Guide to ctDNA Use in Molecular Diagnostics

An important and rapidly evolving advance in cancer diagnostics and surveillance is circulating tumor DNA (ctDNA) profiling. This approach offers a minimal invasive technique for measuring diagnostically significant tumor-derived markers from blood, or other clinical liquid biopsy samples. ctDNA profiling can be used for cancer detection, therapeutic strategy, monitoring therapeutic response, and post-treatment surveillance. It represents an emerging alternative to tumor biopsy, especially when such biopsy is associated with significant risk, the tumor tissue is poorly accessible, or repeat sampling is needed to monitor and tailor treatment.

ctDNA diagnostic technology

ctDNA is the fragmented freely circulating DNA that is derived from cancer tissues. It can be present at very low levels and suffers from a relatively short half-life (30 minutes to 2 hours). Recent advances in digital PCR and next-generation sequencing (NGS) have helped accelerate the development and adoption of ctDNA diagnostics. Techniques used for detecting and analyzing ctDNA include:

Droplet digital polymerase chain reaction (DDPCR)—uses established water-in-oil emulsion droplet technology to permit quick turnaround, scalability, and a high sensitivity profile. The technology only permits detection of characterized sequences and known mutations and is not adaptable for multiplexing across multiple targets.

Beads emulsion, amplification, and magnetics (BEAMing)—an adaption of DDPCR that combines digital PCR with magnetic beads and flow cytometry to deliver clinical assays for the accurate detection of ctDNA. Similar to DDPCR it provides high sensitivity detection of known alterations.

Tagged-amplicon deep sequencing (TAm-Seq)—allows targeted sequencing across the whole genome to detect mutations and can also be scrutinized for new or uncharacterized alterations and mutations. Parallel singleplex reactions derived from the same clinical sample permit profiling across multiple loci.

Cancer personalized profiling by deep sequencing (CAPP-Seq)—utilizes large genomic libraries combined with individual patient sample sequence signatures to identify alterations within ctDNA. The technology is driven by selector probes, the design of which is crucial to identify recurrent mutations in a particular cancer type using publicly available NGS data.

Whole exome sequencing (WES)—permits the comprehensive analysis and characterization of potentially all tumor mutations through sequencing of all protein-encoding genes within the genome. The focus on exome elements has the advantage of reduced costs compared to whole genome sequencing.

Whole genome sequencing (WGS)—delivers comprehensive sequencing profiles across the entire tumor genome. Although more expensive than WES, WGS provides information on non-coding regions, including untranslated regions, introns, promoters, regulatory elements, non-coding functional RNA, repetitive regions, and mitochondrial genomes, which make up 98% of the human genome.

Advantages of ctDNA profiling

The minimally invasive sampling of patient blood (or other liquid biopsies) enables a rapid and cost-effective route to clinically enabling data. The appeal of the technology is rooted in the potential applicability across the whole process of cancer diagnosis and management, to aid the delivery of precision therapy and maximize healthcare impacts. ctDNA profiling not only brings the potential for early detection, and improved outcomes, but benefits from the option of frequent sampling, which allows the therapeutic response to be monitored and adapted. Crucially, the turnaround of analyzed data is compatible with clinical management cycles and is likely to improve further as the technology becomes even more established.

Disadvantages and limitations of ctDNA profiling

One limitation of the approach is the dependency on a single type of analyte, ctDNA, which needs to be abundant enough for detection and analysis. The required quantities can present a challenge in detection of early-stage cancers, where therapeutic intervention can have the biggest impact.

Additionally, the ctDNA-derived cancer profile needs to be representative of the primary tumor; this issue of concordance has been the focus of significant research, with degree of concordance reported from 50–95%. Where discrepancies exist, these may be due to factors including the site of ctDNA shedding into bloodstream, suppression of ctDNA by drug treatment, and comparative tissue sample from a small and poorly representative tumour section.

Clinical utility

As outlined, the diagnostic power of ctDNA profiling promises to significantly impact the clinical management of cancer. From early detection, through cancer characterization, informing treatment strategy and monitoring treatment response, the technology is well placed to optimize and personalize cancer management for improved patient outcomes. A number of ctDNA diagnostic kits have now achieved FDA approval; the forerunners are FoundationOne Liquid CDx from Foundation Medicine, and Guardant360 CDx from Guardant Health, which can sequence 311 genes and 55 genes, respectively. Both these assays are approved for detection of defined mutations in specific cancer types to help inform treatment selection and both rely on hybridization to capture ctDNA fragments for genes of interest from plasma for subsequent sequencing analysis. The profiling is performed on 2x10mL blood samples and returns results 1–2 weeks following sample receipt.

Outlook

The concept of liquid biopsy is still in its relative infancy but has already opened up new avenues in cancer management.

Next generation sequencing is now high throughput, sensitive, specific, and relatively cost effective and is therefore likely to further establish its role in mutation profiling of ctDNA and cancer diagnostics.

A growing body of data supports the potential of other non-blood-based liquid biopsy approaches (saliva, urine, spinal fluid) to further the diagnostic utility of ctDNA.

The concept of liquid biopsy ctDNA analysis has provided novel opportunities for cancer diagnostics and precision therapy. Further clinical trials and technology advancements will further extend this field to cement its role in cancer management.

References

Lin C, Liu X, Zheng B, Ke R, Tzeng CM. Liquid Biopsy, ctDNA Diagnosis through NGS. Life (Basel). 2021 Aug 28;11(9):890.  

Said R, Guibert N, Oxnard GR, Tsimberidou AM. Circulating tumor DNA analysis in the era of precision oncology. Oncotarget. 2020 Jan 14;11(2):188-211. .

Adashek JJ, Janku F, Kurzrock R. Signed in Blood: Circulating Tumor DNA in Cancer Diagnosis, Treatment and Screening. Cancers (Basel). 2021 Jul 18;13(14):3600. 

Keller L, Belloum Y, Wikman H, Pantel K. Clinical relevance of blood-based ctDNA analysis: mutation detection and beyond. Br J Cancer. 2021 Jan;124(2):345-358.