Pathophysiology of ctDNA Release into the Circulation and Its Characteristics: What Is Important for Clinical Applications

Abstract

The clinical implications of being able to accurately detect tumor-derived DNA in the circulation, termed circulating tumor DNA (ctDNA), could be enormous. Already, a plethora of clinical applications is under validation that include detection of minimal residual disease and predicting recurrence, monitoring response and resistance to treatment, identifying targets for therapies, and early detection. ctDNA is only a fraction of the total cell-free DNA (cfDNA) which confounds its detection and sometimes conceals its properties. To use ctDNA as a cancer biomarker with confidence, we need to understand its nature. Its characteristics, including size, half-life, and amount, are critical for the development of tests for its detection and discrimination from the rest of the cfDNA. Technological advances have enabled the detection and quantification of individual fragments of cfDNA, which is pivotal for clinical applications. Understanding the causes, the source of and the mechanisms of release of ctDNA are important for the interpretation of test results. Despite the many advances in understanding the nature and biology of ctDNA, we do not yet have a clear appreciation of the processes that govern its presence and levels in the circulation. ctDNA is not detectable in the blood of every cancer patient, and there is not a directly proportional relationship to tumor type, size, or stage. It is not clear if the lack of correlation with these specific clinical parameters is strictly due to technical or biological challenges. Better understanding of the pathophysiology of ctDNA is therefore important for the improvement of clinical applications and interpretation of their results.

Epilogue

ctDNA is no longer buried in the annals of molecular research. The field has come long way, and clinical applications utilizing ctDNA have even gained FDA approval. ctDNA detection and its applications are moving fast. However, mechanisms of release and/or increase of ctDNA in the circulation remain somewhat ill-defined; it is still not clear if some tumors do not shed ctDNA, or that the analytical methods are not sensitive enough. Can such knowledge help us to safely and transiently increase the levels of ctDNA, so we can detect it more efficiently?

There are several proposals to increase sensitivity, including collecting more blood or interrogating larger areas of the genome. Each has advantages, but also disadvantages. Some of them are practical, and some based on biology. More blood certainly will increase sensitivity, but may not be acceptable in some situations and result in reduced compliance. Large genome areas could provide the advantage of having multiple trials to identify a signal and potentially avoid stochastic events that can be missed when a single mutation is used as the biomarker. On the other hand, this approach will increase background errors and create issues with the interpretation of the results. The false discovery rate increases with increased numbers of bases included in the analysis as well (Wan et al. 2017). In addition, such an approach might rely on the analysis of non-driver mutations making the interpretation of the results difficult, especially in older individuals who already might harbor cfDNA detectable mutations that are not of cancer origin. Such approaches assume that the issue lies in the inefficiency of the analytical approach to capture and interrogate fragments with genetic alterations derived from cancer cells. However, this may not be always the case, or the desired sensitivity may not be achievable within acceptable levels of specificity.

To this end, other approaches are being explored. We mentioned earlier the possibility of combining somatic mutations and epigenetic changes as biomarkers for the detection of cancer. In a study, mentioned earlier, the sensitivity in detecting head and neck cancers varied based on the anatomical location of the cancer. To test the synergy between different bodily fluids in increasing the sensitivity of detecting mutations, both saliva and blood were collected from people with head and neck cancer. The sensitivity of detecting cancers present in the oral cavity and hypopharynx was 100 and 67%, respectively, when DNA isolated from saliva was used, while the sensitivities were 80 and 100% when ctDNA prepared from blood was used (Wang et al. 2015a). Similar results have been observed for ovarian cancer in samples collected from Pap smears and plasma from the same individuals (Wang et al. 2018). While all these combinations include tumor-derived DNA, a recent study explored the combination of ctDNA and protein markers for the early detection of cancer, providing very encouraging results (Cohen et al. 2018). We can imagine that other types of analytes, like metabolites, could augment sensitivity when combined with ctDNA.

There are many studies trying to address the issues associated with ctDNA detection. But, there are not any systematic studies yet to address if location within an organ, vascularization, or the number of mitotic figures correlates better with the amount of released ctDNA. The combination of improved methods for the detection of cancer and improved understanding between the relationship of ctDNA release and the pathological state of the cancer could help in providing better quality information for the management of cancer patients, by providing more significant quantitative and qualitative measurements of ctDNA.

An intersection of current knowledge, technical capabilities, and the requirements of clinical applications (some requiring superb sensitivity and some requiring superb specificity), even with our current status of incomplete knowledge, could help develop better ways of managing cancer patients. Detecting ctDNA for clinical applications should not be viewed in the absence of other parameters or as the sole determinant of a clinical decision. Studies, especially prospective, with accurate clinical information about the participants and their tumors, performed under IRB-approved protocols, will reveal relationships between ctDNA, its potential synergy with other analytes or other modalities, the status of the patients, and tumor characteristics. Deeper investigation into the answered questions about ctDNA will help us relate fundamental biological questions to real clinical situations.