Oncomine Dx Target Test Technical and Validation Information

Gene targets included for NSCLC

Figure 1. Complete gene list. *The test reports fusion/translocation variants for ROS1 and RET only. The test only reports ALK and MET mutations. ** Performance for the additional gene target variants has been validated based on a representative method.

Method comparison studies evaluated the accuracy of the Oncomine Dx Target Test for the detection of BRAF V600E, EGFR exon 19 deletions and L858R, ROS1 fusions, and RET fusions, using a BRAF V600E PCR assay, therascreen™ EGFR PCR Kit, ROS1 FISH assay, and Archer assay respectively. The concordance studies show overall percent agreement (OPA) of 100% for BRAF, 99% for EGFR, and 100% for ROS1, and 92% for RET between the Oncomine Dx Target Test and reference methods, excluding invalids and no-calls. A summary of the concordance studies results are included in Table 1. For details see the User Manual.

* No-calls are for DNA variants and unknowns are for RNA fusions

Table 1. Method comparison between Oncomine Dx Target Test and reference methods for three companion diagnostic biomarkers in NSCLC.

NSCLC

The Oncomine Dx Target Test also detects DNA sequence variations in an additional 19 genes in NSCLC. The variants for KRAS, MET, and PIK3CA have been analytically validated. Safety and effectiveness of these three genes have not been established and they are not intended to be used to direct therapy. The performance of all other variants identified by the test, other than clinically validated therapeutic variants and analytically validated variants, has not been directly demonstrated and was validated based on a representative method.

Limit of detection

Three LoD studies were performed to evaluate DNA variants, ROS1 fusions, and RET fusions.

Study I: The limit of detection (LoD) was evaluated for 14 representative DNA variants representing 3 variant categories detected by the Oncomine Dx Target Test. The LoD is the lowest allele frequency of SNV, multi-nucleotide polymorphism (MNP), or deletion variants, that can be detected at least 95% of the time. The study demonstrated that the Oncomine Dx Target Test can detect DNA variants with allele frequencies between 6 and 8%.

Study II: The LoD was calculated for 2 clinical ROS1 RNA fusion variants using the updated RNA library preparation workflow, and determined at 516 fusion reads.

Study III: The LoD was calculated for 2 clinical RET fusion variants using the updated RNA library preparation workflow, and determined at 405 fusion reads.

Accuracy

To evaluate the ability of the Oncomine Dx Target Test DNA and RNA panels to identify somatic variants in human specimens, 290 FFPE tumor samples were analyzed using the Oncomine Dx Target Test to demonstrate positive percent agreement (PPA) and negative percent agreement (NPA) concordance with validated reference detection methods.

The following reference detection methods were used:

• Validated NGS method, to detect SNV and deletion hotspot variants
• Validated ROS1 FISH test, to detect ROS1 fusions

The study demonstrated variant level PPA of 98.5%, NPA of 100%, and OPA of 100%, excluding invalids no-calls; and PPA level of 98.5%, NPA of 96.8%, and OPA of 96.8% including no-calls. A summary of the data are included in Table 2. For details see the User Manual.

Table 2. Variant level accuracy study results

Reproducibility

Three reproducibility studies were performed to evaluate DNA variants, ROS1 fusions, and RET fusions.

Study I:

The reproducibility and repeatability of the Oncomine Dx Target Test was evaluated for 30 representative variants from 18 DNA samples. The study was designed to evaluate within-run precision performance (repeatability) and variability across sites, operators, and instruments (reproducibility). Due to the large number of variants detected by the test and the rarity of some of the variants, a representative variant approach was used. Variants were selected in the following categories:

• Simple SNVs
• Complex SNVs and MNPs, including SNVs in di- or tri-nucleotide repeat regions and SNVs in high-GC (>60%) or low-GC (<40%) content regions
• Deletions (including deletions of 6, 9, 15, and 18 bp)

Excluding no calls, the percent of correct calls is >96%. The estimate of repeatability at each DNA variant location across all the samples was ≥98.8% (95% CI lower limit of ≥97.5%). A summary of results of the assay reproducibility study are included in Table 3. For details see the User Manual.

Table 3. Assay reproducibility study I.

Study II: 

An additional study was performed to evaluate the reproducibility and repeatability of the Oncomine Dx Target Test for 6 representative variants from 11 DNA samples and 4 RNA samples. 1 WT DNA sample and 4 WT RNA samples were included in the study.

