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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.

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.

Here's a breakdown of the acceptance criteria and the study that proves the device meets them, based on the provided text:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are generally framed around Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) compared to orthogonal methods, as well as satisfactory performance in areas like Limit of Detection (LoD), Limit of Blank (LoB), and reproducibility.

• SNVs/indels with Clinical Significance in Plasma: 97.6% - 100% PPA, 99.9% NPA
• SNVs/indels with Clinical Significance in Tissue: 95.7% PPA, 99.99% NPA
• SNVs/indels with Potential Clinical Significance: 94.3% PPA, 99.99% NPA
  All Variants (Primary Analysis):
• SNVs/indels with Clinical Significance in Plasma: 63.8% - 100% PPA, 99.6% - 100% NPA
• SNVs/indels with Clinical Significance in Tissue: 60.5% PPA, 99.99% NPA
• SNVs/indels with Potential Clinical Significance: 53.4% PPA, 99.99% NPA | Device meets and often exceeds these criteria for variants at or above LoD. Lower PPA for "all variants" is acknowledged and attributed to variants below LoD. |
  | Accuracy (PCCPTerms) | Accuracy point estimates of ≥ 80% PPA and ≥ 95% NPA for copy number amplifications and translocations when considering variants at or above LoD of the PGDx elio plasma focus Dx only. Accuracy point estimates of > 60% PPA and > 95% NPA for all copy number amplifications and translocations irrespective of variant level. | Variants ≥ LoD of Orthogonal Method:
• Amplifications (CCND1, CD274, ERBB2, FGFR2, MET): 65.6% - 100% PPA, 99.4% - 99.8% NPA
• Translocations (ALK, NTRK1, RET): 75% - 92.3% PPA, 99.63% - 100% NPA
  All Variants (Primary Analysis):
• Amplifications: 57.9% - 100% PPA, 99.3% - 99.8% NPA
• Translocations: 50% - 92.9% PPA, 99.63% - 100% NPA | Device meets and often exceeds these criteria for variants at or above LoD of the orthogonal method. |
  | False Positive Rate (FPR) | 95% PPA for mutation positive samples with alteration at 1-1.5x LoD and > 95% NPA for mutation negative samples for precision evaluation (PCCP terms) | Interlaboratory Reproducibility (at 1-1.5x LoD):
• Clinical Blends (Plasma): 97.2% PPA
• Cell Line Blends: 90.3% PPA
  End-to-end Precision (at >1x LoD):
• SNVs: 97.4% APA
• Indels: 100.0% APA
• Aggregate variants: 97.8% APA
• All structural variants: 100% APA
  Lot-to-Lot Precision:
• SNVs: 90.5% PPA
• Deletions: 86.6% PPA
• Amplifications & Translocations: 100% PPA | PPA for 1-1.5x LoD is generally >95% for clinical samples but slightly lower for cell-line blends in interlaboratory. End-to-end and lot-to-lot also demonstrate high agreement. |
  | Interfering Substances | Assay results robust in presence of interfering substances. | Across 11 potential interferents (16 conditions), concordance of variant calls was ≥92.6% PPA and ≥99.9% NPA. Overall success rate ≥ 85.7%, with 2 failures attributed to operator error. | Performance demonstrated as robust. |
  | Hybrid Capture Probe Specificity | Probes uniquely map to intended targets, with off-target hits showing low mapping quality. Minimal risk of false positives from mis-mapping. | 97.1% of probes uniquely mapped. Zero of simulated off-target reads mapped to on-target positions. | Demonstrated high specificity. |
  | DNA Input Robustness | Performance maintained across reasonable DNA input variations. | PPAs >96% and NPAs ≥99.9% across input levels for clinical plasma samples (5ng - 50ng LP, 150ng - 600ng CAP). Overall success rate >47.5%, with failures at very low input (5ng LP) impacting cell lines. | Robust performance around the recommended 25ng input. Acknowledged reduced performance at very low inputs. |
  | Assay Guard Banding | Robustness to variations in assay protocol (volume/time for reagents). | PPAs >94% and NPAs >99.9% for all test conditions, except -25% ligation master mix (PPA 88.6%). Supplemental study improved -15% ligation to 93.2% PPA. | Generally robust, with one identified sensitivity point (Ligation Master Mix volume) leading to a precaution in labeling. |
  | Whole Blood Stability | Performance maintained after storage in BCTs under varying conditions. | PPAs 100% and NPAs ≥99.9% across storage conditions (Room Temp, Summer, Winter for 8 days). Overall success rates >86.7%, with failures attributed to low cfDNA yield from patient. | Blood stable for 7 days in Streck cfDNA BCTs. |
  | Plasma Stability | Performance maintained after storage of isolated plasma. | PPAs 95.0% - 100% and NPAs ≥99.9% across storage conditions (25 hours at 2-8°C, 46 days at -80°C, 1 year at -80°C). Overall success rates >92%. | Plasma stable for up to 1 year at -80°C. |
  | cfDNA Stability | Performance maintained after storage of isolated cfDNA. | Modal PPA ≥ 93% and modal NPA ≥ 99% for 3.5 months at -20°C. PPAs 93.1% - 97.1% for up to >250 days. | cfDNA stable for extended periods at -20°C. |
  | Sample Carryover and Cross-Contamination | No evidence of carryover or cross-contamination. | No evidence of contamination in 100% of mutation-negative samples within and between sequencing runs. | Demonstrated to be contamination-free. |
  | Lot Interchangeability | Performance maintained after exchanging reagents between unique lots. | PPA > 91% and NPA ≥ 99.9% across all aggregated variant types. | Reagents from different lots are interchangeable. |
  | Lane Combination | Performance maintained when using filling material for partial batches. | 100% PPA and ≥ 99.99% NPA for all variants at all lane ratio test conditions. | Device performs consistently even with partial batch filling. |
  | BCT Incomplete Mixing & Underfilled Tube | Performance maintained with variations in mixing and filling of BCTs. | PPAs 100% and NPAs >99% for all conditions except 5mL underfilled tube (73.3% success rate). | Performance maintained for proper usage, but a limitation for underfilled tubes is noted. |
  | BCT Lot-to-Lot Reproducibility | Consistent performance across different BCT lots. | APA between and within lots >93% and ANA ≥99%. | Consistent performance across BCT lots. |
  | BCT Concordance | Concordance between Streck cfDNA BCTs and BD Vacutainer K2 EDTA BCTs. | PPA 95.8% and NPA 99.9% for aggregated variant types. | High concordance between BCT types. |

