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. This test only reports mutations for ALK and MET. ** Performance for the additional gene target variants has been validated based on a representative method. Only IDH1 is reported for CC. Only RET mutations are reported for MTC and only RET fusions are reported for TC.

Method comparison studies evaluated the accuracy of the Oncomine Dx Target Test for the detection of BRAF V600E, EGFR exon 19 deletions, L858R, and exon 20 insertions, ERBB2/HER2 activating mutations (SNVs and exon 20 insertions), ROS1 fusions, and RET fusions, using a BRAF V600E PCR assay, therascreen™ EGFR PCR Kit, ROS1 FISH assay, and validated NGS assays. 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 five companion diagnostic biomarkers in 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

Six LoD studies were performed to evaluate DNA variants, ROS1 fusions, RET fusions, EGFR exon 20 insertions, and ERBB2/HER2 SNVs and exon 20 insertions.

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 (AF) 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 6–8% allele frequencies.

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

Study IV: The LoD was calculated for two clinical EGFR exon 20 insertion-positive samples and determined to be 4.8–5.2% allele frequencies.

Study V: The LoD was calculated for 2 clinical ERBB2/HER2 exon 20 insertion positive samples, and determined to be 4.8-5.0% allele frequencies.

Study VI: The LoD was calculated for 2 clinical ERBB2/HER2 SNV positive samples, and determined to be 4.5-5.8% allele frequencies.

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

Six reproducibility studies were performed to evaluate DNA variants, ROS1 fusions, RET fusions, EGFR fusions, EGFR exon 20 insertions, and ERBB2/HER2 SNVs and exon 20 insertions.

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 wild-type (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)

Study IV:

A study was performed to evaluate the reproducibility and repeatability of the Oncomine Dx Target Test for detection of EGFR exon 20 insertion variants using FFPE DNA from two EGFR variant-positive samples (blended with WT clinical samples) and two EGFR variant-negative (WT) samples. The study was designed to evaluate within-run precision performance (repeatability) and variability across sites, operators, and instrument platforms (reproducibility). Excluding no-calls, estimates of the repeatability is 100% for both EGFR exon 20 insertion variants. A summary of the results of Study IV is included in Table 7.

Table 7. Study IV - assay reproducibility results (EGFR exon 20 insertions)

Study V:

A study was performed to evaluate the reproducibility and repeatability of the Oncomine Dx Target Test for detection of ERBB2/HER2 exon 20 insertion variants using FFPE DNA from two ERBB2/HER2 variant-positive samples. The study was designed to evaluate within-run precision performance (repeatability) and variability across sites, operators, and instrument platforms (reproducibility). Excluding no-calls, estimates of the repeatability is 100% for both ERBB2/HER2 exon 20 insertion variants. A summary of the results of Study V is included in Table 8.

Table 8. Study V - assay reproducibility study results (ERBB2/HER2 exon 20 insertions)

Study VI:

A study was performed to evaluate the reproducibility and repeatability of the Oncomine Dx Target Test for detection of ERBB2/HER2 SNV variants using FFPE DNA from three ERBB2/HER2 variant-positive samples and four negative samples. The study was designed to evaluate within-run precision performance (repeatability) and variability across sites, operators, and instrument platforms (reproducibility). Excluding no-calls, estimates of the repeatability is 100% for four ERBB2/HER2 SNV variants. A summary of the results of Study VI is included in Table 9.

Table 9. Study VI - assay reproducibility study results (ERBB2/HER2 SNVs)

A clinical concordance study was conducted to evaluate the ability of the Oncomine Dx Target Test to identify five IDH1 biomarkers in FFPE cholangiocarcinoma tumor specimen compared to a validated Sanger assay. The study demonstrated OPA of 97.9%, excluding invalids and no calls. A summary of the data is included in Table 10.

Table 10. Concordance between the Oncomine Dx Target Test and reference method for IDH1 R132

Limit of detection

The limit of detection (LoD) was evaluated for 5 IDH1 R132 variants detected by the Oncomine Dx Target Test. The LoD is the lowest allele frequency of SNV that can be detected at least 95% of the time. The study demonstrated LoD of the 5 IDH1 R132 variants ranged from 4.5–5.7% allele frequencies, including 4.5% for R132C, 5.7% for R132G, 4.9% for R132H, 5.1% for R132L, and 5.3% for R132S.

