 97.5) |
Between Plasma Isolation | |||
Operators ANA (%) | 99.9% (99.9, 100.0) | 100.0% (99.99, 100.0) | 99.9% (99.9, 100.0) |
Between Paired Replicates | |||
APA (%) | 98.0% (95.5, 100.0) | 100.0% (89.3, 100.0) | 98.4% (96.3, 100.0) |
Between Paired Replicates | |||
ANA (%) | 99.9% (99.9, 100.0) | 100.0% (99.99, 100.0) | 99.9% (99.9, 100.0) |
c) Lot-to-lot Precision
Performance of the PGDx elio plasma focus Dx assay was assessed across three unique reagent lots by assessing variant concordance using two clinical blends and one cell line blend. Each of the three reagent lots were used to prepare the samples in quadruplicate for a total of 12 observations per lot. The APA and ANA between and within reagent lots, inclusive of all variants identified in the samples, stratified by variant type and gene for amplifications and translocations is summarized in Table 18.
23
| Variant Type | Between Lot APA
(%) (95%CI) | Between Lot ANA
(%) (95%CI) | Within Lot APA
(%) (95%CI) | Within Lot ANA
(%) (95%CI) |
|---------------|--------------------------------|--------------------------------|-------------------------------|-------------------------------|
| SNVs | 90.5%
(89.5, 91.5) | 99.9%
(99.9, 100.0) | 90.9%
(89.2, 92.4) | 99.9%
(99.9, 100.0) |
| Insertions | n/a | 100.0%
(99.99, 100.0) | n/a | 100.0%
(99.99, 100.0) |
| Deletions | 86.6%
(83.7, 89.3) | 99.9%
(99.9, 100.0) | 85.8%
(80.8, 90.2) | 99.9%
(99.9, 100.0) |
| Amplification | 100.0%
(96.2, 100.0) | 100.0%
(98.0, 100.0) | 100.0%
(90.4, 100.0) | 100.0%
(94.9, 100.0) |
| Translocation | 100.0%
(96.2, 100.0) | 100.0%
(98.0, 100.0) | 100.0%
(90.4, 100.0) | 100.0%
(94.9, 100.0) |
Table 18. Lot-to-lot precision by variant type
5. Linearity:
Not applicable
6. Analytical Specificity/Interference:
- a) Interfering Substances
To evaluate the robustness of the assay results in the presence of potentially interfering exogeneous and endogenous substances, a total of 11 potential interferents were evaluated. These included six endogenous substances (triglycerides, hemoglobin, conjugated and unconjugated bilirubin, total albumin, and cholesterol) and five exogenous substances (ethanol, proteinase K, EDTA, Staphylococcus epidermidis, and molecular index barcodes). Seven contrived samples (i.e., cell line DNA spiked into plasma) were spiked with each interfering substance before, during, or after DNA extraction, as appropriate, and tested in triplicate (single extraction).
The overall success rate in the test conditions were compared to that of the reference condition (no interferent). The overall success rate of the assay across test conditions was 100%, except for two test conditions (proteinase K at + 0.3 mg/mL and total albumin at 60 g/L) (Table 19). For both test conditions, all three replicates failed from a single sample due to operator error at the extraction step and not due to the interfering substances.
The DEC coverage fold change was determined by taking the median of the coverage of each test condition over the coverage of the reference condition at the sample and exon level. The fold change analysis demonstrated high comparability with the reference condition across all test conditions, with median values ranging from 0.74 to 1.04 at the sample level, and 0.74 and 1.11 at the exon level. All test conditions had sufficient coverage needed to maintain assay performance. The percentage of exons with ≥1050x DEC and bases covered at ≥300x DEC remained consistent across the test conditions, ranging from 97.78% to 99.26% and 96.47% to 97.05%, respectively (Table 20).
24
| Condition | Overall
Success
Rate (n/N) | Sample-level
DEC Coverage
Fold Change
(median) | Exon-level
DEC Coverage
Fold Change
(median) | Median %
Exons ≥
1050x
DEC | Median %
Bases
Covered >
300x DEC |
|--------------------------------------|----------------------------------|---------------------------------------------------------|-------------------------------------------------------|-------------------------------------|--------------------------------------------|
| Reference | 100%
(21/21) | N/A | N/A | 98.82 | 96.73 |
| Conjugated Bilirubin at
0.2 g/L | 100%
(21/21) | 0.82 | 0.87 | 98.97 | 96.66 |
| Cholesterol at 150 mg/dL | 100%
(21/21) | 0.74 | 0.74 | 97.93 | 96.52 |
| Cholesterol at 250 mg/dL | 100%
(21/21) | 0.92 | 0.95 | 99.11 | 97.05 |
| Hemoglobin at 2.0 g/L | 100%
(21/21) | 0.91 | 0.94 | 99.11 | 96.69 |
| Total Albumin at 60 g/L | 85.7%
(18/21) | 1.01 | 0.99 | 99.04 | 96.83 |
| Triglycerides at 33 g/L | 100%
(21/21) | 0.99 | 1.00 | 98.67 | 96.64 |
| Unconjugated Bilirubin at
0.2 g/L | 100%
(21/21) | 0.89 | 0.87 | 98.52 | 96.47 |
| Ethanol at +2.5% (v/v) | 100%
(21/21) | 0.85 | 0.91 | 98.82 | 96.83 |
| Ethanol at +5% (v/v) | 100%
(21/21) | 0.94 | 0.99 | 99.11 | 96.83 |
| Molecular Index Barcodes
at +5% | 100%
(21/21) | 1.00 | 1.06 | 99.04 | 96.83 |
| Molecular Index
Barcodes at +15% | 100%
(21/21) | 1.04 | 1.03 | 99.11 | 96.94 |
| Molecular Index
Barcodes at +30%) | 100%
(21/21) | 1.04 | 1.11 | 99.26 | 96.94 |
| Proteinase K at +0.3
mg/mL | 85.7%
(18/21) | 0.96 | 0.93 | 98.38 | 96.76 |
| Proteinase K at +0.6
mg/mL | 100%
(21/21) | 0.82 | 0.84 | 97.78 | 96.47 |
| S. epidermidis at 1e6
CFU/mL | 100%
(21/21) | 0.99 | 1.00 | 98.67 | 96.72 |
| EDTA at 9.0 mg/ml | 100%
(21/21) | 0.84 | 0.84 | 98.97 | 96.87 |
Table 19. Summary of interference results - pass rate and QC metrics
For concordance assessments, reference status was assigned to each variant if all replicates harbored the variant (positive), or no replicates harbored the variant (negative), in the neat sample. For each potential interferent, concordance of variant calls was assessed between the test conditions and reference condition without interferent. Across the 11 potential interferents tested across 16 conditions, concordance across each variant type was ≥92.6% PPA and ≥99.9% NPA compared to the reference condition (Tables 20 and 21). Please also refer to the underfilled tube/short draw study in Section VI.F.1.a for an additional assessment of interference based on increase BCT additive concentration.
