Cell-Free DNA Kinetics in a Pre-Clinical Model of Head and Neck Cancer

Abstract

In cancer patients, circulating tumour-derived DNA (ctDNA) levels imperfectly reflect disease burden apparent on medical imaging. Further evaluation of ctDNA levels over time is needed to better understand the correlation with tumour growth and therapeutic response. We describe ctDNA kinetics within an orthotopic, immunocompetent preclinical rabbit model of local-regionally advanced head and neck squamous cell carcinoma (HNSCC). Monitoring primary tumour and metastatic lymph node volume by computed tomography (CT), we observed a correlation between ctDNA levels and tumour burden. We found that ctDNA detection could precede evidence of tumour on CT. Sensitivity and specificity of ctDNA detection in this model was 90.2% (95% C.I.: 76.9–97.3%) and 85.7% (95% C.I.: 67.3–96.0%), respectively. Rapid tumour growth followed by auto-necrosis and tumour volume contraction produced a spike in ctDNA levels, suggesting that viable tumour cells may be required for sustained ctDNA release. Following surgical resection, both ctDNA and total plasma DNA were correlated with recurrent tumour volume. Our results reveal the complex kinetic behaviour of ctDNA and total plasma DNA upon tumour growth or surgery. This pre-clinical model could be useful for future studies focused on elucidating mechanisms of ctDNA release into the circulation from primary and metastatic sites.

Life and death of circulating cell-free DNA

“…The estimated half-life of cfDNA in circulating blood varies from several minutes (e.g., 4 min after hemodialysis cessation) to 1-2 h. [103][104][105][106] Interestingly, clearance of fetal DNA from maternal blood occurs in a bi-phasic manner: first, a rapid phase occurs with a mean half-life of~10 min to 1 h; then, a second slow phase occurs with a mean half-life of~13 h. 107 The half-life of the ctDNA level after surgical tumor resection in a pre-clinical rabbit model of head and neck cancer was 23-52 min. 25 A serial analysis of ctDNA in patients with colorectal cancer showed a halflife of 114 min. 108 The short half-life of cfDNA is convenient for "real-time" analyses of cfDNA; it facilitates treatment response evaluations and dynamic tissue status assessments in various pathophysiological conditions; for example, tissue damage or regeneration.…”

“…23,24 Some studies suggested that analyses of cfDNA outperformed instrumental methods (such as computed tomography), required a lower tumor burden, and prolonged the time window for adopting clinical decisions. 23,25 Studies of cfDNA in oncology are predominantly devoted to clinical applications of cfDNA as a tumor biomarker. An association of cfDNA level with treatment outcome, low invasiveness of an assay, implementation of high-throughput techniques make liquid biopsy using extracellular DNA an attractive candidate for a routine test in cancer management.…”

“…16,56,65,76 Longer DNA fragments (i.e., >10 kb) are considered to result from necrotic cell death; for example, from cells in necrotized parts of tumors. 25 Experiments in vitro have demonstrated that the amount of extracted DNA fragments depended on the type of necrosis-inducing agent applied. 77,78 Moreover, blood sampling can affect the type of cell death; indeed, the collection of serum stimulates a release of necrotic DNA from blood cells; this mechanism could account for the higher levels of total cfDNA found in serum compared to levels found in plasma samples.…”

Cell-Free DNA and Circulating Tumor Cell Kinetics in a Pre-Clinical Head and Neck Cancer Model Undergoing Radiation Therapy

“…For the purpose of this study, plasma ctDNA was detected by means of a previously validated qPCR assay based on a 58-bp E6 assay derived from the E6 open reading frame (ORF). (14) From prior studies, the 58-bp E6 assay has shown to be sensitive enough to achieve robust detection of CRPV DNA sequences form as little as 3.7 pg of VX2 genomic DNA. Speci cities for this test also remain high showing no evidence in samples of rabbits that were not inoculated with VX2.…”

“…With our prior work in kinetics of ctDNA following surgery as well as a novel nanoparticle based detection of CTC in a preclinical model, we sought to develop a similar model to determine the kinetics of CTC and ctDNA in response to radiation therapy. (13,14) The establishment of such a preclinical model will allow for greater understanding of the kinetics of liquid biopsies in response to radiation, which may be of signi cance as a biomarker in further development of combination therapy.…”