An international team has reported findings from an independent assessment of five commercially available ctDNA assays as a method to diagnose and monitor cancer.
When cancer cells undergo apoptosis or necrosis, they release fragments of DNA into the circulatory system. These circulating tumour DNA (ctDNA) fragments can harbour various somatic mutations from their tumour of origin and their abundance typically correlates with tumour size and stage. As a result, ctDNA can act an accessible biomarker to inform cancer detection, stratify cancer types and patients, and monitor the effects of treatment and potential relapse.
Assays that measure ctDNA have several advantages over current methods. In general, ctDNA analysis is fast, cheap and minimally invasive and can be performed serially. In addition, unlike tissue biopsy, ctDNA assays can identify evidence of unknown lesions. This can enable the detection of minimal residual disease after treatment or even amongst healthy populations.
Nonetheless, ctDNA sequencing assays face several major technical challenges. For example, cell-free DNA exists as small fragments and at low concentrations. Therefore, the detection of rare somatic mutations is highly challenging. In addition, ctDNA sequencing assays can be affected by a range of experimental variables and artefacts, such as extensive PCR.
Evaluating available ctDNA assays
Despite the existing challenges, experts are developing ctDNA assays with increasing resolution and clinical adoption is already underway. As a result, it is vital that the community understand the sensitivity, accuracy and reproducibility of these assays and any potential variables that may impact their analytical performance.
In this study, published in Nature Biotechnology, researchers from 12 participating laboratories across Europe, Asia and the United States evaluated the performance of five industry-leading ctDNA assays. These assays were from Roche Sequencing Solutions, Illumina, Integrated DNA Technologies, Burning Rock Dx and Thermo Fisher Scientific. The team specifically evaluated each stage of the ctDNA sequencing workflow.
The researchers found that all assays could reliably detect ctDNA mutations above 0.5% variant allele frequency. Below this limit, the team found that the detection rate became unreliable. It also varied widely between assays, particularly when the researchers had limited input material. They also found that missed mutations were more common than incorrect candidates. This reinforces that reliably sampling rare ctDNA fragments is a key challenge for ctDNA assays.
This evaluation serves to inform best practice guidelines and provides a key resource for precision oncology. The team hope that their findings clear a path for more advanced clinical trials of ctDNA assays.
Image credit: By Mohammed Haneefa Nizamudeen – canva.com