25
| | | Triglycerides | Hemoglobin | Bilirubin | | Total
Albumin | Cholesterol | |
|---------------------|------------------|------------------------|------------------------|-------------------------|------------------------|---------------------------|----------------------------------|----------------------------------|
| Variant
Type | Metric
(CI95) | 37 mmol/L
(33 g/L) | 2.0 g/L | Unconjugated
0.2 g/L | Conjugated
0.2 g/L | 60 g/L | 3.88
mmol/L
(150
mg/dL) | 6.47
mmol/L
(250
mg/dL) |
| SNVs | PPA | 96.9%
(94.5, 98.3) | 95.2%
(92.4, 97.0) | 95.2%
(92.4, 97.0) | 95.5%
(92.8, 97.2) | 94.5%
(91.3,
96.6) | 95.8%
(93.1,
97.4) | 96.0%
(93.5,
97.6) |
| SNVs | NPA | 99.99%
(99.9, 99.9) | 99.99%
(99.9, 99.9) | 99.99%
(99.9, 99.9) | 99.99%
(99.9, 99.9) | 99.99%
(99.9,
99.9) | 99.99%
(99.9,
99.9) | 99.99%
(99.9,
99.9) |
| Indels | PPA | 93.3%
(78.7, 98.2) | 93.3%
(78.7, 98.2) | 93.3%
(78.7, 98.2) | 96.7%
(83.3, 99.4) | 95.2%
(77.3,
99.2) | 100%
(88.6,
100) | 100%
(88.6,
100) |
| Indels | NPA | 100%
(99.9, 100) | 100%
(99.9, 100) | 100%
(99.9, 100) | 100%
(99.9, 100) | 100%
(99.9,
100) | 100%
(99.9,
100) | 100%
(99.9,
100) |
| Amp-
lifications | PPA | 100%
(79.6, 100) | 100%
(79.6, 100) | 100%
(79.6, 100) | 93.3%
(70.2, 98.8) | 100%
(79.6,
100) | 100%
(79.6,
100) | 100%
(79.6,
100) |
| Amp-
lifications | NPA | 100%
(97.6, 100) | 100%
(97.6, 100) | 100%
(97.6, 100) | 100%
(97.6, 100) | 100%
(97.6,
100) | 100%
(97.6,
100) | 100%
(97.6,
100) |
| Trans-
locations | PPA | 95.2%
(77.3, 99.2) | 95.2%
(77.3, 99.2) | 95.2%
(77.3, 99.2) | 95.2%
(77.3, 99.2) | 100%
(82.4,
100) | 95.2%
(77.3,
99.2) | 95.2%
(77.3,
99.2) |
| | NPA | 100%
(95.6, 100) | 100%
(95.6, 100) | 100%
(95.6, 100) | 100%
(95.6, 100) | 100%
(95.6,
100) | 100%
(95.6,
100) | 100%
(95.6,
100) |
Table 20. Summary of endogenous interference results - concordance
Table 21. Summary of exogenous interference results - concordance | |||||
---|---|---|---|---|---|
| Variant
Type | Metric
(CI95) | EDTA
9
mg/mL | S.
epidermi
dis
1e6
CFU/mL | Proteinase K | | Ethanol | | Adaptor | | |
|---------------------|------------------|---------------------------|-------------------------------------------------------------|---------------------------|---------------------------|---------------------------|---------------------------|---------------------------|---------------------------|---------------------------|
| | | | | +0.3
mg/mL | +0.6
mg/mL | 2.5%
(v/v) | 5.0%
(v/v) | +5% | +15% | +30% |
| SNVs | PPA | 93.8%
(90.8,
95.9) | 95.2%
(92.4,
97.0) | 97.1%
(94.6,
98.5) | 94.1%
(91.1,
96.1) | 96.7%
(94.1,
98.2) | 97.1%
(94.5,
98.4) | 97.1%
(94.5,
98.4) | 95.4%
(92.5,
97.3) | 95.8%
(92.9,
97.5) |
| SNVs | NPA | 99.99%
(99.9,
99.9) | 99.99%
(99.9,
99.9) | 99.99%
(99.9,
99.9) | 99.99%
(99.9,
99.9) | 99.99%
(99.9,
99.9) | 99.99%
(99.9,
99.9) | 99.99%
(99.9,
99.9) | 99.99%
(99.9,
99.9) | 99.99%
(99.9,
99.9) |
| Indels | PPA | 96.7%
(83.3,
99.4) | 96.7%
(83.3,
99.4) | 100%
(87.5,
100) | 93.3%
(78.7,
98.2) | 92.6%
(76.6,
97.9) | 100%
(87.5,
100) | 96.3%
(81.7,
99.3) | 100%
(87.5,
100) | 92.6%
(76.6,
97.9) |
| | NPA | 100%
(99.9,
100) | 100%
(99.9,
100) | 100%
(99.9,
100) | 100%
(99.9,
100) | 100%
(99.9,
100) | 100%
(99.9,
100) | 100%
(99.9,
100) | 100%
(99.9,
100) | 100%
(99.9,
100) |
| | | | | | | | | | | |
| Amp-
lifications | PPA | 100%
(79.6,
100) | 100%
(79.6,
100) | 100%
(79.6,
100) | 100%
(79.6,
100) | 100%
(75.8,
100) | 100%
(75.8,
100) | 100%
(75.8,
100) | 100%
(75.8,
100) | 100%
(75.8,
100) |
| | NPA | 100%
(97.6,
100) | 100%
(97.6,
100) | 100%
(97.1,
100) | 100%
(97.6,
100) | 100%
(97.2,
100) | 100%
(97.2,
100) | 100%
(97.2,
100) | 100%
(97.2,
100) | 100%
(95.8,
99.9) |
| | | | | | | | | | | |
| Trans- | PPA | 100% | 90.5% | 95.2% | 100% | 94.4% | 100% | 88.9%1 | 94.4% | 100% |
26
| Variant
Type | Metric
(CI95) | EDTA
9
mg/mL | S.
epidermi
dis
1e6
CFU/mL | Proteinase K | | Ethanol | | Adaptor | | |
|-----------------|------------------|------------------------|----------------------------------------|------------------------|------------------------|------------------------|------------------------|------------------------|------------------------|------------------------|
| | | | | +0.3
mg/mL | +0.6
mg/mL | 2.5%
(v/v) | 5.0%
(v/v) | +5% | +15% | +30% |
| locations | | (84.5,
100) | (71.7,
97.3) | (77.3,
99.2) | (84.5,
100) | (74.2,
99.0) | (82.4,
100) | (67.2,
96.9) | (74.2,
99.0) | (82.4,
100) |
| NPA | | 100%
(95.6,
100) | 100%
(95.6,
100) | 100%
(94.7,
100) | 100%
(95.6,
100) | 100%
(94.9,
100) | 100%
(94.9,
100) | 100%
(94.9,
100) | 100%
(94.9,
100) | 100%
(94.9,
100) |
Expected ALK translocations were not detected in two replicates, two fusion reads per replicate were detected, which is below the calling threshold.
