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The Invitae Common Hereditary Cancers Panel is a qualitative high-throughput sequencing based in vitro diagnostic test system intended for analysis of germline human genomic DNA extracted from whole blood for detection of substitutions, small insertion and deletion alterations and copy number variants (CNV) in a panel of targeted genes.

This test system is intended to provide information for use by qualified health care professionals, in accordance with professional guidelines, for hereditary cancer predisposition assessment and to aid in identifying hereditary genetic variants potentially associated with a diagnosed cancer.

The test is not intended for cancer screening or prenatal testing. Results are intended to be interpreted within the context of additional laboratory results, family history, and clinical findings.

The test is a single-site assay performed at Invitae Corporation.

The Invitae Common Hereditary Cancers Panel uses hybridization-based capture, nextgeneration sequencing (NGS), and a custom-built bioinformatics pipeline to compare all positions in targeted regions of 47 genes to a reference sequence and identify variants, including single nucleotide variants (SNVs), insertions and deletions (Indels), and copy number variants (CNVs). Sequence analysis covers clinically important regions of each gene, including coding exons and 10 to 20 base pairs of adjacent intronic sequence on either side of the coding exons in the transcript listed in Table 1. Genes of "high clinical significance" are defined as those for which the test result(s) may lead to prophylactic screening, confirmatory procedures, or treatment that may incur morbidity or mortality to the patient and are shown in bold text. In addition, the analysis covers the select non-coding variants specifically defined in the table. Any variants that fall outside these regions are not analyzed. Identified variants are assessed by clinical professionals using currently available literature and data from public genetic variant databases. Variants are assigned a score, calculated according to an algorithm that weights the available clinical evidence. Possible outcomes include the following, which are based on joint ACMG/AMP Committee guidelines: Benign (not reported), Likely benign (not reported), Likely pathogenic, Pathogenic, Variant of Uncertain Significance. Variants are reported using HGVS nomenclature and the human reference genome GRCh37.

The Invitae Common Hereditary Cancers Panel is a high-throughput sequencing-based in vitro diagnostic test system designed for detecting germline substitutions, small insertions and deletions, and copy number variants (CNVs) in 47 targeted genes. It is intended for use by qualified healthcare professionals for hereditary cancer predisposition assessment and to aid in identifying hereditary genetic variants potentially associated with diagnosed cancer.

Here's a breakdown of the acceptance criteria and the study proving the device meets them:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria for the Invitae Common Hereditary Cancers Panel are primarily based on the analytical performance metrics of Positive Percent Agreement (PPA), Negative Percent Agreement (NPA), and Technical Positive Predictive Value (TPPV), and Technical Negative Predictive Value (TNPV).

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

• Precision/Reproducibility Test Sets:

  • Set 1: 25 clinical samples.
  • Set 2: 18 samples enriched for Indels and CNVs.
  • Each sample was tested with 14 replicates.
  • Data Provenance: Clinical samples (exact country of origin not specified, but the applicant is Invitae Corporation, a US-based company, suggesting data is likely from the US or a similar regulatory environment). The studies were retrospective for established clinical samples.

• DNA Input Study Test Set:

  • 8 whole blood clinical specimens.
  • Each specimen tested at 5 input levels (1, 5, 10, 23, and 46 ng/uL), each in triplicate, for a total of 120 samples.
  • Data Provenance: Clinical specimens.

• Analytical Specificity/Interference Study Test Set:

  • Study 1: 7 interfering substances, each spiked into 5 specimens, tested in 3 replicates. (Plus 10 donor blood samples, 2 replicates each).
  • Study 2: 7 interfering substances, each spiked into 5-6 samples, tested in 2 replicates.
  • Data Provenance: Specimens sourced from a blood bank.

• Accuracy (Orthogonal Comparison) Test Sets:

  • Non-Clinical Samples:
    • 5 Genome in a Bottle (GIAB) samples.
    • 92 supplemental cell line samples.
  • Clinical Specimens:
    • SNVs and Indels: 6014 clinical samples.
    • CNVs: 3542 clinical samples.
    • Additional 106 clinical specimens with prior negative CNV results for TNPV evaluation.
  • Data Provenance: GIAB samples are publicly available reference materials. Cell line samples are commercial. Clinical specimens "tested at Invitae" or "from patients diagnosed with cancer and individuals tested for predisposition assessment," indicating retrospective clinical data.

