Abstract PR05: Radiation-assisted Amplification Sequencing (RAMP-Seq): Evaluating the use of stereotactic body radiation therapy (SBRT) for enriching circulating tumor DNA in liquid biopsies
Authors: Christopher Boniface, Kathryn Baker, Chris Dieg, Carol Halsey, Ramtin Rahmani, Mark Deffenbach, Charles Thomas, Garth Tormoen, Nima Nabavizadeh, Paul Spellman
Journal: Clinical Cancer Research (2020)

Abstract Purpose: To determine if the administration of stereotactic body radiation therapy (SBRT) to known tumor masses temporarily elevates levels of circulating tumor DNA (ctDNA) derived from target or distal masses and to identify an optimal period of peak ctDNA enrichment for liquid biopsy. Experimental Design and Procedures: We used deep, whole-exome sequencing (WES) or 100-gene capture with UMI-based error corrected sequencing of cell-free DNA (cfDNA) of individuals undergoing SBRT as standard-of-care (SOC) to determine (a) whether SBRT treatment induces elevated levels of ctDNA in blood plasma as confirmed by biopsy-proven cases, and (b) the optimal window of ctDNA enrichment for sample collection. We collected blood draws every 24-72 hours from each patient prior to and during SBRT treatment (n=20), as well as for two weeks following the final dose (n=3). CfDNA and genomic DNA were isolated from blood plasma and solid tissue biopsies, respectively, and subjected to whole-exome capture and next-generation sequencing with the Illumina HiSeq 2500 platform. Sequencing data were aligned and mutations were called using BWA MEM and Mutect pipelines as previously described (Butler et al., 2019). Genomic DNA extracted from buffy coat was prepared and used as a matched normal in mutation calling. Variant allele frequencies (VAF) of Mutect calls were compared between baseline and post-treatment samples to determine enrichment levels. WES of bulk tumor DNA from pathology-proven cancer patients was used to confirm that the variants identified in the cfDNA sequencing originated in the target tissue. Data Summary: Preliminary data suggest that SBRT induces enrichment of ctDNA in blood plasma by at least 10-fold within 24-96 hours following treatment. When variants identified in separate biopsies of multiple tumors in a single individual were analyzed after SBRT, we found that increases were not uniform among variant clusters, suggesting an abscopal response from nonradiated tumor tissues found elsewhere in the patient. Conclusions: The ability to detect tumor-derived genomic variants in cfDNA has been a major challenge to liquid biopsy applications, particularly in early-stage cancers and recurrence settings. This novel detection strategy, RAMP-Seq, utilizes highly conformational radiation to induce ctDNA enrichment from tumor-cell death, making previously undetectable levels of ctDNA well within the range of standard sequencing and analysis methods. Such an approach has valuable applications from diagnosis of early-stage cancer to genotyping lesions normally inaccessible by a traditional biopsy. Abscopal effects have also been observed, suggesting that induction of ctDNA enrichment by radiating the target lesion can also result in the increase of ctDNA derived from distal lesions. Taken together, these findings offer promising new approaches to both early- and advanced-stage cancer characterization and intervention. This abstract is also being presented as Poster A44. Citation Format: Christopher Boniface, Kathryn Baker, Chris Dieg, Carol Halsey, Ramtin Rahmani, Mark Deffenbach, Charles Thomas, Garth Tormoen, Nima Nabavizadeh, Paul Spellman. Radiation-assisted Amplification Sequencing (RAMP-Seq): Evaluating the use of stereotactic body radiation therapy (SBRT) for enriching circulating tumor DNA in liquid biopsies [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 PR05.