Search Thermo Fisher Scientific
Search Thermo Fisher Scientific
The Ion Torrent 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 targeted therapies for non-small cell lung cancer (NSCLC), cholangiocarcinoma (CC), anaplastic thyroid cancer (ATC), astrocytoma (AC), oligodendroglioma (OG), medullary thyroid cancer (MTC), and thyroid cancer (TC) patients. Concordance with FDA-approved or validated reference methods based on fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR), Sanger sequencing, or NGS was established for all CDx biomarkers included in the test. The variants for KRAS, MET and PIK3CA have been analytically validated. Performance of all other variants identified by the test, other than the clinically validated therapeutic variants and analytically validated variants, has not been directly demonstrated.
Gene targets for therapeutic use | ||||
---|---|---|---|---|
BRAF: V600E | EGFR: L858R, exon 19 deletions, and exon 20 insertions | ERBB2/HER2: activating mutations (SNVs and exon 20 insertions) | RET: fusions | ROS1: fusions |
Analytically validated targets | ||||||
---|---|---|---|---|---|---|
KRAS | MET* | PIK3CA |
Additional targets** | ||||
---|---|---|---|---|
AKT1 | HRAS | MTOR | RET | ROS1 |
ALK* | ERBB3 | KIT | NRAS | |
CDK4 | FGFR2 | MAP2K1 | PDGFRA | |
DDR2 | FGFR3 | MAP2K2 | RAF1 |
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.
Select analytical and clinical studies are summarized below. For complete studies and results, refer to the Oncomine Dx Target Test Part I: Test Description and Performance Characteristics User Guide
The LoD was evaluated for 5 IDH1 R132 variants detected by the Oncomine Dx Target Test. The LoD is the lowest allelic frequency (AF) 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% AF, including 4.5% AF for R132C, 5.7% AF for R132G, 4.9% AF for R132H, 5.1% AF for R132L, and 5.3% AF for R132S.
The reproducibility and repeatability of IDH1 R132 variant detection using Oncomine Dx Target Test were assessed with 1 IDH1 WT sample and three IDH1 R132 variant-positive samples at 2 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 1).
Table 1. Reproducibility results for IDH1 in cholangiocarcinoma
Sample
|
Variant ID
| Positive call rate +95% Cl | |||||||
---|---|---|---|---|---|---|---|---|---|
Variant (amino acid change) | # of valid sample results (N) | # of positive calls (A) | # of positive calls (B) | # of positive calls (C) | Including no calls (A/N) | Excluding no calls (A/(A+B)) | Relative LoD | ||
D1 | COSM28747 | R132C | 36 | 36 | 0 | 0 | 100% (90.3%, 100%) | 100% (90.3%, 100%) | 2.1–2.7X |
D2 | COSM28747 | R132C | 36 | 35 | 0 | 1 | 97.2% (85.5%, 99.9%) | 100% (90.0%, 100%) | 0.98–1.4X |
D3 | COSM28749 | R132G | 36 | 36 | 0 | 0 | 100% (90.3%, 100%) | 100% (90.3%, 100%) | 1.9–2.5X |
D4 | COSM28749 | R132G | 36 | 36 | 0 | 0 | 100% (90.3%, 100%) | 100% (90.3%, 100%) | 0.9–1.3X |
D5 | COSM28750 | R132L | 36 | 36 | 0 | 0 | 100% (90.3%, 100%) | 100% (90.3%, 100%) | 1.4–1.8X |
D6 | COSM28750 | R132L | 35 | 20 | 6 | 9[1] | 57.1% (39.4%, 73.7%) | 76.9% (56.4%, 91.0%) | 0.65–0.94X |
D7 | Wild-type (WT) | N/A | 36 | 0 | 0 | 0 | 0% (0%, 9.17%) | 0% (90.3%, 100%) | N/A |
A clinical concordance study was conducted to evaluate the ability of the Oncomine Dx Target Test to identify 5 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 2.