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

The document does not explicitly state the number of experts used to establish the ground truth for the test sets in terms of individual review. Instead:

• For the Precision/Reproducibility and DNA Input studies, "variants" are stated to have been identified in the samples. The ground truth for these samples (clinical and reference materials) is based on their characterized genetic profiles.
• For the Accuracy (Orthogonal Comparison) study with non-clinical samples, ground truth was established by "well characterized genome sequence data" for GIAB samples and previous identification for the "at least one variant" in supplemental cell lines. These reference standards typically have their ground truth established by consensus of multiple methods and experts, but the exact number of experts involved in the initial characterization of these reference materials is not detailed in this document.
• For the Accuracy (Orthogonal Comparison) study with clinical specimens, the ground truth was established by "a validated high-throughput sequencing platform" or "a validated multiplexed PCR based test or a validated microarray." This implies that the ground truth in these cases relied on the established accuracy and validation of these orthogonal methods.
• For Interpretation Agreement, Invitae's internal classifications were compared to "independently generated prior clinical laboratory testing results" and "ClinVar classifications." ClinVar classifications are curated by various groups, including "Expert Panel submissions" (which imply expert consensus).

The document states that identified variants "are assessed by clinical professionals using currently available literature and data from public genetic variant databases" and that "Variant interpretation and curation is performed according to controlled SOPs by trained individuals who have passed a competency assessment." This implies that highly qualified professionals are involved in the overall process of variant interpretation, which indirectly contributes to the establishment and verification of ground truth in their internal processes. These professionals are described as "PhD level scientists, genetic counselors, as well as licensed, board-certified clinical molecular geneticists or licensed, board-certified molecular pathologists." No specific number of experts used for each ground truth assessment within the test sets is provided.

4. Adjudication Method for the Test Set

The document does not describe an explicit "adjudication method" (like 2+1 or 3+1 for human readers) for establishing the ground truth for the test sets. Instead, it relies on:

• Reference Standards: For non-clinical samples (GIAB), the ground truth is pre-established "well-characterized genome sequence data," implying a high level of confidence through consensus or advanced methods.
• Orthogonal Validated Methods: For clinical samples, the comparison study uses "validated" orthogonal methods as the de-facto ground truth. This means the ground truth relies on the established accuracy of those methods, which would have undergone their own validation.
• Internal Review/SOPs: The internal process for variant interpretation and curation involves review by "PhD level scientists, genetic counselors, as well as licensed, board-certified clinical molecular geneticists or licensed, board-certified molecular pathologists." This implies an implicit adjudication or consensus within their established clinical laboratory practices, but not a formalized adjudication by independent experts for each test set sample specifically for this study.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not done. This study is for a genetic diagnostic panel, not for an AI-assisted diagnostic imaging system that typically involves human readers interpreting images. The evaluation focuses on the analytical performance of the automated sequencing pipeline.

6. Standalone (Algorithm Only Without Human-in-the-Loop Performance) Study

Yes, the studies described are essentially standalone evaluations of the assay's analytical performance, which includes the bioinformatics pipeline (algorithm) that calls variants. The "Invitae Common Hereditary Cancers Panel uses hybridization-based capture, next-generation sequencing (NGS), and a custom-built bioinformatics pipeline to compare all positions in targeted regions... and identify variants." The precision, DNA input, analytical specificity, and accuracy studies directly assess the output of this automated system, comparing it against known ground truths or orthogonal methods. While "Variant Interpretation and Review" does involve human professionals, the core performance metrics (PPA, NPA, TPPV, TNPV) are evaluating the technical ability of the system (including the bioinformatics pipeline) to accurately detect and call variants.

7. Type of Ground Truth Used

The ground truth used in the studies includes:

• Reference Genome Sequence Data: "Genome in a Bottle (GIAB) samples with well characterized genome sequence data."
• Cell Line Samples: Supplemental cell line samples with "at least one variant that has been identified and reported."
• Orthogonal Validated Methods: Comparison to results from "a validated high-throughput sequencing platform," "a validated multiplexed PCR based test," or "a validated microarray."
• Prior Clinical Laboratory Testing Results: For variant interpretation agreement, comparison was made to "independently generated prior clinical laboratory testing results" and "ClinVar classifications" (which are curated by expert panels).

8. Sample Size for the Training Set

The document does not provide a specific sample size for a "training set." The listed studies are for the validation of the device. The development of the "custom-built bioinformatics pipeline" and the "algorithm that weights the available clinical evidence" would have implicitly involved training data, but that data and its size are not detailed in this document, which focuses on validation data.

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

Since no specific "training set" is described with sample sizes, the method for establishing its ground truth is also not explicitly stated. However, the general approaches described for variant assessment and interpretation ("assessed by clinical professionals using currently available literature and data from public genetic variant databases," "curated by qualified Invitae staff" according to SOPs, consultation of "external databases") would logically form the basis for establishing ground truth for any data used in the development or "training" of the bioinformatics pipeline and variant interpretation rules.

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