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

Ion Torrent Oncomine cell-free nucleic acids assays are tumor type–specific, multibiomarker next-generation sequencing (NGS) assays that enable detection of somatic mutations in plasma, down to a level of 0.1% in genes relevant to solid tumors.

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 cancer samples for research. 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 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, a cancer genomics data resource, and reviewed by clinical researchers.

Oncomine cell-free assay gene content

Assay

DNA/RNA

Gene

Selected SNV hotspots

CNVs

Fusions

Extras

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

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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

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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

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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

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Oncomine cell-free assays leverage Ion Torrent sequencing, enabling researchers to develop future applications

  • 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

 

Enabling results from more of your liquid biopsy research samples

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.

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.

Oncomine cell-free research workflow

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 S5™ XL sequencing system.  


All the tools you need for liquid biopsy clinical research

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 S5 System Series

Ion S5 System Series supports four 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 about the Ion S5 System ›

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