Assay reproducibility

The reproducibility and repeatability of IDH1 R132 variant detection using Oncomine Dx Target Test were assessed with one IDH1 WT sample and three IDH1 R132 variant-positive samples at two allele frequency levels. Testing was performed at 4 testing sites using 4 lots of reagents, and each site had 2 PGM Dx instrument systems and 2 operators. The overall positive call rate for IDH1 R132 variants was 92.6% when including no calls and 97.1% when excluding no calls. The negative call rate for IDH1 WT sample were 100% at all IDH1 R132 variant locations (Table 11).

Table 11. Reproducibility results

Clinical concordance for companion diagnostics markers for targeted therapies selection-TC

A clinical concordance study was conducted to evaluate the ability Oncomine Dx Target Test to identify RET DNA variants in FFPE MTC tumor specimens and RET fusions in FFPE TC tumor specimens compared to validated NGS assay. The study demonstrated overall percent agreement (OPA) of 99% for RET in medullary thyroid cancer (MTC) and 100% in thyroid cancer (TC). A summary of the data is included in table 12.

Table 12. Concordance between the Oncomine Dx Target Test and reference method for RET in MTC and TC

Analytical validation performance-TC limit of detection

Limit of detection (LoD) was evaluated for RET DNA variants and for RET RNA fusions in clinical thyroid cancer samples. The LoD is the lowest allele frequency of RET SNV, MNV, or deletion variants and lowest fusion reads of RET fusions that can be detected at least 95% of the time. The study demonstrated LoD of 4.9–5.5% for RET DNA variants and 236 fusion reads for RET fusions.

Assay reproducibility study

The reproducibility and repeatability of the Oncomine Dx Target Test was evaluated for four RET DNA variants and two RNA fusion variants at two allele frequency (AF) levels. The study was designed to evaluate within-run precision performance (repeatability) and variability across sites, operators, and instruments (reproducibility). Sample libraries were pooled and sequenced at three sites. At each site two operators were assigned two instrument systems. Estimates of within-run repeatability were 100% for the RET DNA variants tested, with one WT blend showing a 97.9% repeatability with no calls included. Repeatability estimates for the RET RNA fusion blends tested ranged from 88.9% to 100%. A summary of the data is included in Table 13 and Table 14.

Table 13. RET DNA variants assay reproducibility study results.

Table 14. RET RNA fusions variants assay reproducibility study results.

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), deletions, and insertions in 23 genes from DNA and fusions in ROS1 and RET from RNA isolated from formalin-fixed, paraffin-embedded (FFPE) tumor tissue samples from patients with non-small cell lung cancer (NSCLC); IDH1 R132 mutations from FFPE tumor tissue samples from patients with cholangiocarcinoma (CC); RET SNVs, MNVs, and deletions from DNA isolated from FFPE tumor tissue samples from patients with medullary thyroid cancer (MTC); and RET fusions from RNA isolated from FFPE tumor tissue samples from patients with thyroid cancer (TC) 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.
• Oncomine Dx Target Test has only been validated for use with NSCLC, CC and TC FFPE tumor slide specimens. The use of fine needle aspirates for thyroid cancer (TC) specimens has not been validated
• 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), and insertions of 3, 6, 9, and 12 base pairs (bps) from DNA.
• 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.
• For NSCLC, the Oncomine Dx Target Test assay definition file includes prevalent but not all rare or newly identified RET isoforms, ROS1 isoforms, EGFR exon 20 insertions and ERBB2/HER2 activating mutations (SNVs and exon 20 insertions). The Oncomine Dx Target Test may miss rare or newly identified:
• RET isoforms carried by a subset of patients who may derive benefit from GAVRETO™ (pralsetinib) and RETEVMO® (selpercatinib)
• ROS1 isoforms carried by a subset of patients who may derive benefit from XALKORI® (crizotinib)
• EGFR exon 20 insertions carried by a subset of patients who may derive benefit from EXKIVITY™ (mobocertinib) or RYBREVANT™ (amivantimab-vmjw)
• ERBB2/HER2 activating mutations (SNVs and exon 20 insertions) carried by a subset of patients who may derive benefit from ENHERTU® (fam-trastuzumab deruxtecan-nxki)

For In Vitro Diagnostic Use