Abstract A59: Biologic rationale for radiation-induced release of circulating tumor DNA
Authors: Rostami Ariana , Waldron John N. , Bratman Scott V. 
Published: June 2020

Abstract
Purpose: Rapid release of circulating tumor DNA (ctDNA) during cytotoxic therapy could provide an early indication of treatment efficacy. However, the biologic basis for treatment-induced ctDNA release is poorly understood. To study this phenomenon, we evaluated ctDNA release in head and neck squamous cell carcinoma (HNSCC) treated with radiotherapy (RT) or chemoradiotherapy (CRT). We sought to characterize the kinetics and biologic underpinnings of this process.
Methods: 33 HPV+ HNSCC patients treated with RT (n=17) or CRT (n=16) had blood collected at baseline, after the 4th fraction of RT (early-RT), mid-RT, and 3 months post-RT. ctDNA was quantified using digital PCR for HPV-16. Nine HNSCC cell lines (3 HPV+, 6 HPV-) were used for in vitro ctDNA kinetic analysis. Apoptosis was measured by luminescent DEVD cleavage assay, triggered by staurosporine and blocked by z-vad-fmk. Senescence was measured by SA-β-galactosidase activity, triggered by HrasG12V-transduction of fibroblast cell lines and blocked by navitoclax. Three HNSCC cell lines (2 HPV+, 1 HPV-) were grown as xenografts in NRG mice for in vivo analysis. Endpoint xenograft tumors were assessed for necrotic area and for markers of apoptosis (cleaved-caspase 3) and senescence (p21) by immunohistochemistry. ctDNA was quantified from media or mouse plasma by quantitative PCR using human-specific LINE-1 primers.
Results: Of 30 patients with detectable HPV-16 ctDNA (90.9%), we observed variable kinetic patterns of release. We grouped patients into three categories: pattern 1, peak at early RT (12, 40%); pattern 2, peak at mid RT (7, 23.3%); pattern 3, no peak (11, 36.7%). Based on these findings, we characterized the mechanisms dictating RT-induced ctDNA release in preclinical models of HNSCC. Following RT, HNSCC cell lines exhibited variable kinetics of ctDNA release in vitro and in vivo with peak release observed after 72-96 hours. RT-induced ctDNA release was not associated with intrinsic radiosensitivity, HPV status, or propensity to undergo apoptosis. Although caspase inhibition resulted in a near-complete reduction in RT-induced caspase activity (-84%±8%), a comparatively minor reduction in ctDNA release (-29%±9%) was observed. There was an inverse association between RT-induced senescence and ctDNA release. HrasG12V-triggered senescence reduced ctDNA release, while navitoclax treatment reversed this effect. HNSCC models with greater RT-induced ctDNA release displayed more necrosis and less senescence in endpoint xenograft tumors.
Conclusion: Dynamic changes in HPV-16 ctDNA are observed during treatment, with variable patterns of release. Preclinical HNSCC models demonstrate RT-induced spikes within 3-4 days of treatment, revealing a complex interplay between distinct mechanisms of cell death and ctDNA release. Necrosis and senescence are the major determinants of RT-induced ctDNA release, while apoptosis has a comparatively minor impact. This study will inform future work on implementing ctDNA as a response biomarker.
Citation Format: Ariana Rostami, John N. Waldron, Scott V. Bratman. Biologic rationale for radiation-induced release of circulating tumor DNA [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A59.