Abstract A55: Circulating tumor DNA in newly diagnosed intermediate-risk rhabdomyosarcoma
Authors: Abbou Samuel , Hall David , Barkauskas Donald A. , Klega Kelly , Nag Anwesha , Thorner Aaron R. , Krailo Mark , Dubois Steven , Hawkins Douglas S. , Crompton Brian D. 
Published: June 2020

Abstract
Background: Patients with intermediate-risk rhabdomyosarcoma (IR RMS) have approximately 60-70% chance of survival with current therapies. However, biomarkers of outcome and response to therapy are lacking for these patients. Circulating tumor DNA (ctDNA) has been shown to be prognostic in a wide range of cancer types, but it is unknown whether patients with IR RMS have detectable levels of ctDNA.
Objective: To study ctDNA prevalence and prognostic impact in newly diagnosed IR RMS, we utilized two next-generation sequencing (NGS) approaches that detect the presence of somatic copy-number changes and oncogenic translocations.
Methods: Cell-free DNA was extracted from serum samples obtained from patients with newly diagnosed IR RMS who enrolled on the COG studies ARST0531 and D9803, including patients with embryonal (ERMS) and alveolar (ARMS) histology subtypes. While both subtypes are characterized by frequent somatic copy-number variants (CNVs), ARMS is also defined by recurrent translocations. To detect CNVs, we performed ultra-low passage whole-genome sequencing (ULP-WGS). Translocations were detected with a validated custom hybrid capture assay (TranSS-Seq).
Results: Serum samples were analyzed from 132 patients with IR RMS, including 75 with ERMS and 57 with ARMS. ctDNA was detected by CNVs in 70% (92/132) of IR RMS patients with similar detection rates in each histology: 65% (49/75) in ERMS and 75% (43/57) in ARMS. Among the ARMS samples sequenced, only 37% (18/49) were positive by TranSS-Seq. Furthermore, copy-number events resulting in amplifications of the somatic translocation frequently resulted in miscalculation of ctDNA content by the TranSS-Seq method. Estimates of event-free and overall survival were lower in patients with detectable ctDNA, though the differences were not statistically significant.
Conclusion: Patients with IR RMS frequently have detectable levels of ctDNA that can be measured by NGS assays designed to detect somatic structural events. This study relied on previously banked serum samples and a relatively small retrospective analysis. These findings provide justification for our current efforts to utilize a large prospective study, COG ARST1431, to collect pretreatment and serial blood samples using procedures optimized for ctDNA assays. Sequencing of matched tumor samples is ongoing to understand the differences in sensitivity between ULP-WGS and TranSS-Seq for ctDNA detection in patients with ARMS.
Citation Format: Samuel Abbou, David Hall, Donald A. Barkauskas, Kelly Klega, Anwesha Nag, Aaron R. Thorner, Mark Krailo, Steven Dubois, Douglas S. Hawkins, Brian D. Crompton. Circulating tumor DNA in newly diagnosed intermediate-risk rhabdomyosarcoma [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 A55.