2. Sample Size Used for the Test Set and Data Provenance

• Test Set (Accuracy Study):
  • Clinical Plasma Specimens: 931 samples across 35 solid tumor types (e.g., NSCLC, Colorectal, Breast, etc.).
  • Contrived Samples: 48 samples (34 spiked cell lines, 13 spiked cell line blends, 1 clinical plasma blend).
  • Total Samples Assessed: 979 (785 passed QC for both PGDx elio plasma focus Dx and at least one orthogonal method).
  • Data Provenance: The text does not explicitly state the country of origin for the clinical samples. It mentions "clinical plasma specimens," implying human-derived samples. It appears to be retrospective as samples were compared to "orthogonal methods" which are likely established assays already used for patient variant detection. The "Orthogonal Method" results served as the reference standard.
  • LoB Study (Test Set):
    • Cohort 1: 29 normal plasma specimens (tested in duplicate)
    • Cohort 2: 38 normal plasma specimens (tested singularly)
    • Total unique donors: 67
    • Data Provenance: Healthy donors (no cancer diagnosis). Retrospective.
  • LoD Study (Test Set):
    • 11 cell line blends (dilution series, 10 replicates at 5 levels)
    • Clinical plasma blends (20 unique pools, 10 replicates, 2 reagent lots)
    • Data Provenance: Contrived and clinical plasma samples.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications of Those Experts

The ground truth for the test set (accuracy study) was established by orthogonal methods, not directly by human experts reviewing the PGDx elio plasma focus Dx results. The orthogonal methods themselves would have undergone their own validation processes, which may or may not have involved expert review for their initial validation. The text does not specify the number of experts or their qualifications who established the ground truth for these orthogonal methods.

4. Adjudication Method for the Test Set

The primary method for establishing ground truth was comparison to orthogonal methods.

• Concordance Assessment: Reference status was assigned to each variant based on the orthogonal method.
• The comparisons were made between the PGDx elio plasma focus Dx output and the orthogonal methods. There is no mention of an "adjudication panel" or specific adjudication method (e.g., 2+1, 3+1) being used to resolve discrepancies between the PGDx elio plasma focus Dx and the orthogonal methods, or between multiple orthogonal methods (though one instance mentions a second orthogonal method was used when a discrepancy occurred for NTRKI translocation).

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was done

No, a multi-reader multi-case (MRMC) comparative effectiveness study was not done. The performance evaluation focuses on the analytical performance of the device itself (standalone) against orthogonal methods, not on how human readers' performance might improve with or without AI assistance from this specific device.

6. If a Standalone Study (i.e., algorithm only without human-in-the-loop performance) was done

Yes, a standalone study was done. The entire "Analytical Performance" section (VI.A) describes the performance of the PGDx elio plasma focus Dx assay, which is a "next generation sequencing based tumor profiling assay" with automated data analysis software. The evaluation metrics (PPA, NPA, LoD, LoB, reproducibility) are all measures of the assay's ability to detect genetic variants autonomously. There is no mention of human-in-the-loop performance in any of the analytical or clinical validation sections.

7. The Type of Ground Truth Used

The primary type of ground truth used was:

• Orthogonal Methods: For the accuracy study, variants detected by established and validated independent assays served as the reference.
• Known Mutations in Cell Lines/Blends: For LoD and reproducibility studies, where contrived samples (cell line DNA spiked into plasma) were used, the "expected variants" in these materials were the ground truth.
• Absence of Mutations in Healthy Donors: For LoB studies, a "mutation negative" status based on pre-screening with an externally validated orthogonal method or sequencing of matched buffy coat (to exclude CHIP and germline variants) was used as ground truth.
• Modal Status: For precision/reproducibility studies across replicates, the majority call (modal status) served as a form of internal reference to assess consistency.

8. The Sample Size for the Training Set

The document does not explicitly describe a separate "training set" for the PGDx elio plasma focus Dx algorithm. Phrases like "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" imply an internal development and optimization phase. However, a distinct, quantified "training set" with specific sample sizes for algorithm development (in the machine learning sense) is not provided. The study design focuses on the analytical validation (test set) of the final locked assay.

9. How the Ground Truth for the Training Set was Established

As noted above, a formal "training set" is not explicitly defined. However, the initial establishment and optimization of metrics involved:

• Well-characterized cell lines with known variants: This implies that for initial parameter tuning and threshold setting, variants in these cell lines were used as ground truth, likely established through extensive prior characterization (e.g., Sanger sequencing, other NGS methods, etc.).
• Clinical samples for initial performance measurement: These samples would have had ground truth established by orthogonal methods, similar to the main accuracy study.

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