COL14670_FB-270-195
Comprehensive liquid biopsy workflow for cell-free nucleic acids, from blood sample to variant data

Ion Torrent Oncomine cell-free nucleic acids assays are multibiomarker next-generation sequencing (NGS) assays that enable reproducible detection of somatic mutations in plasma, down to a level of 0.1% in genes relevant to solid tumors. When used  as part of a complete liquid biopsy NGS workflow, the road from research blood sample to variant data only takes two days.

Each assay contains the reagents for library construction and a single pool of primers used to perform multiplex PCR for preparation of amplicon libraries from cell-free DNA (cfDNA) and cell-free RNA (cfRNA) found in the plasma fraction of whole blood, and each is part of a complete research solution to detect tumor-derived DNA and RNA from cell-free nucleic acids.

Using a single tube of blood, our assays are designed for analysis of single nucleotide variants, short indels, copy number variations, and fusions that are frequently mutated in research cancer samples. The assays enable a limit of detection (LOD) down to 0.1%.

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Liquid biopsies offer several advantages over conventional solid tumor samples: 

  • Less invasive to obtain, enabling tumor content to be sampled multiple times
  • Lower cost compared to traditional tissue samples
  • Faster turnaround time from sample to results
  • Help capture more of the heterogeneity of the tumor
  • May lead to improvements in standard of care in the future

We now know that cancer is a molecular disease. To better advance cancer research in the future, we need a clearer understanding of how those oncogenes and oncoproteins change in both time and space. Liquid biopsy applications enable clinical researchers to investigate tumors in ways not previously possible.

In the past, clinical cancer research on cfDNA focused on digital PCR or droplet digital PCR applications to analyze samples with low-frequency mutations. These approaches, while sensitive, limit the number of target genes, thereby limiting the field of vision of the researcher.

The Oncomine Cell-Free Research Assays and the Ion GeneStudio S5 Systems enable tumor heterogeneity research, therapy selection research, therapy monitoring research, and reoccurrence research studies from as little as 1ng of sample input.

Detect mutations like EGFR T790M

“Using the Oncomine Lung cfDNA Assay, we were able to amplify more difficult samples. The assay allowed us the detection of mutations like EGFR T790M down to 0.14% allelic fraction.”

José Luis Costa, PhD
Senior Researcher
Institute of Molecular Pathology and Immunology of the University of Porto, Portugal


Optimized for liquid biopsy clinical research focused on primary driver and resistance mutations in cell-free nucleic acids

Oncomine cell-free research assays include targets identified by the Oncomine Knowledgebase Reporter, a cancer genomics data resource, and reviewed by clinical researchers.

Oncomine cell-free research assay gene content

Assay

DNA/RNA

Gene

Selected SNV hotspots

CNVs

Fusions

Extra

Oncomine Pan-Cancer Cell-Free Assay

DNA & RNA

AKT1
ALK
APC
AR
ARAF
BRAF
CCND1
CCND2
CCND3
CDK4
CDK6
CHEK2
CTNNB1
DDR2
EGFR
ERBB2
ERBB3
ERG
ESR1
ETV1
FBXW7
FGFR1
FGFR2
FGFR3
FGFR4
FLT3
GNA11
GNAQ
GNAS
HRAS
IDH1
IDH2
KIT
KRAS
MAP2K1
MAP2K2
MET
MTOR
MYC
NRAS
NTRK1
NTRK3
PDGFRA
PIK3CA
PTEN
RAF1
RET
ROS1
SF3B1
SMAD4
SMO
TP53

>900 hotspots including:

BRAF 
EGFR
HRAS, KRAS, NRAS

 

CCND1
CCND2
CCND3
CDK4
CDK6
EGFR
ERBB2
FGFR1
FGFR2
FGFR3
MET
MYC
ALK
BRAF
ERG
ETV1
FGFR1
FGFR2
FGFR3
MET
NTRK1
NTRK3
RET
ROS1

MET exon
14 skipping

Tumor
suppressor
genes:
APC
FBXW7
PTEN
TP53

Oncomine Lung cfTNA Assay

DNA & RNA

ALK BRAF EGFR ERBB2 KRAS MAP2K1 MET NRAS PIK3CA RET ROS1 TP53

>150 hotspots including:

EGFR: T790M, C797S,
L858R, Exon 19 del KRAS: G12X, G13X, Q61X

BRAF
: V600E

ALK
: Exon 21-25

PIK3CA
: E545K, H1047R, E542K

MET

ALK, RET,ROS1

MET exon 14 skipping

Oncomine Lung cfDNA Assay

DNA

ALK BRAF EGFR ERBB2 KRAS MAP2K1 MET NRAS PIK3CA ROS1 TP53

>150 hotspots including:

EGFR: T790M, C797S, L858R, Exon 19 del

KRAS: G12X, G13X, Q61X

BRAF
: V600E

ALK
: Exon 21-25

PIK3CA
: E545K, H1047R, E542K

---

---

---

Oncomine Breast cfDNA Assay v2

DNA

AKT1 CCND1 EGFR ERBB2 ERBB3 ESR1 FBXW7 FGFR1 KRAS PIK3CA SF3B1 TP53

>150 hotspots including:

PIK3CA
: E545K, H1047R

AKT1: E17K

ESR1: mutations associated with anti- estrogen resistance TP53: mutations associated with loss of function

ERBB2: mutations associated with sensitivity to anti-ERBB2 therapies

CCND1, ERBB2,
FGFR1

---

Expanded coverage of TP53

Oncomine Breast cfDNA Assay DNA AKT1 EGFR ERBB2 ERBB3 ESR1 FBXW7 KRAS PIK3CA SF3B1 TP53

>150 hotspots including: PIK3CA: E545K, H1047R AKT1: E17K

ESR1: mutations associated with anti- estrogen resistance TP53: mutations associated with loss of function

ERBB2: mutations associated with sensitivity to anti-ERBB2 therapies

--- --- ---
Oncomine Colon cfDNA Assay DNA AKT1 APC BRAF CTNNB1 EGFR ERBB2 FBXW7 GNAS KRAS MAP2K1 NRAS PIK3CA SMAD4 TP53

>240 hotspots including:

KRAS/NRAS: G12/G13/Q61 BRAF: V600E

PIK3CA: E545K, H1047R

TP53: R175H R273H/C/L

Recurrent deleterious APC mutations (including p.R876*, p.R1114*, p.Q1378*, p.R1450*) SMAD4: R361C/H CTNNB1: S45F, T41A

--- --- ---
Hotspot Genes Tumor Suppressor Genes Copy Number Genes Gene Fusions
AKT1
ALK
AR
ARAF
BRAF
CHEK2
CTNNB1
DDR2
EGFR
ERBB2
ERBB3
ESR1
FGFR1
FGFR2
FGFR3
FGFR4
FLT3
GNA11
GNAQ
GNAS

HRAS
IDH1
IDH2
KIT
KRAS
MAP2K1
MAP2K2
MET
MTOR
NRAS
NTRK1
NTRK3
PDGFRA
PIK3CA
RAF1
RET
ROS1
SF3B1
SMAD4
SMO

APC
FBXW7
PTEN
TP53

CCND1
CCND2
CCND3
CDK4
CDK6
EGFR
ERBB2
FGFR1
FGFR2
FGFR3
MET
MYC

ALK
BRAF
ERG
ETV1
FGFR1
FGFR2
FGFR3
MET
NTRK1
NTRK3
RET
ROS1

  • Single tube of blood—the end-to-end, two-day NGS workflow is enabled from a single tube of blood
  • High-value content—including key targets selected and verified by the OncoNetwork consortium and industry experts, the assays enable analysis of all types of key mutations (SNV, indels, CNVs, fusions)
  • Sample tolerance—flexible input amounts and tolerance of sample input variability to accommodate more of your samples
  • Low limit of detection—variant detection down to 0.1% for SNV hotspots and indels
  • Reduced cost—uniform coverage of tumor type–specific amplicons enables more samples per sequencing run
  • Optimized analysis—the variant caller helps to increase sensitivity and specificity

Since cell-free nucleic acids can vary from sample to sample, flexibility in supporting varying amounts of input DNA, especially small amounts, is critical. Oncomine cell-free assays have the ability to support varying input amounts (Figure 1). Getting data from as many samples as possible is a crucial step in advancing liquid biopsy research.

Figure 1. Amplicon coverage and input material determine limit of detection using Oncomine cfDNA assays.