Based on these study results. PGDx elio plasma focus Dx is robust to potential specimenrelated endogenous substances and exogenous contaminants or interferents.
- b) Hybrid Capture Probe Specificity
To verify that the oligonucleotide hybrid capture probes used in PGDx elio plasma focus Dx are specific to the target genomic regions, the sequence homology of the capture probes to the human reference genome, hg19, was assessed. First, probes were considered specific if they uniquely mapped to the intended targets in the human reference genome. Second, probes were considered specific if sequence homology of off-target hits showed low mapping quality. Sequence homology was defined as reference genome alignments of ≥ 95% identity over ≥ 90% of the probe length. Low mapping quality was specified as 90% of sequence reads Mapping Quality Score (MapQ) ≤ 40.
Of the 9,093 target probes evaluated, 8,831 (97.1%) uniquely mapped to intended targets in the human reference genome. The remaining probes were then evaluated to determine if off-target hits would result in sequence reads with high mapping quality. Sequence reads were simulated in 50 noncancerous samples for all potential off-target regions. Zero of the simulated off-target sequence reads mapped to the on-target position, showing the probes to be specific to their intended targets. Based on the results of this study, there is minimal risk of PGDx elio plasma focus Dx producing a false positive patient result due to mis-mapping of hybrid capture probes to off-target sequences in the human genome.
-
- Robustness Studies
- a) DNA Input
The purpose of this study was to evaluate the robustness of PGDx elio plasma focus Dx across DNA input requirements guard banded around the recommended inputs of 25 ng into library preparation and 300 ng into hybrid capture. To assess the impact of varying input concentrations at these steps, five contrived samples (i.e., cell line DNA spiked into plasma) and three clinical plasma samples were run at five replicates per condition. The samples contained representative variants across all variant types reported by the assay (i.e., SNVs, indels, translocations, and amplifications).
The overall success rate in the test conditions were compared to that of the reference condition (25 ng input into library preparation and 300 ng input into hybrid capture). The overall success rate of the assay across test conditions was 100%, except for two test
27
conditions (5 ng and 10 ng input into library prep) (Table 22). For both test conditions, the cell line samples contributed to the lower success rate.
The fold change analysis demonstrated high comparability with the reference condition across all test conditions, with median values ranging from 0.52 to 1.26 at the sample level, and 0.55 and 1.22 at the exon level. The percentage of exons with ≥1050x DEC and bases covered at >300x DEC remained consistent across the reference and test conditions, ranging from 87.15% to 99.87% and 95.57% to 97.11%, respectively (Table 22).
| Condition | Input | Overall
Success
Rate
(95%CI) | Sample-level
DEC Coverage
Fold Change
(median) | Exon-level
DEC Coverage
Fold Change
(median) | Median %
Exons ≥
1050x
DEC | Median %
Bases
Covered ≥
300x DEC |
|----------------------------|----------------------------------------|---------------------------------------|---------------------------------------------------------|-------------------------------------------------------|-------------------------------------|--------------------------------------------|
| Library Prep
(LP) | Reference
(25 ng LP; 300 ng
CAP) | 100%
(91.2, 100) | N/A | N/A | 98.31 | 97.24 |
| Library Prep
(LP) | 5 ng | 47.5%
(32.9, 62.5) | 0.52 | 0.55 | 87.15 | 95.57 |
| Library Prep
(LP) | 10 ng | 85.0%
(70.9, 92.9) | 0.66 | 0.65 | 93.73 | 96.20 |
| | 15 ng | 100%
(91.2, 100) | 0.84 | 0.89 | 97.64 | 96.82 |
| | 20 ng | 100%
(91.2, 100) | 0.94 | 0.96 | 98.16 | 96.81 |
| | 50 ng | 100%
(90.6, 100) | 1.26 | 1.22 | 99.87 | 97.11 |
| Hybrid
Capture
(CAP) | 150 ng | 100%
(90.8, 100) | 0.88 | 0.87 | 97.71 | 96.71 |
| | 225 ng | 100%
(91.2, 100) | 0.92 | 0.93 | 97.78 | 96.84 |
| | 600 ng | 100%
(90.8, 100) | 1.04 | 1.03 | 98.45 | 96.94 |
Table 22. Summary of DNA input guardbanding results - pass rate and QC metrics
Concordance was assessed for observed variants using the positive or negative modal status of each variant in the reference condition as truth across sample replicates. Each test condition was compared to the reference condition and assessed by evaluating the concordance of variants detected by calculating PPA and NPA between varying input levels, stratified by sample type (i.e., clinical plasma v. all sample types). When considering all variant types identified in clinical plasma samples, the PPAs were >96% and NPAs ≥99.9% across input levels (Table 23).
