Techniques of using circulating tumor DNA as a liquid biopsy component in cancer management

Abstract

Precision medicine in the clinical management of cancer may be achieved through the diagnostic platform called “liquid biopsy”. This method utilizes the detection of biomarkers in blood for prognostic and predictive purposes. One of the latest blood born markers under investigation in the field of liquid biopsy in cancer patients is circulating tumor DNA (ctDNA). ctDNA is released by tumor cells through different mechanisms and can therefore provide information about the genomic make-up of the tumor currently present in the patient. Through longitudinal ctDNA-based liquid biopsies, tumor dynamics may be monitored to predict and assess drug response and/or resistance. However, because ctDNA is highly fragmented and because its concentration can be extremely low in a high background of normal circulating DNA, screening for clinical relevant mutations is challenging. Although significant progress has been made in advancing the detection and analysis of ctDNA in the last few years, the current challenges include standardization and increasing current techniques to single molecule sensitivity in combination with perfect specificity. This review focuses on the potential role of ctDNA in the clinical management of cancer patients, the current technologies that are being employed, and the hurdles that still need to be taken to achieve ctDNA-based liquid biopsy towards precision medicine.

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

“…This step enriched the cfDNA sample, allowing ddPCR and sequencing to become more feasible due to the higher amount of DNA template required. Numerous NGS technologies such as BEAMing and Safe-SeqS have been used to detect known breast cancer mutations using ctDNA with sensitivity higher than 99% 30 . Our own studies have shown for primary patients that recurred, patient specific ctDNA profiling detected molecular relapse up to 2 years ahead of clinical relapse with 89% sensitivity and 100% specificity 31 .…”

Low Abundance of Circulating Tumor DNA in Localized Prostate Cancer

“…To address this challenge, we reconstructed tumor phylogenies from genome and exome sequencing of tissue in order to select alleles representing the major subclones that would be present at the time of surgery and further mediate relapse. Prior to executing these experiments, we developed and tested a bespoke patient-specific, allele-specific sequencing assay that satisfied requirements for reproducibility, accuracy, sensitivity, and specificity [16,30,31]. This assay consistently detected spiked-in alleles and showed 100% concordance to unbiased whole exome sequencing of the same sample at very high coverage.…”

Performance of the OncomineTM Lung cfDNA Assay for Liquid Biopsy by NGS of NSCLC Patients in Routine Laboratory Practice

“…Molecular barcoding is one of the possible solutions when dealing with challenging clinical samples since unique molecular tags, also known as unique molecular index (UMI), are added into every DNA molecule, prior to library amplification [5][6][7][8][9][10]. This has the great advantage to increase the sensitivity by keeping track of the DNA molecules intercepted, meanwhile reducing polymerase chain reaction (PCR) and/or sequencing artefacts [11][12][13][14], also when compared to amplicon-based NGS [15].…”