Abstract LB112: Liquid biopsy monitoring reveals high molecular weight cfDNA fragments associated with radiotherapy dose-intensity
Authors: Balazs Zsolt , Ivankovic Ivna , Balermpas Panagiotis , Gitchev Todor , Willmann Jonas , Krauthammer Michael , Andratschke Nicolaus 
Published: June 2022

Introduction: Radiotherapy aims at delivering a high dose of radiation to a tumor while minimizing the dose absorbed by healthy tissues, minimizing side effects or toxicities. Radiotherapy monitoring currently largely relies on longitudinal imaging with spatial and limited functional information on therapy response. Particularly, discrimination of radiotherapy effect on the tumor versus healthy tissue can be challenging and imaging response may lag behind actual response on the tumor cell level. Liquid biopsy monitoring promises to complement imaging by providing systemic biological information about the effects of radiotherapy. Cell-free DNA (cfDNA) in the blood plasma contains genetic and epigenetic information about cell death processes, and cfDNA monitoring may thus lend itself to observing both tumor response and normal-tissue toxicity during radiotherapy.

Methods: Seven polymetastatic patients, seven healthy volunteers, seven oligometastatic patients (OMD) and seven head-and neck cancer patients (H&N) were recruited for the study. OMD patients received stereotactic body radiation therapy in 5 fractions of 5-10 Gy, heterogeneously prescribed, while H&N underwent homogeneously 2 Gy radiation to a total dose of 70 Gy. Blood samples were collected from all patients at baseline and from patients undergoing radiotherapy twice during and twice after treatment, with samples collected up to six months after treatment. cfDNA was isolated and low-pass (7x) whole-genome sequencing was performed to examine fragment-length distribution and copy number alterations. We applied in silico size selection for 90 to 150 bp long fragments to improve copy number variant detection. Clinical measures and imaging findings were compared to cfDNA fragment analysis.

Results: Samples showing high circulating tumor DNA content in copy number analysis (tMAD&gt;0.03) also showed a higher proportion of short fragments (80-100 bp) compared to the samples of healthy individuals (ANOVA: p=0.0216, Tukey-pairwise: p=0.0287), whereas samples with low circulating tumor content did not (Tukey-pairwise: p=0.7540). We observed rapidly declining tumor fraction under treatment in a patient with prostate cancer and a high tMAD value at baseline. However, in the follow-up sample, residual tumor was detectable in the cfDNA copy number analysis, which was further confirmed by increasing PSA values. We observed a significant increase in the fraction of fragments between 200 and 250 bp in response to the treatment in oligometastatic patients receiving high-intensity radiotherapy, which returned to baseline in the follow-up measurements (ANOVA: p=0.0252, Tukey-pairwise: p=0.3138). A similar trend was observable (ANOVA: p=0.37) in head-and-neck patients, who received lower fractions of radiotherapy.

Conclusions: Applying low-pass whole-genome cfDNA-sequencing, we were able to monitor radiotherapy response in oligometastatic and head-and-neck cancer patients. We have also observed short-term increase in the number fragments between 200 and 250 bp during treatment, which we hypothesize to be a necrotic signal. The minimal invasive nature and the relatively low cost of low-pass cfDNA-sequencing makes it a viable method for frequent monitoring, which may allow prediction of tumor response and patient outcome.

Citation Format: Zsolt Balazs, Ivna Ivankovic, Panagiotis Balermpas, Todor Gitchev, Jonas Willmann, Michael Krauthammer, Nicolaus Andratschke. Liquid biopsy monitoring reveals high molecular weight cfDNA fragments associated with radiotherapy dose-intensity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB112.