Table 23. Summary of DNA input guardbanding results - concordance
Clinical Plasma | All Samples | ||||
---|---|---|---|---|---|
Condition | Input | PPA | |||
(95%CI) | NPA | ||||
(95%CI) | PPA | ||||
(95%CI) | NPA | ||||
(95%CI) | |||||
5 ng | 100% | ||||
(94.0, 99.9) | 99.9% | ||||
(99.9, 99.9) | 89.8% | ||||
(82.2, 94.4) | 99.9% | ||||
(99.9, 99.9) | |||||
Library Prep | |||||
(LP) | 10 ng | 100% | |||
(92.6, 100) | 99.9% | ||||
(99.9, 99.9) | 87.3% | ||||
(83.7, 90.3) | 99.9% | ||||
(99.9, 99.9) | |||||
15 ng | 100% | ||||
(92.9, 99.9) | 100% | ||||
(99.9, 100) | 97.0% | ||||
(95.0, 98.3) | 99.9% | ||||
(99.9, 99.9) |
28
Clinical Plasma | All Samples | ||||
---|---|---|---|---|---|
Condition | Input | PPA | |||
(95%CI) | NPA | ||||
(95%CI) | PPA | ||||
(95%CI) | NPA | ||||
(95%CI) | |||||
Hybrid Capture | |||||
(CAP) | 20 ng | 100% | |||
(92.9, 99.9) | 99.9% | ||||
(99.9, 99.9) | 95.2% | ||||
(92.7, 96.8) | 99.9% | ||||
(99.9, 99.9) | |||||
50 ng | 100% | ||||
(92.0, 100) | 100% | ||||
(99.9, 100) | 97.2% | ||||
(95.2, 98.4) | 99.9% | ||||
(99.9, 99.9) | |||||
150 ng | 100% | ||||
(92.6, 100) | 99.9% | ||||
(99.9, 99.9) | 95.6% | ||||
(93.2, 97.2) | 99.9% | ||||
(99.9, 99.9) | |||||
225 ng | 100% | ||||
(92.9, 100) | 99.9% | ||||
(99.9, 99.9) | 98.6% | ||||
(97.0, 99.4) | 99.9% | ||||
(99.9, 99.9) | |||||
600 ng | 100% | ||||
(92.6, 100) | 100% | ||||
(99.9, 100) | 98.6% | ||||
(97.0, 99.4) | 99.9% | ||||
(99.9, 99.9) |
Based on these study results, PGDx elio plasma focus Dx performance is maintained around the required 25 ng DNA input.
- b) Assay Guard Banding
The purpose of this study was to evaluate the impact of variations in the assay protocol on the performance of PGDx elio plasma focus Dx. Three contrived samples (i.e., cell line DNA blend spiked into plasma) were run in triplicate, wherein critical steps in the assay protocol were varied, including the end repair/a-tailing enzyme, ligation master mix, post-ligation bead cleanup, library PCR master mix, RNA bait/hybridization master mix, and post-capture PCR master mix. All steps were evaluated at +/- 25% the recommended volume or time except for the end repair/a-tailing enzyme which was instead assessed at +/- 33% the recommended volume, due to low volume pipetting requirements. The contrived samples contained representative variants across all variant types reported by the assay (i.e., SNVs, indels, translocations, and amplifications).
The overall success rate in the test conditions were compared to that of the reference condition. The overall success rate of the assay across test conditions was 100%.
For concordance assessments, reference status was assigned to each variant if all replicates harbored the variant (positive), or no replicates harbored the variant (negative). Each test condition was compared to the reference condition and assessed by evaluating the concordance of variants detected by calculating PPA and NPA.
When considering all variant types, the PPAs were >94% and NPAs >99.9% for all test conditions except for the -25% ligation master mix condition, which demonstrated a PPA of 88.6%. A supplemental study at -15% ligation master mix volume demonstrated a PPA of 93.2%. A precaution is included in the device labeling to warn users that an insufficient volume of ligation master mix can impact device performance.
-
- Sample Stability
- a) Whole Blood Stability
The objective of this study was to evaluate the stability of whole blood specimens used for PGDx elio plasma focus Dx collected in Streck Cell-Free DNA BCTs. Whole blood
29
was drawn from 15 cancer patients, representative of five tumor types with low to high shedding status, into four BCTs. For the reference condition tube, plasma was isolated within 24 hours of blood collection. The remaining three tubes (test condition tubes) filled with whole blood were stored at varying temperatures and time points before plasma was isolated on the 8th day after blood collection (Table 24).
Condition | Description |
---|---|
Reference | Plasma isolated within 24 hours of blood collection. |
Room Temperature | Blood stored 8 days at room temperature (18-25°C). Plasma isolated on |
eighth day. | |
Summer Condition | Blood stored 4h at 22°C, 6h at 37°C, 56h at 22°C, 6h at 37°C, and the |
remaining 5 days at 18-25℃. Plasma isolated on eighth day. | |
Winter Condition | Blood stored 4h at 18°C, 6h at 0°C, 56h at 10°C, 6h at 0°C, remaining 5 |
days at 18-25°C. Plasma isolated on eighth day. |
Table 24. Whole blood storage conditions
The overall success rate in the test conditions were compared to that of the reference condition (plasma isolated within 24 hours of blood collection). The overall success rate of the assay was 93.3% in the Room Temperature and Summer Condition and 86.7% in the Winter Condition. The sample failures were due insufficient DNA input into hybrid capture. A single patient contributed to a failure in each of the test conditions, indicating these failures were more likely caused by the patient having low cfDNA yield rather than the condition being tested. The average plasma volume, cfDNA concentration, and cfDNA yield were calculated for each condition, which showed comparable yield in test conditions relative to the reference (Table 25).
Table 25. Summary of whole blood stability results - pass rate, plasma volume, and cfDNA vield by condition
| Condition | Overall Success
Rate
(95%CI) | Mean Volume of
Plasma Isolated
(mL) | Mean cfDNA
concentration
(ng/uL) | Mean Total
cfDNA Yield
(ng) |
|------------------|------------------------------------|-------------------------------------------|----------------------------------------|-----------------------------------|
| Reference | 100%
(79.6, 100) | 5.1 | 1.1 | 58.8 |
| Room Temp | 93.3%
(70.2, 98.8) | 4.4 | 2.8 | 97.6 |
| Summer Condition | 93.3%
(70.2, 98.8) | 3.8 | 2.5 | 88.2 |
| Winter Condition | 86.7%
(62.1, 96.3) | 3.9 | 2.7 | 93.4 |
In addition to the sample-level DEC coverage fold change, the relative exon-level sequencing coverage was assessed by computing 95% CIs of the sequencing coverage for all exon in the PGDx elio plasma focus Dx panel across the reference condition replicates. The sequencing coverage of each exon across each of the test condition replicates was averaged, and the percentage of exons that were within the 95% CI of the reference condition (within 2 standard deviations) were calculated. The DEC coverage fold change analysis demonstrated high comparability with the reference condition across all test conditions, with median values ranging from 0.85 to 0.90 at the sample level. The percentage of exons within 95% CIs of the reference condition was 98.97% for the Room
30
Temperature condition, 94.53% for the Summer Condition and 88.63% for the Winter Condition (Table 26).