Table 2. Concordance results for IDH1
Agreement measure | Excluding invalid and no calls | Including invalid results and no-calls | ||
---|---|---|---|---|
Percent agreement | 95% Cl | Percent agreement | 95% Cl | |
PPA | 99.4% (163/164) | (96.7%, 100.0%) | 97.0% (163/168) | (93.2%, 99.0%) |
NPA | 96.5% (164/170) | (92.5%, 98.7%) | 90.6% (164/181) | (85.4%, 94.4%) |
OPA | 97.9% (327/334) | (95.7%, 99.2%) | 93.7% (327/349) | (90.6%, 96.0%) |
The LoD of the Oncomine™ Dx Target Test for 10 IDH1 and IDH2 SNVs was determined using various FFPE clinical samples and DNA sample blends across 6 dilution levels. The LoD for the 5 IDH1 SNVs ranged from 4.6% to 7.0% AF, while the LoD for the 5 IDH2 SNVs ranged from 4.1% to 5.8% AF.
The reproducibility and repeatability of detecting IDH1 and IDH2 variants in astrocytoma and oligodendroglioma samples were assessed across three test sites. The study involved 12 positive sample blends and one negative blend, tested by 2 operators per site using 2 Ion PGM Dx systems and 3 reagent lots. Each operator performed 12 replicates per sample blend, resulting in a total of 156 valid sequencing runs. The overall positive call rate for IDH1 variants was 100%, and for IDH2 variants, it was 97.5% including no calls and 99.9% excluding no calls. The negative call rate and within-run repeatability for IDH1 variants were both 100%, while IDH2 repeatability ranged from 95.8% to 100% excluding no calls.
A clinical concordance study was to evaluate the concordance between the Oncomine Dx Target Test and local laboratory tests (LLT) for IDH1/IDH2 mutations in glioma specimens. A summary of the results is detailed in Tables 3 and 4.
Table 3. Concordance between Oncomine Dx Target Test and NGS LLT assay for IDH1/IDH2 mutation
Oncomine Dx Target Test (IDH1/IDH2) | ||||
---|---|---|---|---|
NGS LLT assay (IDH1/IDH2) | ||||
Frequency | Positive | Negative | Unknown | Total |
Positive | 306 | 0 | 21 | 327 |
Negative | 1 | 34 | 1 | 36 |
Unknown | 0 | 3 | 0 | 3 |
Total | 307 | 37 | 22 | 366 |
Table 4. Clinical concordance of the Oncomine™ Dx Target Test in reference to NGS LLT assay for IDH1/IDH2 mutation
Agreement measure | Estimate | 95% Cl |
---|---|---|
PPA | 99.7% | (98.2%, 99.9%) |
NPA | 100% | (89.8%,100.0%) |
Adjusted PPV | 100% | (98.8%, 100.0%) |
Adjusted NPV | 100% | (89.8%, 100.0%) |
The LoD of the Oncomine Dx Target Test was evaluated across several studies to determine the lowest AF that can be detected at least 95% of the time.
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 including:
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 is included in Table 5. For details see the User Manual.
Table 5. Variant level accuracy study results
Variant level measure of agreement | Percent agreement (N) | Percent agreement (N) |
---|---|---|
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) |
The reproducibility and repeatability were validated across multiple studies for various DNA and RNA variants, including SNVs, deletions, and fusions.
Study I:
Reproducibility and repeatability were evaluated using 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:
The study demonstrated a call rate of >96% excluding no calls, and the estimate of repeatability was >98.8%, excluding no calls.
Study II:
Study II assessed reproducibility and repeatability for 6 representative variants from 11 DNA and 4 RNA clinical samples across multiple sites, operators, and instruments. The call rate for variant-positive DNA was 99%, while WT DNA had a call rate of 100%, excluding no calls. For ROS1 fusion-positive RNA, the call rate was 100%, and WT RNA had a call rate of 99%, excluding no calls.
Study III:
The reproducibility and repeatability for detecting RET fusions were evaluated using 4 RET fusion-positive and 2 RET fusion-negative RNA samples. The study showed a call rate of 99% for RET fusion-positive RNA and 100% for WT RNA, excluding no calls.
Study IV:
Reproducibility and repeatability for detecting EGFR exon 20 insertion variants were assessed using 2 EGFR variant-positive and 2 EGFR variant-negative DNA samples. The call rate for EGFR exon 20 insertion-positive DNA was 100%, and WT DNA had a call rate of 100%, excluding no calls.
Study V:
Reproducibility and repeatability for detecting ERBB2/HER2 exon 20 insertion variants was evaluated using 2 ERBB2/HER2 variant-positive and 2 ERBB2/HER2 variant-negative DNA samples. The call rate for ERBB2/HER2 exon 20 insertion-positive DNA was 100%, and WT DNA had a call rate of 100%, excluding no calls.
