Oncomine cfNA assays

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%.

Why are pathologists and oncologists interested in liquid biopsy clinical cancer research?

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

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.

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. 

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.

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 GeneStudio S5 Prime system.