Study Endpoint | Metric | Room Temperature | Summer Condition | Winter Condition |
---|---|---|---|---|
Aggregate Variant | ||||
Concordance | PPA (95%CI) | 100% | ||
(72.2, 100) | 100% | |||
(72.2, 100) | 100% | |||
(72.2, 100) | ||||
Concordance | NPA (95%CI) | 99.9% | ||
(99.9, 99.9) | 99.9% | |||
(99.9, 99.9) | 99.9% | |||
(99.9, 99.9) | ||||
Sample-level DEC | ||||
Coverage Fold | ||||
Change (median) | n/a | 0.88 | 0.90 | 0.85 |
Relative Exon- | ||||
Level Coverage | % of exons within | |||
95% CI of | ||||
reference | ||||
condition | 98.97 | 94.53 | 88.63% |
Table 26. Summary of whole blood stability results - QC metrics and concordance (≥ 1x LoD)
Reference status was assigned to each variant detected in the reference condition at ≥ 1x the LoD. PPA and NPA were calculated for each variant type under each condition. The patient specimens contained representative SNVs and amplifications. When considering all variant types evaluated in the study, the PPAs were 100% and NPA was ≥99% compared to the reference condition (Table 26).
Based on these results, whole blood may be stored in Streck cfDNA BCTs for up to seven days after blood collection prior to plasma isolation and can withstand winter and summer shipping conditions.
- b) Plasma Stability
The objective of this study was to evaluate the stability of plasma isolated from peripheral whole blood. A total of 42 cancer patients, representing >10 turnor types with low to high shedding status, were enrolled into the study. The stability of plasma from each patient was assessed at defined temperature and storage conditions (Table 27).
Plasma Tube Number | Time Point | Condition |
---|---|---|
1 | T0 | Reference, cfDNA extracted on same day as plasma isolation |
2 | 25 hours | Store at 2-8°C for 25 hours before cfDNA extraction |
3 | 46 days | Store at -80°C for 46 days, including 2 freeze/thaw cycles |
before cfDNA extraction | ||
4 | 1 year | Store at -80°C for 1 year including 2 freeze/thaw cycles |
before cfDNA extraction |
Table 27. Plasma storage conditions
The overall success rate in the test conditions were compared to that of the reference condition (cfDNA extracted on the same day as plasma isolation). The overall success rate of the assay across test conditions was >92%, which was comparable to the reference condition (Table 28).
31
The fold change analysis also demonstrated high comparability with the reference condition across all test conditions, with median values ranging from 0.93 to 0.99 at the sample level, and 0.91 and 1.01 at the exon level. The percentage of exons with >1050x DEC and bases covered at ≥300x DEC remained consistent across the reference and test conditions, ranging from 91.22% to 97.05% and 94.23% to 96.63%, respectively (Table 28).
| Condition | Overall
Success Rate
(95%CI) | Sample-
level DEC
Coverage
Fold
Change
(median) | Exon-level
DEC
Coverage
Fold
Change
(median) | Median %
Exons ≥
1050x DEC | Median %
Bases
Covered ≥
300x DEC | PPA
(95%CI) | NPA
(95%CI) |
|-----------|------------------------------------|----------------------------------------------------------------|-------------------------------------------------------------|----------------------------------|--------------------------------------------|-----------------------|-----------------------|
| Reference | 92.9%
(81.0%, 97.5%) | n/a | n/a | 96.16% | 92.26% | n/a | n/a |
| 25 hours | 92.3%
(79.7%,97.3%) | 0.93 | 0.91 | 93.72% | 95.80% | 100%
(80.6, 100) | 99.9%
(99.9, 99.9) |
| 46 days | 100%
(90.8%, 100%) | 0.99 | 0.98 | 91.22% | 94.23% | 100%
(82.4, 100) | 99.9%
(99.9, 99.9) |
| 1 year | 100%
(90.4%, 100%) | 0.99 | 1.01 | 97.05% | 96.63% | 95.0%
(76.4, 99.1) | 99.9%
(99.9, 99.9) |
Table 28. Summary of plasma stability results - pass rate, QC metrics, and concordance (≥ 1x LoD)
Reference status was assigned to each variant detected in the reference condition at ≥ 1x the LoD. PPA and NPA were calculated for each variant type at each time point. The patient specimens contained representative variants across all variant types reported by the assay, except for translocations. When considering all variant types evaluated in the study, the PPA was 100% for the 25-hour timepoint, 100% for the 46 day timepoint, and 95.0% for the 1 year timepoint, with NPAs ≥ 99% compared to the reference condition (Table 28).
Based on these results, plasma may be stored at 2-8℃ for up to 24 hours or at -80℃ for up to 1 year prior to cfDNA extraction.
c) cfDNA Stability
An assessment of seven clinical samples stored at -20℃ from the interlaboratory reproducibility study were used to evaluate cfDNA stability. Variant calls from 84 replicates (seven samples x 12 replicates) at site three of the interlaboratory reproducibility study were compared to the prequalification (seven samples x 12 replicates) results obtained 3.5 months apart. Concordance was assessed for observed variants using the positive or negative modal status of each variant in the prequalification as truth across sample replicates. All variant types had a modal PPA ≥ 93% and modal NPA ≥ 99% (Table 29).
Table 29. Assessment of interlaboratory reproducibility site 3 concordance to prequalification results
Variant Type | Modal PPA | Modal NPA |
---|---|---|
All Sequence Mutations | 93.41% | |
(91.7, 94.8) | 99.99% | |
(99.9, 99.9) |
32
| SNVs | 92.75%
(90.9, 94.3) | 99.99%
(99.9, 99.9) |
|-----------------------------|------------------------|------------------------|
| Insertions | 95.83%
(79.8, 99.3) | 99.99%
(99.9, 99.9) |
| Deletions | 100%
(95.6, 100) | 99.99%
(99.9, 99.9) |
| ERBB2 Amplifications | 100%
(75.8, 100) | 100%
(94.9, 100) |
| MET Amplifications | 100%
(75.8, 100) | 100%
(94.9, 100) |
| CCND1 Amplifications | 100%
(75.8, 100) | 100%
(94.9, 100) |
| CD274 Amplifications | n/a | 100%
(95.6, 100) |
| FGFR2 Amplifications | 100%
(75.8, 100) | 100%
(94.9, 100) |
| ALK Translocations | n/a | 100%
(95.6, 100) |
| NTRK1 Translocations | 100%
(75.8, 100) | 100%
(94.9, 100) |
| RET Translocations | n/a | 100%
(95.6.100) |
An additional assessment was conducted to evaluate PGDx elio plasma focus Dx success rate by cfDNA sample age using clinical plasma samples enrolled in analvtical performance studies. The cfDNA age was defined as the amount of time elapsed from cfDNA extraction to start of library preparation when stored at -20℃. Early cfDNA age bin was defined as 1-100 days. Mid cfDNA age bin was defined as 101-250 days. Late cfDNA age bin was defined as >250 days. The overall pass rates were similar for all early, mid, and late cfDNA age bins, 96.7%, 100%, and 97.3%, respectively.
