Ko Tun Kiat
What can we learn by analysing cell-free DNA (cfDNA) from cancer patients?
What is cfDNA?
cfDNA is composed of mainly short (50bp-250bp) double-stranded DNA fragments. cfDNA can be extracted from body fluids (e.g., blood & urine) & is low in abundance (1-10ng/ml plasma) (1, 2). In normal individuals, most cfDNA is derived from dead leukocytes (1, 2). In cancer patients, a proportion of cfDNA is derived from cancer cells & is termed circulating tumour DNA (ctDNA). Depending on the tumour burden, the proportion of cfDNA from a cancer patient that is ctDNA can range from less than 1% to as high as 90% (2). Therefore, the total amount of cfDNA tends to increase with increased tumour size. Compared to tissue biopsy, the collection of body fluid is generally less intrusive, less costly & safer. Additionally, data from cfDNA may better reflect the molecular heterogeneity of a tumour that may have different clonal populations that are dispersed in several locations.
What are the potential applications of cfDNA in oncology?
The figure below highlights the potential applications of cfDNA as a cancer biomarker. For single sampling taken at a particular time-point or disease stage, the detection of known cancer-specific mutation & DNA methylation signatures can be applied to screening, early detection, diagnosis as well as to guide treatment. For serial sampling at different disease or treatment stages, one can monitor dynamic changes to cfDNA level & clonal evolution. Furthermore, serial sampling would allow detection of novel mutations & DNA methylation changes. This information can guide future treatment strategy as well as to provide insights on disease relapse & minimum residual disease.
What analysis platform is available to study cfDNA at Cancer Discovery Hub?
Currently, we provide 2 different analysis platforms that are based on next generation sequencing (NGS). The first platform detects SNV (single nucleotide variant) & short indels (insertions & deletions) and it is based on a customised targeted panel (ArcherDx® Liquidplex™ from Invitae). This platform interrogates the mutational status of 29 well-known oncogenes (e.g., KIT, EGFR & BRAF) & tumour suppressors (e.g., TP53).
The second platform is based on evaluating the DNA methylation profile of cfDNA. It is called cfMeDIP-seq (cell-free methylated DNA immunoprecipitation & high-throughput sequencing) (3). cfMeDIP-seq uses an anti-methylated cytosine antibody that can enrich for methylated cfDNAs. cfMeDIP-seq is designed for low DNA input (1-10ng) and that makes it suitable for studying cfDNA. cfMeDIP-seq is more sensitive than a mutation-based assay because a DNA methylation profile is more extensive and varied as there are more than 28,000 CpG islands in the human genome that can be methylated. cfMeDIP-seq has been successfully applied in early detection and stratification of a number of cancers (3-5).
1. Wan JCM et al. Liquid biopsies come of age: towards implementation of circulating tumour DNA. Nat Rev Cancer. 2017 Apr;17(4):223-238.
2. Corcoran RB, Chabner BA. Application of Cell-free DNA Analysis to Cancer Treatment. N Engl J Med. 2018 Nov 1;379(18):1754-1765.
3. Shen SY et al. Sensitive tumour detection and classification using plasma cell-free DNA methylomes. Nature. 2018 Nov;563(7732):579-583.
4. Nuzzo PV et al. Detection of renal cell carcinoma using plasma and urine cell-free DNA methylomes. Nat Med. 2020 Jul;26(7):1041-1043.
5. Nassiri, F. et al. Detection and discrimination of intracranial tumors using plasma cell- free DNA methylomes. Nat. Med. 2020 Jul;26(7), 1044–1047.
Dr Ko Tun Kiat
NGS Lead, Cancer Discovery Hub
National Cancer Centre Singapore