Ultrasensitive circulating tumor DNA analysis enables precision medicine: experimental workflow considerations

Abstract

Introduction: Circulating tumor DNA (ctDNA) has become a relevant biomarker in cancer management, allowing tumor assessment through analysis of minimally invasive liquid biopsies. Applications include screening, diagnostics, monitoring of treatment efficacy and detection of minimal residual disease as well as relapse. The potential of ctDNA analysis is significant, but several biological and technical challenges need to be addressed before widespread clinical implementation. Areas covered: Several clinical applications where ctDNA analysis may be beneficial require detection of individual DNA molecules. Consequently, to acquire accurate and informative data the entire workflow from sampling to final data interpretation needs to be optimized. In this review, we discuss the biological and technical challenges of ctDNA analysis and how preanalytical and analytical approaches affect different cancer applications. Expert opinion: While numerous studies have demonstrated the potential of using ctDNA in cancer applications, yet few reports about true clinical utility exist. Despite encouraging data, the sensitivity of ctDNA analyses, i.e. the probability to detect presence of cancer in liquid biopsies, is still an issue. Analysis of multiple mutations in combination with simultaneous assessment of other analytes is one solution. Improved standardization and guidelines will also facilitate the introduction of ctDNA analysis into clinical routine.

Circulating Tumor DNA in Pediatric Cancer

“…This is because ddPCR employs absolute quantitation and end-point measurement of lower concentrations of starting material sample input of 1 ng/reaction is possible with varying levels of multiplexing ( Fitarelli-Kiehl et al, 2018 ) and Poisson statistics. In a study by Klega et al, ddPCR paired with NGS in pediatric Ewing’s sarcoma showed that ctDNA levels correlate with treatment response and disease-specific biomarkers ( Klega et al, 2018 ) as ddPCR alone is not sufficient to identify all clinically relevant mutations and it is recommended to complement untargeted approaches ( Andersson et al, 2021 ).…”

“…Sensitivities can be limited by the dilution of fragmented ctDNA with normal germline cfDNA ( Sherwood et al, 2016 ). Andersson et al discussed the potential for the lysis of normal blood cells, which may contaminate the sample ( Andersson et al, 2021 ). Plasma samples must typically be centrifuged and separated within 1–4 h of collection.…”

Counting unique molecular identifiers in sequencing using a multitype branching process with immigration

“…1 involving six random inputs ξ 0 ∼ Ber(r 1 ), ξ 1 ∼ Ber(r 2 ), ξ (1) ∼ Ber(r 3 ), ξ (2) ∼ Ber(r 4 ), ξ (3) , ξ (4) ∼ Ber(r).…”

“…Finally, libraries are purified and sequenced. lower than 0.1% in clinically relevant samples [1,7,11]. The main source of sequencing noise is due to the polymerase induced errors that occur during library construction and sequencing [5].…”

Assessment of Circulating Nucleic Acids in Cancer: From Current Status to Future Perspectives and Potential Clinical Applications

“…The use of unique molecular identifiers (UMIs or UIDs), applied to this type of sequencing, has dramatically improved the results from such technologies. UMIs and UIDs are molecular barcodes, usually short random nucleotide sequences, that are ligated to amplicons or hybridized sequences during library preparation, allowing for a reduction in background signals, and correcting both DNA polymerase-induced errors and uneven amplification when used in combination with deep sequencing [ 65 , 66 ]. There is a wide range of NGS-based technologies performing targeted sequencing through the use of gene panels aimed at mutation detection.…”