Concordance of PGDx elio plasma focus Dx to an orthogonal method was also assessed by cfDNA age. Variant detected at ≥ 1x the LoD were considered in the concordance assessment. When considering all variant types, the PPAs for early, mid, and late cfDNA age bins were 93.1% (27/29), 97.2% (105/108), and 97.1% (238/245), respectively.
Together. these results demonstrated that cfDNA stored at -20°C for extended periods of time is stable and does not impact the performance of PGDx elio plasma focus Dx.
-
- Assay Reportable Range:
Not applicable
- Assay Reportable Range:
10. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):
-
a) Traceability
The PGDx elio plasma focus Dx assay is not traceable to any known standard. Controls and quality metrics are described in Section IV.A. -
b) Reagent Stability/Shelf life
33
The objective of this study was to evaluate the stability and establish the shelf-life of the PGDx elio plasma focus Dx assay reagent kit. The study tested two clinical and one contrived sample (i.e., cell line DNA spiked into plasma), which contained representative variants across all variant types reported by the assay. The reagent kits were aged and used to test samples across multiple timepoints (i.e., 0-month (reference). 6-months. 10months, and 19-months). At each timepoint, three unique kit lots and two kits from each lot were evaluated for two transport conditions (i.e., simulated hot and cold days) and underwent four freeze/thaw cycles.
The overall success rate of the assay across test conditions was 100%. For concordance assessments, reference status was assigned to each variant if all replicates harbored the variant (positive), or no replicates harbored the variant (negative). Each test condition was compared to the reference condition and assessed by evaluating the concordance of variants detected by calculating PPA and NPA.
When considering all variant types, the PPAs were ≥98.3% and NPAs ≥99.9% for all test conditions. Based on these study results. PGDx elio plasma focus Dx reagents may be stored for a maximum 18 months and may be thawed and re-refrozen three times when stored at -20℃ and -80℃.
- c) Intermediate Sample/In-Process Stability
The objective of this study was to evaluate the robustness of PGDx elio plasma focus Dx during optional stopping points and storage conditions throughout library preparation and hybrid capture. The study tested three contrived samples (i.e., cell line DNA spiked into plasma), which contained representative variants across all variant types reported by the assay. Three replicates of each test condition (Table 30) were compared to seven replicates of the reference condition (i.e., completing all protocol steps without using any optional stopping points or storage conditions).
Process Step | Standard Condition | Test Condition |
---|---|---|
V1: Post-Ligation Clean-up | Proceed to Library Preparation | |
Amplification immediately | Store Post-Ligation cleanup | |
overnight (16h ± 2h) at | ||
-20°C before proceeding to | ||
Library Preparation | ||
Amplification | ||
V2: Library Preparation PCR | Proceed to Post-PCR Bead Clean | |
up immediately | Store Library overnight (16h ± | |
2h) at 4°C on thermocycler | ||
before proceeding with Post-PCR | ||
Bead Clean up | ||
V3: Genomic Libraries | Store genomic libraries at -20°C | |
ON (16h ± 2h) before proceeding | ||
to Hybridization | Store genomic libraries at -20°C | |
for 4 weeks ± 2 days before | ||
proceeding with the | ||
Hybridization | ||
V4: Hybridization | Immediately proceed to next step | |
following Hybridization PCR | ||
cycling | Following Hybridization PCR | |
cycling HOLD hybridization | ||
reaction and allow incubation at | ||
65°C for 1h | ||
Proceed to Post-PCR purification | ||
bead Clean-Up immediately | Store Capture PCR overnight | |
(16h ± 2h) at 4°C on |
Table 30. Intermediate sample/in-process storage conditions
34
Process Step | Standard Condition | Test Condition |
---|---|---|
V5: Capture PCR | thermocycler prior to proceeding | |
with Post-PCR purification bead | ||
Clean-Up | ||
V6: Sequencing Pools | Proceed to Library Normalization | |
and Pooling immediately | Store Sequencing Pools 4 weeks | |
±2 days at -20°C prior to | ||
proceeding with Library | ||
Normalization and Pooling | ||
V7: All 6 conditions tested at | ||
once | Proceed with all standard | |
conditions | Proceed with all test conditions | |
(±2 days) |
The overall success rate of the assay across test conditions was 100%. For concordance assessments, reference status was assigned to each variant if all replicates harbored the variant (positive), or no replicates harbored the variant (negative). Each test condition was compared to the reference condition and assessed by evaluating the concordance of variants detected by calculating PPA and NPA.
When considering all variant types, the PPAs were ≥95% and NPAs ≥99.9% for all test conditions. Based on these study results. PGDx elio plasma focus Dx is robust to suggested library preparation and hybrid capture process stopping points and storage conditions.
-
d) Expected values (controls, calibrators, or methods)
Controls are described in Section IV.A. -
- Detection Limit:
Not applicable
- Detection Limit:
-
- Assay Cut-Off:
Not applicable
- Assay Cut-Off:
13. Sample Carryover and Cross-Contamination
The objective of this study was to assess sample carryover for PGDx elio plasma focus Dx within and between sample runs. A total of 15 cases, 14 mutation-positive samples harboring high-positive variants (including germline) and one mutation-negative reference cell line, were used for this study. Four sequencing batches were processed through PGDx elio plasma focus Dx in a checkerboard configuration, alternating between mutation-positive and mutation-negative samples.
One operator processed batches 1 and 2 to assess contamination within sequencing runs. A second operator processed batches 3 and 4 by shifting the corresponding adapter assigned in the previous run from the positive to the negative samples to assess contamination between. sequencing runs. Two sequencing runs were performed sequentially on one sequencer, and the remaining two runs on a second sequencer. The presence of contaminating positive variants was assessed in each mutation-negative sample.
35
There was no evidence of contamination due to sample carryover in 100% of mutationnegative samples within and between sequencing runs. In addition, positive clinical samples demonstrated no evidence of contamination compared to expected positive variants.
14. Lot Interchangeability
The performance of PGDx elio plasma focus Dx after exchanging reagents between unique lots was evaluated using three contrived samples (i.e., cell line DNA spiked into plasma). Each sample was tested in duplicate using two different lots each of library preparation and hybrid capture reagents. The study was designed to mimic the most probable scenarios of reagent exchange.
There are two main components of the PGDx elio plasma focus Dx assay kit: (i) reagents for library preparation and (ii) reagents for hybrid capture. The library preparation and hybrid capture steps are performed in separate laboratory areas (pre-amplification and postamplification areas) to avoid potential contamination. It is possible that an operator could use reagents for library preparation from one assay kit lot, and reagents for hybrid capture from another kit lot.
Capture bait is the key component in the hybrid capture step. The RNA capture bait is directly related to the assay quality and requires storage at -80°C. Therefore, the capture baits are removed from the other reagents in storage which could result in a possible exchange between unique lots during assay execution.
