Factors Affecting Plasma DNA Analysis Evaluated

There is vast potential in precision-medicine methods of both detecting and monitoring disease by looking for indications of cancer mutations in cell-free DNA (cfDNA), circulating in the blood. However, there are many factors that can significantly alter these samples as they are collected and analyzed.

Circulating tumor DNA (ctDNA) analysis relies on detection and quantification of these mutations, against a background of cfDNA contributed by peripheral blood cells and other tissues. Total cfDNA and tumor-specific ctDNA levels in plasma vary considerably across cancer patients, cancer types and disease stages as well as during longitudinal follow-up of each patient.

Scientists from the Translational Genomics Research Institute (Phoenix, AZ, USA) designed a multiplexed droplet digital polymerase chain reaction (ddPCR) assay targeting nine single copy genomic loci. They prepared digital PCR reactions and performed thermocycling using DNA Engine Tetrad 2 and they measured droplet fluorescence using RainDrop Digital PCR Sense Instrument and analyzed results using accompanying software. To evaluate whether their ddPCR assay performed as expected, they analyzed known quantities of sheared DNA fragments with predetermined size and compared the results with expected theoretical yields.

Healthy volunteer and clinical plasma samples included in comparison of immediately processed and archived samples were collected. Samples from seven independent clinical cohorts were included in this analysis, including patients diagnosed with melanoma, cholangiocarcinoma and rectal cancer as well as healthy volunteers. For assessment of sequencing performance guided by ddPCR results, the team quantified cfDNA from commercially obtained control plasma samples using ddPCR and prepared whole genome sequencing libraries using ThruPLEX DNA-Seq.

The scientists evaluated effects of DNA extraction methods and blood collection tubes on cfDNA yield and fragment size, and developed a multiplexed droplet digital PCR (ddPCR) assay with five short and four long amplicons targeting single copy genomic loci. Using this assay, they compared seven cfDNA extraction kits and found cfDNA yield and fragment size vary significantly. They also compared three blood collection protocols using plasma samples from 23 healthy volunteers (EDTA tubes processed within one hour and Cell-free DNA Blood Collection Tubes processed within 24 and 72 hours) and found no significant differences in cfDNA yield, fragment size and background noise between these protocols. In 219 clinical samples, cfDNA fragments were shorter in plasma samples processed immediately after venipuncture compared to archived samples, suggesting contribution of background DNA by lysed peripheral blood cells.

Muhammed Murtaza, MD, PhD, the senior author of the study, said, “In order for us to rely on sequencing results and evidence of cancer mutations from these samples and to make valid recommendations for treating physicians, we must ensure they have been collected and processed appropriately. We developed this new quality-control assay to ensure we can confirm reliability using a very small volume of a patient’s blood sample.” The study was published on May 9, 2018, in the journal Scientific Reports.

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Translational Genomics Research Institute