ctDNA and Cancer: Diagnostics, Treatments, and Monitoring

Cell-free DNA (cfDNA) was first detected in human blood samples as long ago as 1948,¹ although it has been cfDNA derived from tumors, or circulating tumor DNA (ctDNA), that has gained significantly in promise over the past decade, namely due to its potential to provide minimally invasive testing. ctDNA, or cell-free molecules of DNA shed by tumor cells into the bloodstream that circulate in the body, are detectable in several liquid biopsy sample types such as blood, plasma, urine, and cerebrospinal fluid (CSF). When analyzed with genomic technologies such as PCR, exome sequencing, and whole-genome sequencing, they offer many potential advantages over traditional biopsies.

“ctDNA is minimally invasive, able to be collected serially over multiple timepoints throughout treatment, and may provide a more comprehensive snapshot of the genetic landscape of a patient’s cancer,” advises Stephen Wong, senior research officer at the Dawson Laboratory, Peter MacCallum Cancer Centre. “Importantly, the use of ctDNA analysis is emerging as an important tool in many facets of oncology with applications in early detection, minimal residual disease detection, molecular profiling, and the characterization of treatment resistance.”

The potential for ctDNA to transform both research and clinical approaches is vast, but to make it a reality several challenges need to be addressed, including the need for standardization. This article will look at some of these challenges, as well as current and future approaches that could help realize the use of routine ctDNA testing for cancer care.

Assessing clinical potential

Among the numerous potential clinical applications of ctDNA that is already making an impact is in molecular profiling. Wong comments: “While tumor biopsies will never be replaced, ctDNA analysis provides a useful and important alternative strategy to identify actionable genomic changes to guide treatment, particularly when there is insufficient tumor material available for testing or when tumor tissue testing has failed for various reasons.” The Dawson group, which is currently studying the use of ctDNA for a wide variety of cancer types, is also working on using ctDNA to help define tumor response kinetics and the mutational landscape following treatment.

Aldo Mele, Senior Scientist 2 at Horizon Discovery, a Revvity company, thinks that ctDNA analysis through liquid biopsy has also enhanced our capabilities in precision medicine. “This has led to the development of pan-cancer tests and the ability to detect molecular biomarkers when primary tumor location is not known. It also allows for the detection of disease relapse or minimal residual disease in cancer patients, as well as the ability to adapt therapies to the needs of an individual patient.”

Besides being simpler and often painless in terms of biopsy collection, ctDNA also could be used for broader surveillance in otherwise healthy populations, according to Amar Kamath, VP and General Manager, Diagnostics at Revvity.

ctDNA sources and detection systems

ctDNA can be derived from a variety of different sources, whether directly from tumor tissue as well as blood, CSF, saliva, and urine. The detection sensitivity from these various sources can vary, however, and can depend upon the specific cancer as well as the tissue type(s) involved. An example of this is with primary brain tumors, including gliomas, central nervous system lymphomas, and some pediatric solid tumors, where it has been reported that CSF has higher sensitivity when compared with peripheral blood.^2,3 For tumors of the upper aerodigestive tract, saliva, sputum, or pleural effusions have been found to be effective alternatives to blood.^4,5

New research indicates that blood-based liquid biopsies using ctDNA are well positioned for clinical adoption as blood is collected as part of routine cancer patient clinical management. “There is currently less evidence for the clinical use of other types of liquid biopsies, such as urine or cerebrospinal fluid, compared to blood-based assays, although for certain cancer types they may have distinct advantages,” Wong comments.

Kamath meanwhile points out urine’s advantage: “A larger amount can be collected and used from a patient on a daily basis—compared to a source like blood where the daily sample volume is restricted—making it suitable for monitoring purposes.” However, it should be noted that with urine, ctDNA concentration is comparatively low compared to other sample types.

In addition to the complexities involved in using different sample types for ctDNA analysis, there is a plethora of different detection approaches available. “The most suitable methodology really depends on the research question or clinical application. This includes digital PCR, which provides detection at high sensitivity but only for single loci or a limited number of mutations, through to next-generation sequencing (NGS) approaches including targeted and whole-genome sequencing with much broader genomic coverage,” advises Wong.

Mele notes that, as whole-genome NGS methods continue to evolve and costs continue to fall, they have the potential to cast the widest net to capture and identify relevant pan-cancer biomarkers.

Addressing barriers to adoption—standardization is key

With so many advantages to using ctDNA for cancer diagnosis, treatment, and monitoring, it’s also important to acknowledge and understand the series of challenges that it also faces, which could slow down and/or impede its translation and reliability for routine clinical use.

“There continues to be technical challenges associated with the analysis of ctDNA with an ongoing need to improve the sensitivity of detection with new technologies and approaches,” comments Wong. Kamath agrees: “Cancer-related mutations can be present in very low percentages of extracted total ctDNA.”

Standardization was a key focus for the experts interviewed for this article. Wong elaborates: “There is a need to demonstrate the clinical utility of ctDNA in various clinical applications, but also a requirement to harmonize and standardize methodologies between laboratories. Standardizing the wide range of assays currently available will help make the interpretation and reporting of results more consistent between different laboratories.”

The good news is that there are a number of initiatives trying to promote the use of ctDNA in the clinic by establishing more widely recognized and/or international standards, and providing guidelines for data interpretation, including the European Liquid Biopsy Society, CANCER-ID, and the European Committee for Standardization based in Europe, and in the U.S., BloodPAC and the FNIH Biomarkers Consortium.

The future outlook is positive

While the current prospective of ctDNA is significant, its future is even more promising. Mele predicts an important role for machine learning alongside the rise of ctDNA’s routine use in the clinic: “A combination of multi-analyte assays with algorithmic analysis will allow the application of machine learning by combining multiple biochemical or molecular markers, identified through liquid biopsy (ctDNA, cfRNA, protein), with patient demographics and clinical information to predict patient diagnosis and prognosis,” he states.

Another key area in current ctDNA research involves epigenetics, where gaining an improved understanding of methylation patterns and nucleosome positioning, and how this affects ctDNA features and fragment size, could support the development of improved cancer detection methods. Circulating viral DNA, and its connection to certain cancers, is also currently being explored.¹ “We still have much to learn about the biology and features of ctDNA in different clinical contexts,” Wong aptly concludes.

References

1. Keller, L., Belloum, Y., Wikman, H. et al. Clinical relevance of blood-based ctDNA analysis: mutation detection and beyond. Br J Cancer 124, 345–358 (2021). 

2. Boire, A., Brandsma, D., Brastianos, P. K., Le Rhun, E., Ahluwalia, M., Junck, L. et al. Liquid biopsy in central nervous system metastases: a RANO review and proposals for clinical applications. Neuro Oncol. 21, 571–584 (2019).

3. Abbou, S. D., Shulman, D. S., DuBois, S. G. & Crompton, B. D. Assessment of circulating tumor DNA in pediatric solid tumors: the promise of liquid biopsies. Pediatr. Blood Cancer 66, e27595 (2019).

4. Cristaldi, M., Mauceri, R., Di Fede, O., Giuliana, G., Campisi, G. & Panzarella, V. Salivary biomarkers for oral squamous cell carcinoma diagnosis and follow-up: current status and perspectives. Front. Physiol. 10, 1476 (2019).

5. Ribeiro, I. P., de Melo, J. B. & Carreira, I. M. Head and neck cancer: searching for genomic and epigenetic biomarkers in body fluids—the state of art. Mol. Cytogenet. 12, 33 (2019).