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.
Highlights
The recent revolution in massively parallel DNA sequencing is unravelling the genomic basis of primary cancer[1] and providing important examples of genomic evolution and the hierarchal order of cancer mutations[2], convergent evolution[3], and the development of drug resistance[4]
CfDNA is a poor template for multiple displacement amplification (MDA)[17], even though MDA is proficient for single-cell analysis of single genomes (~6.6 pg) comprising long and intact genomic DNA
Following PlasmaClip-based sample extraction and WG-RCA, the concordance of somatic mutation detection was evaluated by droplet digital PCR (ddPCR) between fingerstick and venous cell-free DNA (cfDNA) samples
Summary
The recent revolution in massively parallel DNA sequencing is unravelling the genomic basis of primary cancer[1] and providing important examples of genomic evolution and the hierarchal order of cancer mutations[2], convergent evolution[3], and the development of drug resistance[4]. For some patients, yields of cfDNA from milliliters of blood may not be sufficient to support workflows that require high amounts of template, such as DNA sequencing and PCR with technical replicates These volumetric and concentration demands often present a challenge to existing liquid biopsy paradigms. We have previously shown that targeted pre-amplification of a stretch of cfDNA containing a mutation enables generation of sufficient template amounts for droplet digital PCR (ddPCR), while preserving the linearity of mutant allele detection[15]. This targeted PCR-based approach is low throughput and multiplexable for only a few known mutations. This study provides a clinical proof-of-concept for fingerstick-detection of somatic variants from advanced cancer patients, which upon further development such as improvement in allele frequency quantification may enable simpler and less-invasive methods for longitudinal or theranostic surveillance of metastatic cancer
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