The study was designed to evaluate within-run precision performance (repeatability) and variability across sites, operators, and instrument platforms (reproducibility). The updated RNA library preparation workflow was used. Due to the large number of variants detected by the test and the rarity of some variants, a representative variant approach was used.

 Variants were selected in the following categories:

• 15 bp deletion
• Simple SNVs
• Complex SNVs and MNPs
• Fusions

Excluding no calls, the estimate of repeatability at each DNA variant location across all the samples was ≥94.4% (95% CI lower limit of ≥72.7%). The estimate of repeatability at each RNA clinical variant location was 100%. A summary of the results of Study II is included in Table 4 and 5.

Table 4. Study II—assay reproducibility study results (DNA variants)

Table 5. Study II—assay reproducibility study results (ROS1 fusions)

Study III:

An additional study was performed to evaluate the reproducibility and repeatability of the Oncomine Dx Target Test for 4 RET fusion positive samples and 2 RET fusion-negative samples. The study was designed to evaluate within-run precision performance (repeatability) and variability across sites, operators, and instrument platforms (reproducibility). The updated RNA library preparation workflow was used. Excluding unknowns, estimates of the repeatability ranged from 98.1% to 100% for two RET variants. A summary of the results of Study III is included in Table 6.

Table 6. Study III—assay reproducibility study results (RET fusions)

A complete and flexible system

The Oncomine Dx Target Test is used in conjunction with the Ion PGM Dx System, which includes a complete NGS system of instruments, reagents, and software. The Ion PGM Dx System was initially validated using challenging germline variants and is now additionally validated with the Oncomine Dx Target Test for somatic mutation reporting for FFPE tissue samples. The Ion PGM Dx sequencing system is a Class II 510 K Medical Device and incorporates combined functionality, with both "IVD Mode" for molecular diagnostic tests and "Assay Development Mode" for clinical research. The system also facilitates 21CFR Part 11 compliance, role-based workflows, sample and reagent tracking, QC metrics, and audit trails.

The Oncomine Dx Target Test workflow—all results in 4 days

The Oncomine Dx Target Test workflow is a fully validated IVD workflow from beginning to end and includes all the reagents, consumables, instruments, and software to perform the test. It is possible to run 1–6 samples per run, plus 2 controls within 4 days (Figure 2).

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Abbreviated Intended Use: The Oncomine Dx Target Test is a qualitative in vitro diagnostic test that uses targeted high-throughput, parallel-sequencing technology to detect single nucleotide variants (SNVs) and deletions in 23 genes from DNA and fusions in ROS1 and RET from RNA isolated from formalin-fixed, paraffin-embedded (FFPE) tumor samples from patients with non-small cell lung cancer (NSCLC), using the Ion PGM Dx System.

Test limitations and warnings

• Use of this product must be limited to personnel trained in the techniques of PCR, NGS, and the use of the Oncomine Dx Target Test and the Ion PGM Dx System.
• The Oncomine Dx Target Test has only been validated for use with NSCLC FFPE tumor slide specimens.
• The Oncomine Dx Target Test has been validated to detect the following somatic mutations: RNA fusions, single-nucleotide variations (SNVs), multi-nucleotide variations (MNVs), and deletions of 3, 6, 9, 12, 15, and 18 base pairs (bps).
• The Oncomine Dx Target Test is only validated for use with the Ion PGM Dx System and the Veriti Dx 96-Well Thermal Cycler.
• The Oncomine Dx Target Test is only validated for use with 10 ng each of DNA and RNA per sample. Input amounts lower or higher than 10 ng are not recommended.
• Both the DNA and RNA from a single sample extraction must meet the concentration requirements specified in the procedure. Do not use DNA from one extraction with RNA from a different extraction.
• The effects of potential variations in FFPE specimen fixation have not been evaluated.
• Extraction from FFPE sample curls has not been evaluated.
• A potential source of contamination in the procedure is nucleic acid from previous sample processing steps. Follow good laboratory practices and all precautions and guidelines in these user guides to avoid cross-contamination between samples.
• The Oncomine Dx Target Test is a qualitative test. The test is not for quantitative measurements of percent mutation.
• The Ion OneTouch Rack Kit has only been designed to work with GeneMate SnapStrip 8-Strip 0.2 mL PCR Tubes. Tubes from other manufacturers may not fit properly in the rack, resulting in a higher risk of user error.
• The Oncomine Dx Target Test assay definition file includes prevalent RET isoforms, but not all rare or newly identified RET isoforms. The Oncomine Dx Target Test may miss a subset of patients carrying rare or newly identified RET isoforms who may derive benefit from pralsetinib.