Study VI:
Reproducibility and repeatability for detecting ERBB2/HER2 SNVs were evaluated using 3 ERBB2/HER2 SNV-positive and 4 ERBB2/HER2 SNV-negative DNA samples. The call rate for ERBB2/HER2 SNV-positive DNA was 100%, and WT DNA had a call rate of 100%, excluding no calls.
Study VII:
Reproducibility and repeatability for detecting RET fusions near the LoD were evaluated using 4 RET fusion-positive and 2 WT RNA samples. The study demonstrated positive call rates of 77.8% for the KIF5B-RET fusion isoform and 100% for the CCDC6-RET fusion isoform, excluding no calls.
Study VIII:
Reproducibility and repeatability for the KIF5B-RET fusion at 1X LoD was confirmed with a call rate 94.4% for the KIF5B-RET fusion isoform, excluding no calls.
Study IX:
Reproducibility and repeatability for 3 EGFR exon 20 insertion variants (3 bp, 9 bp, and 12 bp) was confirmed with a call rate of 100% for EGFR exon 20 insertion-positive DNA variants, excluding no calls
The accuracy of the Oncomine Dx Target Test for detecting various biomarkers in NSCLC was evaluated with method comparison studies against several reference assays including PCR, NGS, and fluorescence in situ hybridization (FISH). A summary of the results is shown below. For details, refer to the Oncomine Dx Target Test Part I: Test Description and Performance Characteristics User Guide.
Table 6. Concordance of the Oncomine Dx Target Test with various reference assays
Variants for therapy selection
|
Validated comparator methods
| Excluding no calls or unknowns* | Including no calls or unknowns* | ||||
---|---|---|---|---|---|---|---|
Positive percent agreement | Negative percent agreement | Overall percent agreement | Positive percent agreement | Negative percent agreement | Overall percent agreement | ||
BRAF V600E | Validated BRAF | 100% (67/67) | 100% (114/114) | 100% (181/181) | 91.8% (67/73) | 97.4% (114/117) | 95.3% (181/190) |
EGFR | Therascreen™ EGFR PCR Kit | 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) | |
EGFR exon 20 insertions | Validated NGS assay 1 | 100% (54/54) | 100% (95/95) | 100% (149/149) | 98.2% (54/55) | 90.5% (95/105) | 93.1% (149/160) |
Validated NGS Assay 2 | 100% (46/46) | 100% (63/63) | 100% (109/109) | 97.9% (46/47) | 91.3% (63/69) | 94.0% (109/116) | |
ROS1 fusions | Validated ROS1 FISH test | 100% (9/9) | 100% | 100% | 90.0% (9/10) | 88.6% (62/70) | 88.8% (71/80) |
ERBB2/HER2 activating mutations (SNVs and exon 20 insertions) | Validated NGS Assay | 100% (38/38) | 99.1% (108/109) | 99.3% (146/147) | 97.4% (38/39) | 92.3% (108/117) | 93.6% (146/156) |
RET fusions | Validated NGS Assay 1 | 90.9% (40/44) | 91.8% (101/110) | 91.6% (141/154) | 90.9% (40/44) | 91.8% (101/110) | 91.6% (141/154) |
RET fusions | Validated NGS Assay 2 | 92.3% (84/91) | 96.8% (121/125) | 94.9% (205/216) | 92.3% (84/91) | 96.0% (121/126) | 94.5% (205/217) |
* No-calls are for DNA variants and unknowns are for RNA fusions
The LoD of the Oncomine Dx Target Test for detection of BRAF V600E mutation in thyroid cancer FFPE tissue was 6.4% AF, which confirmed the established LoD for BRAF V600E in NSCLC tissues.
Reproducibility and repeatability for BRAF V600E was evaluated with two variant-positive and one variant-negative thyroid cancer sample. The results demonstrated 100% agreement for average positive and average negative agreements (APA, ANA), across all replicates, operators, instruments, and reagent lots.
Concordance of the Oncomine Dx Target Test with a clinical trial PCR assay in the detection of BRAF V600E variants in ATC was evaluated using 199 samples, which included 32 from the ATC clinical trial and 167 commercially sourced thyroid cancer tissue samples of various histologies including follicular (FTC), papillary (PTC), and medullary thyroid cancer (MTC). The PPA, NPA, and overall percent agreement (OPA) results are shown in Table 7.