Reagent lot interchangeability was assessed by evaluating concordance between reference conditions in which no reagents were exchanged to library preparation and hybrid capture reagent lot exchange configurations, inclusive of all SNVs, indels, amplifications, and translocations identified in the reference condition. PGDx elio plasma focus Dx demonstrated a PPA > 91% and NPA ≥ 99.9% across all aggregated variant types after exchanging reagents between unique lots. In addition, no impact to assay performance was observed based on library vields post-library preparation and post-hybrid capture when samples underwent reagent lot exchange were processed through the assay.
15. Lane Combination
The objective of this study was to evaluate the impact of using PGDx elio diversiPhi to fill partial sequencing batches on PGDx elio plasma focus Dx performance. A full batch is classified as eight samples (seven samples and one external control). This study used seven variant-positive clinical plasma samples from full sequencing batches as the reference. The same samples were then processed using PGDx elio diversiPhi as filler and sequenced at six ratios of sample to PGDx elio diversiPhi (i.e., 7:0 [reference], 1:6, 2:5, 3:4, 4:3, 5:2, 6:1).
Reference status was assigned to SNVs, indels, amplifications, and translocations > 1x LoD within the reference samples run without diversiPhi. Results obtained from the samples sequenced with different lane ratios of diversiPhi were compared to the reference status for concordance, using PPA and NPA calculations. PGDx elio plasma focus Dx demonstrated 100% PPA and ≥ 99.99% NPA for all variants at all lane ratio test conditions, indicating that samples yield reproducible variant calls when processed in partial batches.
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B Comparison Studies:
-
- Method Comparison
Not applicable
- Method Comparison
-
- Matrix Comparison:
Not applicable
- Matrix Comparison:
C Clinical Studies:
Other Clinical Supportive Data
Not applicable
D Clinical Cut-Off:
Not applicable
Expected Values/Reference Range: E
Not applicable
Other Supportive Performance Characteristics Data: F
-
- Blood Collection Tube Characterization and Concordance Studies:
- a) Incomplete Mixing and Underfilled Tube
The objective of this study was to evaluate the impact of 1) variations in mixing after blood collection, and 2) potential interference caused by underfilling the Streck cfDNA BCTs. Whole blood was collected from 15 cancer patients, representative of >> tumor types with low to high shedding status, into five tubes. For the reference condition tube, 10 mL of whole blood was collected and inverted 10 times per the Streck cfDNA BCT Instructions for Use. The remaining four tubes (test condition tubes) were filled with varying amounts of whole blood and inverted a specified number of times before plasma was extracted within eight days of blood collection (Table 31).
Table 31. BCT incomplete mixing and underfilled/short draw sample processing conditions
Number of Subjects | Reference Condition | Test Variable | Test Condition |
---|---|---|---|
15 | 10 mL blood draw with | ||
10 inversions | Incomplete Mixing | 10 mL blood draw with | |
5 inversions | |||
10 mL blood draw with | |||
15 inversions | |||
Underfilled tube | 5 mL blood draw with | ||
10 inversions | |||
8 mL blood draw with | |||
10 inversions |
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The overall success rate in the test conditions were compared to that of the reference condition (10 mL blood draw with 10 inversions). The overall success rate of the assay across test conditions was >93%, except for the 5mL underfilled tube test condition which had an overall success rate of 73.3% (Table 32). This lower success rate was largely due to percentage of bases ≥ 300X DEC being below the QC threshold. The average plasma volume, cfDNA concentration, and cfDNA yield were calculated for each condition, which showed lower yields in the 5mL underfilled tube test condition relative to the reference. A limitation is included in the device labeling to address the risk of reduced assay performance as a result of underfilling the tube (94% across all test conditions (Table 33).
Table 33. Summary of BCT incomplete mixing and underfilled tube study results - |
---|
concordance and QC metrics |
| Study
Endpoint | Metric | 5x
Inversions | 15x
Inversions | 5 mL
Underfilled | 8 mL
Underfilled |
|---------------------------------------------------------|----------------------------------------------------------|------------------------|------------------------|------------------------|------------------------|
| Aggregate
Variant
Concordance | PPA (95%CI) | 100%
(72.2, 100) | 100%
(72.2, 100) | 100%
(70.1, 100) | 100%
(72.2, 100) |
| | NPA (95%CI) | 99.99%
(99.9, 99.9) | 99.99%
(99.9, 99.9) | 99.99%
(99.9, 99.9) | 99.99%
(99.9, 99.9) |
| Sample-level
DEC Coverage
Fold Change
(median) | n/a | 1.01 | 1.04 | 1.01 | 0.99 |
| Relative Exon-
Level Coverage | % of exons
within 95% CI
of reference
condition | 100 | 100 | 94.53 | 96.90 |
Reference status was assigned to each variant detected in the reference condition at > 1x the LoD. PPA and NPA were calculated for all variants across each test condition. The patient specimens contained representative variants across all variant types reported by
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the assay, except for translocations. When considering all variant types evaluated in the study (SNVs, indels, and amplifications), the PPAs were 100% and NPA was >99% (Table 33).
Based on these results, whole blood samples collected in Streck cfDNA BCTs are suitable for use with PGDx elio plasma focus Dx when used as labeled.
- b) BCT Lot-to-Lot Reproducibility
The objective of this study was to evaluate PGDx elio plasma focus Dx performance within and between Streck cfDNA BCT lots. Whole blood was collected from 60 cancer patients into four BCTs per patients with two BCTs per lot processed by two different operators (each operator processed one tube per BCT lot). The BCTs from 59 of 60 specimens were processed into plasma within seven days after whole blood collection, while the remaining one was processed into plasma within eight days. Plasma was stored at -80°C until testing with PGDx elio plasma focus Dx.
