oncomine-dx-target-test-technical-validation-information-270

Established performance

The Oncomine Dx Target Test is the first targeted next-generation sequencing (NGS) in vitro diagnostic test simultaneously delivering multiple biomarker results to aid selection of multiple NSCLC targeted therapies. Concordance with validated comparator methods based on FISH or PCR was established for EGFR, BRAF, and ROS1. The variants for KRAS, MET, and PIK3CA were analytically validated. The performance of all other variants identified by the test, other than clinically validated therapeutic variants and analytically validated variants, was validated based on a representative method.

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The Oncomine Dx Target Test will be available in July 2017. LabCorp, NeoGenomics, and Cancer Genetics Inc. will be among the first customers to implement testing.

Oncomine Dx Target Test content

The cancer-associated gene targets included in the Oncomine Dx Target Test all play an important role in NSCLC pathogenesis. Three of the markers provide an aid in selecting patients for approved targeted therapies, while others are currently being investigated in clinical trials and are potentially actionable in the future, as referenced in Figure 1.

Gene targets included in the Oncomine Dx Target Test

Gene targets for therapeutic use
BRAF: mutation EGFR: mutation ROS1: fusion
Analytically validated targets
KRAS MET* PIK3CA
Additional targets
AKT1 ERBB2 HRAS MTOR RET*
ALK* ERBB3 KIT NRAS ROS1
CDK4 FGFR2 MAP2K1 PDGFRA  
DDR2 FGFR3 MAP2K2 RAF1  

Figure 1. Cancer-associated gene targets included in the Oncomine Dx Target Test.

*The test reports fusions/translocations variants for ROS1 only. The test only reports ALK, MET, and RET mutations.
**The performance for the additional gene targets variants has been validated based on a representative method.


Established performance for three biomarkers for NSCLC targeted therapies selection

Method comparison studies evaluated the accuracy of the Oncomine Dx Target Test for the detection of BRAF V600E, EGFR Exon 19 deletions and L858R, and ROS1 fusion, using a BRAF V600E qPCR assay, Qiagene therascreen EGFR PCR kit, and ROS1 FISH assay. The concordance studies show Overall Percent Agreement (OPA) of 100% for BRAF, 99% for EGFR, and 96.5% for ROS1 between the Oncomine Dx Target Test and comparator methods excluding no calls. A summary of the concordance studies results are included in Table 1. For details see the User Manual.

 

Variants for therapy selection

 

 

Validated comparator methods

 

Excluding no calls Including no calls

Positive percent agreement

Negative percent agreement

Overall percent agreement

Positive percent agreement

Negative percent agreement

Overall percent agreement

 

BRAF V600E

Validated BRAF
V600E qPCR
test

100% (67/67)

100% (114/114)

100% (181/181)

91.8% (67/73)

97.4% (114/117)

95.3% (181/190)

EGFR

Validated EGFR PCR test

98.6% (71/72)

99.2% (120/121)

99.0% (191/193)

81.6% (71/87)

96.8% (120/124)

90.5% (191/211)

EGFR exon 19 deletions

97.6% (41/42)

99.3% (147/148)

99.0% (188/190)

74.6% (41/55)

94.2% (147/156)

89.1% (188/211)

EGFR exon 21 L858R

100% (30/30)

100% (167/167)

100% (197/197)

93.8% (30/32)

93.3% (167/179)

93.4% (197/211)

ROS1 fusions

Validated ROS1 FISH test

80% (20/25)

100% (119/119)

96.5% (139/144)

80% (20/25)

100% (119/119)

96.5% (139/144)

Table 1. Concordance between Oncomine Dx Target Test and reference methods for three companion diagnostic biomarkers, excluding invalids and no calls


Validation of performance for additional 20 gene targets

The Oncomine Dx Target Test also detects DNA sequence variations in an additional 20 genes (approximately 343 targets) that are clinically associated with NSCLC. The variants for KRAS, MET, and PIK3CA have been analytically validated. 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

The limit of detection (LoD) was evaluated for 17 variants representing four variant categories detected by the Oncomine Dx Target Test. The LoD is the lowest allele frequency of SNV, MNP, or deletion variants, and the lowest number of reads of RNA fusion variants that can be detected at least 95% of the time. The study demonstrated that the Oncomine Dx Target Test can detect DNA variants from 6–13% allele frequency, and RNA fusion at 732 reads, with 95% confidence.

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% (Table 2), 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.

Variant level measure of agreement

Percent agreement (N)
Excluding no calls

Percent agreement (N)
Including no calls

Positive percent agreement

98.5% (195/198)

98.5% (195/198)

Negative percent agreement

100.0% (118,155/118,159)

96.8% (118,155/122,012)

Overall percent agreement

100.0% (118,350/118,357)

96.8% (118,350/122,210)

Table 2. Variant level accuracy study results

Reproducibility

The reproducibility and repeatability of the Oncomine Dx Target Test was evaluated for 30 representative variants from 18 DNA and 9 RNA 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)
  • Fusions

Excluding no calls, the percent of correct calls ranged from 95-99%.  The estimate of repeatability at each DNA variant location across all the samples was ≥98.8% (95% CI lower limit of ≥97.5%).  The estimate of repeatability at RNA variant location was 94.4%. A summary of results of the assay reproducibility study are included in Table 3. For details see the User Manual.

 

Description

No. of variant samples

Call rate excluding no calls

Call rate including no calls

Mean

Median

Mean

Median

DNA positive variants (positive calls)

46

96.60%

97.10%

94.50%

95.80%

RNA positive variants (positive calls)

6

94.80%

95.50%

94.80%

95.50%

WT DNA variant locations (negative calls)

872

96.10%

95.00%

96.10%

95.00%

WT DNA variant locations (negative calls)

170

99.30%

99.30%

99.30%

99.30%

Table 3. 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).

dx-target-test-workflow

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 from RNA isolated from formalin-fixed, paraffin-embedded (FFPE) tumor tissue 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: 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.