In verification studies, the assays demonstrate high correlation between the variants called in formalin-fixed, paraffin-embedded (FFPE) samples and those called in plasma (Table 1). As expected, there is a higher mutant allelic fraction in the FFPE tumor sample compared to that measured in plasma, and germline variants are seen at expected levels of ~50% in both FFPE and plasma. 

Sample

Variant

FFPE

Plasma

1

EGFR-L858R

71.42%

2.62%

2

TP53-R158L

51.89%

4.32%

3

MET-T1010I
KRAS-G12C

43.87%
34.62%

51.57%
0.28%

4

N/A

No detection

No detection

5

EGFR-L858R
MET-T1010I
TP53-Y220C

58.44%
41.93%
35.54%

7.28%
48.72%
1.93%

6

TP53-R158L

10.19%

1.26%

Table 1. Correlation of FFPE and matched plasma sample results (late-stage lung cancer samples). Oncomine cell-free assays demonstrate high correlation between variants called in FFPE samples and those called in matched plasma samples. The above data was obtained using the Oncomine Lung cfDNA Assay. Bolded values indicate somatic mutations. Non-bolded values indicate germline mutations.

Overall, Oncomine cell-free assays enable results from more samples, with demonstrated, repeatable results using clinical research samples at 0.1% limit of detection with 90% sensitivity and >98% specificity across our whole portfolio of Oncomine cell-free assays.

Sample

EGFR
E746_A750delELREA

EGFR
L858R

EGFR
T790M

EGFR
V769_D770insASV

KRAS
G12D

NRAS
A59T

NRAS
Q61K

PIK3CA
E545K

0.1% HDX

0.06

0.17

0.06

0.10

0.22

0.17

0.15

0.10

1% HDX

0.72

1.07

0.75

0.74

1.14

1.15

1.15

2.29

5% HDX

4.52

4.86

6.32

3.97

6.34

6.11

6.94

5.29

100% WT

0

0

0

0

0

0

0

0

Table 2. Variants called from Horizon cfDNA Multiplex Reference Set. All 8 mutant hotspots were called at 0.1%. Data obtained using the Oncomine Lung cfDNA Assay.

Running up to 8 samples on an Ion 530 chip or up to 32 samples on an Ion 540 chip, the Oncomine cell-free assays enable efficiently multiplexed PCR analysis of single nucleotide variants (SNVs) , short indels, copy number variations, and fusions across our whole portfolio of Oncomine cell-free assays. frequently mutated in common tumor types, tolerating sample input variability and accommodating flexible input amounts as low as 1 ng.

The entire workflow (Figure 2), from isolation of cell-free nucleic acids using the appropriate Applied Biosystems MagMAX Cell-Free Isolation Kit to analysis of samples, can be accomplished in just 2 days on the Ion GeneStudio S5 Prime system.

oncomine-cell-free-dna-assays-liquid-biopsy-clinical-research-banner

Prep.

  1. Cell-free isolation (MagMAX Cell-Free Isolation Kit)
  2. Library prep (Oncomine Cell-Free Assays)
  3. Template prep (Ion Chef System)

Sequence.

  1. High-throughput sequencing (Ion GeneStudio S5 Series)

Analyze.

  1. Variant caller for optimized analysis (Torrent Suite / Ion Reporter Software)
  2. Labels, guidelines, and global clinical trials (Oncomine Knowledgebase Reporter Software)

Oncomine cell-free assays are part of a complete workflow designed for liquid biopsy clinical research, which includes:

Oncomine cell-free assays

A kit containing reagents for library construction and a single pool of multiplex PCR primers to prepare amplicon libraries from cell-free nucleic acids found in the plasma fraction of whole blood.

Tag Sequencing Barcode Sets 1–24 and 25–48

A set of unique barcode adapters specifically designed for optimal performance with our technology. The unique barcode adapters are compatible with the Ion S5, Ion PGM, and Ion Proton systems. When used in combination with kits, this set enables you to pool up to 24 amplicon libraries and conduct multiplex sequencing analysis, which helps reduce the sequencing cost per sample.

Ion GeneStudio S5 Series

The Ion GeneStudio S5 Series supports five different chip types, so you can run multiple applications on a single sequencer. This flexibility eliminates the need to batch samples in order to achieve the optimum cost efficiency. Just choose the chip type that matches your specific throughput or application needs. 

Learn more

Resources

** Oncomine Knowledgebase Reporter is enabled in Ion Reporter™ Software version 5.2 or later.