Liquid biopsies using ctDNA and CTC have been demonstrated to have potential for a diverse range of diagnostic, prognostic and treatment response monitoring applications. In the clinic, this could allow for an additional biochemical monitoring for patients who are at risk of treatment failure. For the purpose of research and treatment development, these markers are valuable to distinguish treatment accuracy and validate target acquisition particularly with regards to radiation therapy. However, a full understanding of the behavior of these biomarkers with respect to radiation treatment remains to be obtained . A preclinical model has the advantage of being able to control variables to allow for a homogenous cohort, which is essential to effectively demonstrate the effects of radiation on the levels of ctDNA and CTC.
As evident in our study, CTC detection, especially when only EpCAM capture was utilized, was only feasible when tumors were larger and more advanced in contrast to ctDNA. This is not surprising, as CTCs are thought to be harbingers of metastatic deposits in the “seed and soil” theory. Early and reliable detection has a significant utility to determine the aggressive nature of the primary tumor that may aid in treatment decisions. Our experience with CTC for the monitoring of tumor growth in this head and neck model shows an increased sensitivity when utilizing TxViva as a detection method in comparison to EpCAM only capture method. This is noticeable via earlier detection of rare cells, such as EpCAM, Her-2, PSMA and EGFR in the circulation as early as week 1 following inoculation. Elevations of CTC in weeks 2 and 3 further demonstrated a higher sensitivity profile compared to EpCAM only CTC capture methods.
To date, Cellsearch (which uses predominantly EpCAM detection) is the only FDA approved method of CTC enumeration and its sensitivity in detection of CTC has been challenged in several other publications. Techniques that incorporate other microfluidic systems or the use of isolation via size of epithelial tumor cells (ISET) filtration methods have been explored to improve detection of CTC. Direct comparisons between ISET and Cellsearch has shown a greater detection rate in other sites such as hepatocellular carcinoma, pancreatic cancer and non-small cell lung cancers [21,22,23]. In addition to ISET, there have been several other microfluidic chips such as spiral microfluidics and ClearCell Fx System that have also been evaluated for head and neck cancers [24, 25].
Clinically, the identification of CTC has gained significant attention over the past decades. It is hypothesized that CTC may be useful to determine radioresistance and further aid in prognostication of treatment outcomes. However, there is yet to be a reliable method for sensitive detection of these rare cells. Our results here show similar findings with the current literature in the limitations in sensitivity of CTC detection by Cellsearch.
For the purpose of ctDNA detection, we created a design for the treatment of early head and neck cancers using radiation as a single modality treatment. Each animal underwent a total of 40 Gy over 10 fractions, with a 2-day treatment break in the middle of treatment. This multi-fraction approach mimics current standard treatment of early head and neck cancers. As demonstrated in our results, there remains a good correlation between levels of ctDNA with overall tumor burden (Fig. 4A). In the context of early primary tumor, levels of ctDNA closely mirrored the primary tumor volumes in all but 1 rabbit. This was thought to be attributed to nodal metastasis, which corresponds to the transient increase in nodal volume despite the dramatic decrease in size in the tumor volumes. Despite this being an early stage tumor, it is established that up to 20–30% of early staged tumors may be associated with occult nodal metastasis . This would be consistent with our study cohort. Furthermore, the subsequent drop in ctDNA also corresponds to the decline in nodal volumes. We postulate that this may represent an abscopal effect of radiation which can explain the drop in tumor burden and hence the decline in ctDNA. Final histology (Fig. 3B) at 10 days following completion of radiation similarly shows no residual tumor in both primary site and lymph node.
Following treatment with radiation, plasma ctDNA was noted to begin its decline towards the third to fourth day of radiation treatment. Interestingly, all the rabbits demonstrated an acute elevation in plasma ctDNA at the beginning of the second cycle of radiation. It is our interpretation that this sudden surge of plasma ctDNA is likely from acute release of tumor DNA into the circulation following local tumor destruction. It is possible that this is potentiated after the 2 day treatment break due to the influx of macrophages and subsequent increase in phagocytosis, or possible restoration of vasculature within the tumor both of which are essential for tumor DNA’s escape into the circulation . Plasma ctDNA levels however quickly declines following completion of treatment, this is in contrast to imaging which often shows indeterminate features within the treated fields. This is reinforced with histological evidence of complete tumor resolution despite imaging features. In that aspect, ctDNA allows for greater accuracy in predicting complete tumor resolution compared to imaging and allows for a noninvasive method to establish complete clinical response in the early period following radiation treatment.
Plasma ctDNA proves to be a viable biomarker of tumor volume within this model. For the purpose of head and neck research, we postulate this may be of value to assess biological response of treatment within preclinical trials involving combination therapies and future radiation modalities. As this is an immunocompetent model, the possibilities of utilizing this model for combination therapy with immunotherapy and radiation guided by ctDNA levels makes for an interesting avenue to be explored.
From our study, plasma ctDNA declines rapidly following tumor eradication. As such, it may represent a more reliable determinant of complete therapeutic response and may be used as a surrogate in lieu of early post-treatment imaging. Plasma ctDNA may also allow for confirmation of target acquisition during radiation treatment, as evident by the surge of ctDNA seen during initiation of the second cycle. This marker may be further utilized in the context of advanced radiotherapy modalities, as it provides biologic feedback that confirms an on-target response following precision radiotherapy.
Liquid biopsies in the form of CTC and ctDNA have been proposed as an adjunct resource for cancer monitoring. These cancer markers allow monitor of disease activity on the biological front, thereby potentially alerting the clinicians to recurrent/residual disease prior to structural evidence that is picked up by imaging. Early studies have shown the utility of CTC detection in the setting of head and neck cancers in predicting pulmonary metastasis and has been shown prognostic values in patients with recurrent or metastatic head and neck cancers [28, 29]. The value of ctDNA in the setting of EBV related nasopharyngeal cancers and HPV derived oropharyngeal cancers further show great promise in some clinical trials. EBV DNA has shown value for prognostication to treatment response and preclinical detection of tumor recurrence, while detection of plasma HPV DNA has also been successful in assessment of tumor recurrence in oropharyngeal cancers [30,31,32]. These studies show the utility of liquid biopsies in patient care following radiation treatment. As such, better understanding of CTCs and ctDNA in the peri-treatment period is important to comprehend and correlate its post-treatment effects.
From this series of 7 animals, we attempted to evaluate the kinetics of both CTC and ctDNA in their response to radiation treatment. Certainly, there are limitations in drawing conclusions form such a small sample size however, as the aim was to achieve a qualitative analysis of liquid biopsy response to radiation, it was felt that a small cohort of animals would suffice. From our study conduct, it is clear that rabbits evaluated by ctDNA and CTC had different tumor sizes and different treatment regimes. This limitation was largely due to the need for greater tumor progression before CTC could be evaluated in the blood. As these tumors were significantly larger, these rabbits had a shorter follow-up period as they reached humane endpoints earlier than the ctDNA cohort.