Table 7. Concordance between the clinical trial PCR assay and the Oncomine Dx Target Test
Agreement measure | Excluding invalid and no calls | Including invalid results and no-calls | ||
---|---|---|---|---|
Percent agreement | 95% Cl1 | Percent agreement | 95% Cl1 | |
PPA | 99.0% (97/98) | (94.4%, 99.8%) | 77.6% (97/125) | (69.5%, 84.0%) |
NPA | 100% (57/57) | (93.7%, 100.0%) | 77.0% (57/74) | (66.3%, 85.1%) |
OPA | 99.4% (154/155) | (96.4%, 99.9%) | 77.4% (154/199) | (71.1%, 82.6%) |
1The 95% Cl was calculated using the Wilson Score method.
The LoD for the Oncomine Dx Target Test was evaluated for 4 RET DNA variants and 2 RET RNA fusions in clinical thyroid cancer samples. The LoD for RET DNA variants were determined to be allelic frequencies ranging from 4.9% to 5.5% AF, and the LoD for RET RNA fusions was 236 fusion reads.
Reproducibility and repeatability were evaluated for detection of RET DNA variants and RET RNA fusions. The call rate for RET variant-positive DNA was 100%, and WT DNA had a call rate of 100%, excluding no calls. The call rate was 97.4% for RET fusion-positive RNA, and 100% for WT RNA. 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%.
Concordance was evaluated for detecting RET DNA variants and fusions in medullary thyroid cancer (MTC) and other thyroid cancer (TC) specimens through various method comparison studies comparing to validated reference NGS assays and local laboratory tests (LLTs). The concordance of RET DNA variants and RET fusions are shown in Tables 8-10 below.
Table 8. Concordance between Oncomine Dx Target Test and the reference assay—RET DNA variants (MTC)
Agreement measure | Excluding unknowns1 | Including unknowns1 | ||
---|---|---|---|---|
Percent agreement | 95% Cl | Percent agreement | 95% Cl | |
PPA | 100.0% (36/36) | (90.3%, 100.0%) | 100.0% (36/36) | (90.3%, 100.0%) |
NPA | 98.3% (57/58) | (90.8%, 100.0%) | 86.4% (57/66) | (75.7%, 93.6%) |
OPA | 98.9% (93/94) | (94.2%, 100.0%) | 91.2% (93/102) | (83.9%, 95.9%) |
1Unknowns are defined as invalid or no result using the Oncomine Dx Target Test.
Table 9. Concordance between Oncomine™ Dx Target Test and the reference assay—RET fusions (TC)
Agreement measure | Excluding unknowns1 | Including unknowns1 | ||
---|---|---|---|---|
Percent agreement | 95% Cl | Percent agreement | 95% Cl | |
PPA | 100.0% (25/25) | (86.3%, 100.0%) | 100.0% (25/25) | (86.3%, 100.0%) |
NPA | 100.0% (57/57) | (93.7%, 100.0%) | 91.9% (57/62) | (82.2%, 97.3%) |
OPA | 100.0% (82/82) | (95.6%, 100.0%) | 94.3% (82/87) | (87.1%, 98.1%) |
1Unknowns are defined as invalid or no result using the Oncomine Dx Target Test.
Table 10. Concordance between Oncomine™ Dx Target Test and LLT—RET fusions (TC)
Agreement measure | Excluding unknowns1 | Including unknowns1 | ||
---|---|---|---|---|
Percent agreement | 95% Cl | Percent agreement | 95% Cl | |
PPA | 93.33% (42/45) | (81.73%, 98.60%) | 64.62% (42/65) | (51.77%, 76.08%) |
NPA | 100.0% (133/133) | (97.26%, 100.0%) | 87.5% (133/152) | (81.17%, 92.30%) |
OPA | 98.31% (175/178) | (95.15%, 99.65%) | 80.65% (175/217) | (74.75%, 85.68%) |
1Unknowns are defined as invalid or no result using the Oncomine Dx Target Test.
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 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).
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 SNVs from FFPE tumor tissue samples from patients with cholangiocarcinoma (CC), BRAF V600E mutations from FFPE tumor tissue samples from patients with anaplastic thyroid cancer (ATC), IDH1 and IDH2 SNVs from FFPE tumor tissue samples from patients with astrocytoma (AC) or oligodendroglioma (OG), RET SNVs, multi-nucleotide variants (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.
For In Vitro Diagnostic Use