Circulating Tumor DNA Easier to Spot with Size Selection

A bioRxiv preprint from scientists at Cancer Research UK and Cambridge University Hospitals offers a look at how DNA size selection can be used to enhance results of circulating tumor DNA studies. Their analysis indicates that adding a simple sizing step prior to sequencing can provide important insight about tumor genetics from liquid biopsies.

Selecting Short DNA Fragments In Plasma Improves Detection Of Circulating Tumour DNA” comes from lead author Florent Mouliere, senior author Nitzan Rosenfeld, and collaborators. The researchers note that an ongoing challenge in analyzing ctDNA — an increasingly important marker of cancer progression — is detecting these rare fragments amid a background of much more common cell-free DNA from healthy cells. “In patients with advanced cancers, the median concentration of ctDNA can reach 10% or more of the total cfDNA, but this fraction is much lower in earlier stage cancer, and ctDNA may rapidly decrease following initiation of systemic treatment or surgery,” the authors write. “Recent observations that ctDNA fragments may be shorter than non-tumour cfDNA in plasma has led to suggestions that these differences may be exploited to enrich for the tumour-specific signal in plasma DNA.”

For this project, the team aimed to assess the effectiveness of targeting ctDNA by size in an NGS experimental workflow. Since healthy cell-free DNA is known to peak around 167 bp, the scientists targeted fragments ranging from 90 bp to 150 bp using the PippinHT automated DNA size selection platform. In 26 plasma samples collected from 13 patients with advanced ovarian cancer, the scientists determined that adding a size-selection step “yielded enrichment of mutated DNA fraction of up to 11-fold,” they report. “This allowed identification of adverse copy number alterations, including MYC amplification, otherwise not observed.”

Somatic copy number aberrations (SCNAs) that were detected after size selection, but not in a control workflow lacking size selection, included important cancer-associated genes such as NF1 and PARP2, in addition to MYC. “More SCNAs could be detected after size selection in 11/13 patients, and the absolute level of the log2ratio was significantly increased after size selection,” the authors note.

“These results demonstrate a proof-of-principle that by a simple step of filtering of cfDNA and selection of shorter fragments, it is possible to increase the tumour DNA fraction in plasma cell-free DNA samples,” Mouliere et al. conclude, noting that their approach could work with any downstream NGS analysis method. “The compatibility of the cfDNA fragment size selection with wide-scale and sensitive genomic analysis could unlock the potential of liquid biopsies for the diagnosis of cancer at an earlier stage, and for the detection of minimal residual disease.”

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