Whole genome amplification of cell-free DNA enables detection of circulating tumor DNA mutations from fingerstick capillary blood

Abstract

The ability to measure mutations in plasma cell-free DNA (cfDNA) has the potential to revolutionize cancer surveillance and treatment by enabling longitudinal monitoring not possible with solid tumor biopsies. However, obtaining sufficient quantities of cfDNA remains a challenge for assay development and clinical translation; consequently, large volumes of venous blood are typically required. Here, we test proof-of-concept for using smaller volumes via fingerstick collection. Matched venous and fingerstick blood were obtained from seven patients with metastatic breast cancer. Fingerstick blood was separated at point-of-care using a novel paper-based concept to isolate plasma centrifuge-free. Patient cfDNA was then analyzed with or without a new method for whole genome amplification via rolling-circle amplification (WG-RCA). We identified somatic mutations by targeted sequencing and compared the concordance of mutation detection from venous and amplified capillary samples by droplet-digital PCR. Patient mutations were detected with 100% concordance after WG-RCA, although in some samples, allele frequencies showed greater variation likely due to differential amplification or primer inaccessibility. These pilot findings provide physiological evidence that circulating tumor DNA is accessible by fingerstick and sustains presence/absence of mutation detection after whole-genome amplification. Further refinement may enable simpler and less-invasive methods for longitudinal or theranostic surveillance of metastatic cancer.

A novel hotspot specific isothermal amplification method for detection of the common PIK3CA p.H1047R breast cancer mutation

“…In a similar way, Wang et al 28 used non-extendable primer blockers for allele-specific PCR detection of three mutations in cancer K-RAS, B-RAF, and EGFR with a limit of detection of a single copy. A different approach reported by Gyanchandani et al 29 , has been used to demonstrate amplification of cfDNA from liquid biopsies linked to metastatic BC without compromising allelic balance. This step enriched the cfDNA sample, allowing ddPCR and sequencing to become more feasible due to the higher amount of DNA template required.…”

Targeted deep sequencing revealed variants in cell-free DNA of hormone receptor-positive metastatic breast cancer patients

“…Exactly those deep sequencing qualities were met in all 40 cases used for variant analysis, since 18 million read fragments were sequenced on average in each sample, the mean coverage was 22,000×, and the UMI coverage was 6351. This high coverage enabled calling variants with low AFs and resulted in the identification of a high prevalence of variants, which has rarely been described before [20][21][22]. Many studies [2,[23][24][25] that described a low frequency of variants were conducted with a lack of high coverage, missing descriptions of the input amount or sequencing of hotspots rather than all exonic regions of a gene.…”

Ultra-low DNA input into whole genome methylation assays and detection of oncogenic methylation & copy number variants in circulating tumour DNA

“…We hypothesised that as the Illumina Infinium assay requires a whole genome (14) amplification (WGA) step, the theoretical input to this kit could encompass concentrations seen in ctDNA samples. We therefore aimed to study the effect of input concentration of DNA on assay performance.…”