Building a Better Biopsy
Combating the challenges of liquid biopsy with cfDNA synthetic plasma reference standards
Lisa M. Wright | | Longer Read
sponsored by Horizon
Medical professionals in the cancer sphere are all familiar with solid tumor testing for patients. And although valuable, these procedures are also painful, invasive, and costly in multiple ways. Liquid biopsy – the approach of examining fluid samples, usually blood, for biomarkers – holds many advantages over solid tumor testing. It is less invasive for the patient and has improved levels of sensitivity to detect low-frequency somatic driver mutations. In oncology, pathologists often examine circulating free DNA (cfDNA) for markers that indicate the presence of cancer, its molecular characteristics, and the tumor’s susceptibility to treatment.
The industry is pushing to make liquid biopsy the go-to method of collecting clinical DNA samples for oncology genotyping. Liquid biopsies can be taken at the point of diagnosis for routine monitoring during treatment, enabling practitioners to rapidly detect the appearance of resistance mutations that might indicate the need for a change of therapy. One day, liquid biopsy could even be used for preventative cancer screening in the general population. The ultimate goal is to facilitate earlier diagnosis and better treatment outcomes.
As with any new technology, using cfDNA for diagnosis via liquid biopsy has its challenges. Common technical hurdles include:
1. Sample handling
Liquid biopsy workflows involve additional sample handling steps. For example, clinical labs that have been handling robust FFPE blocks for many decades are now faced with processing blood samples, which have shorter shelf lives and require multiple extraction steps. Each step must be properly validated to ensure it does not introduce errors into the final results.
2. Reliability of results
Liquid biopsy assays must operate at much lower limits of detection than previous FFPE-based sequencing. As a result, the technology needs to be rigorously tested to ensure it can accurately call variants down to between 0.1–5 percent allele frequency without calling false positives.
3. Sample variability
Human plasma naturally displays high lot-to-lot variability, making it difficult to control and implement a consistent protocol for your diagnostic assay. Inconsistencies in the clinical blood draw and immediate blood storage process, which can vary between phlebotomists and hospitals, can introduce further sample variation. Controlling for this variation and introducing a consistent protocol is essential for the success of wide-scale liquid biopsy adoption.
The two big challenges
1. Limit of detection and false positive error rates
A key challenge in using cfDNA to detect cancers early is the extremely low quantities of cfDNA in patients’ blood. So how can we be confident in our lower limit of detection and ensure that we’re not seeing false positives? The answer: an appropriate reference standard. Using a reference standard with a range of precisely defined allelic frequencies can help determine a true limit of detection and reduce the risk of false positives.
In this example dataset, the reference standard informs the user that i) the reliable limit of detection for this cfDNA assay is 1 percent allelic frequency, and ii) they are calling a false positive for NRAS A59T.
When you run a reference standard before a patient sample, you can be sure of the limit of detection for your assay. It also allows pipeline optimization; you can recalibrate and amend your workflow to counter any false results, which gives you confidence when handling real patient samples.
2. The variability and instability of human plasma
Using human plasma as a control for your cfDNA assay comes with numerous challenges (see Table 1). Yes, human plasma matches your patient sample behaviors, but this does not always outweigh the challenges that come with using it as a reliable control for diagnosis.
How does this impact performance? In one experiment, we investigated the stability of our cfDNA spiked into either human or synthetic plasma over a 60-week period. Results show that the amount of cfDNA that could be extracted (~40 percent average extraction efficiency) and the ALK1 gene copy number as determined by ddPCR are similar in both human and synthetic plasma after 60 weeks of storage at -80°C (see Figure 1). The results demonstrate that our synthetic plasma behaves like real human plasma and can thus act as a suitable cfDNA reference standard matrix material.
Our approach to testing:
1. 400 ng of cfDNA was spiked into 1 mL of human or synthetic plasma and stored at -80˚C.
2. cfDNA was extracted using a Circulating Nucleic Acid kit (Qiagen); extraction efficiency was measured with Qubit BR Reagents (Molecular Probes).
3. Total AKT1 gene copies were quantified by ddPCR (Biorad).
Take control of your workflow
Having well-characterized cell line-derived reference standards that closely mimic real patient samples, with clinically relevant variants defined by a gold standard mechanism like droplet digital PCR (ddPCR), allows new liquid biopsy assays to be properly validated. Users can:
- check that their workflows accurately detect all of the variants in the control material at the correct allele frequencies without calling false positives
- validate and control for the introduction of errors during the DNA extraction procedure
- ensure that the design of their liquid biopsy sequencing assay functions effectively with no amplicon dropout (liquid biopsy assays need to sequence from smaller fragments of DNA than was previously required in fresh tissue or FFPE assays)
Horizon has developed a range of cell line-derived cfDNA reference standards to help develop, optimize, monitor, and control the accuracy of new patient tests. These materials contain a range of actionable variants in key cancer genes at well-characterized allele frequencies as determined by ddPCR. The variants are located within genomic DNA and have an average fragment size of 160 bp.
Find out more at tp.txp.to/horizon/cfDNA
Our cfDNA material in synthetic plasma helps users to monitor the entire liquid biopsy workflow from DNA extraction to interpretation of results, giving labs confidence in the accuracy of their test.
Find out more at tp.txp.to/synthplasma
The ability to examine and support cancer patients using liquid biopsy is hugely exciting. It promises to make genetic analysis more accessible with only a simple blood draw, and it encourages more frequent testing in all aspects of cancer management – pre-disease preventative monitoring, diagnosis, treatment, tumor evolution, resistance management, and long-term remission surveillance and check-up. For both laboratory professionals and the patients they serve, liquid biopsy with appropriate reference standards is the way to a brighter future.