The overall success rate of the assay for each BCT lot was 99.2%. The cfDNA yield was similar between and within BCT lots (data not shown). The APA and ANA between and within BCT lots, for variants identified at ≥ 1x the LoD, is summarized in Table 34. The patient specimens contained representative variants across all variant types reported by the assay, except for translocations. When considering all variant types evaluated in the study (SNVs, indels, and amplifications), the APA between and within lots was >93% and ANA was ≥99%.
| Study
Endpoint | Metric | Between Lot
1 & 2 | Within Lot 1 | Within Lot 2 |
|-------------------------------------|------------|-----------------------|-----------------------|-----------------------|
| Aggregate
Variant
Concordance | APA (CI95) | 93.3%
(90.4, 95.8) | 94.0%
(87.9, 98.7) | 95.8%
(91.1, 99.1) |
| | ANA (CI95) | 99.99%
(99.9, 100) | 99.99%
(99.9, 100) | 99.99%
(99.9, 100) |
Table 34. Summary of BCT lot-to-lot precision (≥ 1x LoD)
- c) Concordance
The purpose of this study was to establish concordance between the Streck cfDNA BCTs and the BD Vacutainer K2 EDTA (EDTA) BCTs, in which the latter were used for sample collection for a subset of samples in the accuracy study. Whole blood was collected from 60 advanced cancer patients, representative of 16 tumor types and low to high shedding status, in EDTA and Streck cfDNA BCTs. The overall success rates were ≥95% for both tube types. Concordance was assessed across all variant types detected by the assay, with reference status assigned to each variant detected at ≥ 1x the LoD in samples collected in Streck cfDNA BCTs. The PPA and NPA for aggregated variant types was 95.8% and 99.9%, respectively.
VII Proposed Labeling:
The labeling supports the decision to grant the De Novo request for this device.
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VIII Identified Risks and Mitigations:
Risks to Health | Mitigation Measures |
---|---|
Risk of false positive, false negative, or failure | |
to provide a result. | Certain design verification and validation |
activities, including certain analytical studies | |
Certain labeling information, including | |
certain performance information and | |
limitations | |
Incorrect interpretation of test results by the | |
user | Certain design verification and validation |
activities, including certain analytical studies | |
Certain labeling information, including | |
certain performance information and | |
limitations |
IX Benefit/Risk Assessment:
A Summary of the Assessment of Benefit:
The PGDx elio plasma focus Dx provides comprehensive genomic profiling (single nucleotide variants, small insertions and deletions, and translocations and amplifications in a subset of genes) utilizing circulating cell-free DNA (cfDNA) from plasma of peripheral whole blood, in previously diagnosed cancer patients and is for use by qualified health professionals in accordance with professional guidelines, which provides significant probable benefit to the patient. There is probable clinical benefit of the device based on the analytical performance of the device in identifying genomic alterations and for the clinical use of this device in accordance with professional guidelines.
Beyond the now fully recognized benefits of these devices, the PGDx elio plasma focus Dx further facilitates characterization of solid tumors by providing a minimally invasive approach to analysis that complements conventional tissue-based diagnostic methods, due to its use of peripheral blood. The PGDx elio plasma focus Dx provides molecular profiling when tumor tissue is not available, avoids risk of biopsy while allowing rapid results, and enables profiling of intra-tumor heterogeneity and mutations from metastatic sites, which provides clinically meaningful probable benefit.
B Summary of the Assessment of Risk:
The probable risks associated with the use of this device are mainly due to erroneous results and incorrect use and interpretation of test results by the healthcare providers, both of which may adversely impact clinical consultation for patients.
False positive results could result in improper medical management of patients, such as erroneous enrollment in a clinical trial or incorrect management of patients, which represent a significant clinical risk. A false negative result may prevent a patient from accessing medical
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care in accordance with professional guidelines or will otherwise impede identification of appropriate therapy unless additional tissue testing is performed following negative plasma results.
There is a degree of probable risk of mismanagement of patient care (as described above), in accordance with professional guidelines, based on false test results from this test, or incorrect interpretation of test results.
C Patient Perspectives:
This submission did not include specific information on patient perspectives for this device.
D Summary of the Assessment of Benefit-Risk:
The probable benefit of this test was demonstrated by a series of analytical studies (refer to Section VI Performance Characteristics for details), including an analytical accuracy study utilizing more than 900 clinical specimens and additional contrived samples covering a variety of clinically relevant variants across multiple tumor types and variant types (i.e., SNVs, indels, translocations, and amplifications).
The concordance between the PGDx elio plasma focus Dx and the orthogonal methods was high for variants above the LoD of the orthogonal methods, and was more modest for all observations, including samples below the LoD of the orthogonal methods. The following data represents performance in clinical plasma specimens. For SNVs/indels with evidence of clinical significance in plasma, the PPAs ranged from 63.8% to 100% overall, and 97.6% to 100% PPA when considering only those at or above the LoD of the orthogonal methods. For SNV/indels with evidence of clinical significance in tissue, the PPA was 60.5% overall, and 95.7% for those at or above the LoD of the orthogonal methods. For SNVs and indels with evidence of potential clinical significance, the PPA was 53.4% overall and 94.3% at or above the LoD of the orthogonal methods. For all groups discussed thus far, the NPA was between 99.6% and 99.9%. For amplifications, the PPA ranged from 62.5% to 100.0% and was not notably different in the subgroup above the LoD of the orthogonal methods; the NPA, however, was uniformly above 99% for all groups. For ALK, NTRKI and RET translocations, the PPAs ranged from 75% to 92.3% PPA when the data from clinical plasma and contrived samples were combined.
False test results from this test, or incorrect interpretation of test result in improper medical management of patients. However, the results of the device are not prescriptive or conclusive for use of any specific therapeutic product, which partially mitigates this risk. In addition, the NPA for this device was uniformly high, ranging from 99.3% to 100%. However, the PPV is impacted by the prevalence of specific biomarkers, and some degree of false positivity may still be observed with a positive PGDx elio plasma focus Dx result, thus the labeling specifies that confirmation of turnor mutation status using an FDA-approved CDx test is needed for therapeutic use. Finally, proposed reporting will separately tabulate Variants with Evidence of Clinical Significance in Plasma, Variants with Evidence of Clinical Significance in Tissue, and Variants with Potential Clinical Significance.
The risk of false negative results is partially mitigated by inclusion of a statement noting that a negative result from a plasma specimen does not assure that the patient's tumor is negative for
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genomic findings and a limitation noting such results should be followed by additional confirmatory testing. The risk of erroneous results is otherwise partially mitigated by the analytical performance of a device. The analytical accuracy study, however, revealed lower PPA at low analyte levels, below the LoD of the orthogonal methods; nevertheless, the device performed robustly in terms of the PPA above the LoD of the orthogonal methods, and demonstrated robust NPA for all variants. Thus, the probable risk of this device is partially mitigated by the supportive analytical performance for the device, when also considering the clinical limitations and the established special controls, beyond the general controls.
While general controls are insufficient to ensure the safety and effectiveness of the device, in light of the mitigations provided by the special controls, the probable benefits outweigh the probable risks for the use of the PGDx elio plasma focus Dx device.
X Conclusion:
The De Novo request is granted and the device is classified under the following and subject to the special controls identified in the letter granting the De Novo request:
Product Code(s): SBY Device Type: High throughput sequencing based tumor profiling test of circulating cell-free nucleic acids Class: II Regulation: 21 CFR 866.6085