Abstract B49: Quantitating circulating tumor DNA in translocation-positive sarcoma patients using CAPP-Seq
Authors: Shah Avanthi Tayi , Azad Tej D. , Chabon Jake J. , Breese Marcus , Tanasa Bogdan , Spillinger Aviv , Leung Stanley G. , Diehn Maximilian , Alizadeh Ash A. , Sweet-Cordero E. Alejandro 
Published: February 2019

Abstract
A promising tool for noninvasive disease monitoring is analysis of circulating tumor DNA (ctDNA). Healthy individuals carry 1-10 ng/ml of cell-free DNA (cfDNA) in the blood; in oncology patients, ctDNA, which is released from tumor cells, comprises a fraction of the cfDNA and carries tumor-specific alterations, such as mutations, translocations, and copy number alterations. Most ctDNA assays lack robust translocation detection capabilities, since they are designed for adult cancers, which are commonly characterized by mutations and copy number alterations. Even assays that are designed specifically for translocation detection have inherent limitations. They are often PCR-based with laborious methods. First, the patient’s primary tumor sample is sequenced to determine the unique sequence across the translocation breakpoint. This is followed by design of patient-specific primers that can only be used for that individual patient. We sought to design an off-the-shelf, broadly applicable ctDNA assay for translocation detection across pediatric Ewing sarcoma (ES), osteosarcoma (OS), rhabdomyosarcoma (RMS), and synovial sarcoma (SS).
Recent work by our collaborators at Stanford University led to the development of CAncer Personalized Profiling by deep Sequencing (CAPP-Seq), a method capable of ultraspecific and ultrasensitive detection of ctDNA. Utilizing COSMIC and TCGA data, recent sequencing publications defining the landscape of pediatric sarcomas, and our own in-house sequencing data, we designed a pediatric sarcoma CAPP-Seq selector. This selector is comprised of biotinylated oligonucleotides that tile across the introns where translocation breakpoints occur in these pediatric sarcomas. The selector is applied to a sequencing library prepared from patient cfDNA to enrich for the genomic regions of interest via hybrid capture. The resulting enriched library undergoes next-generation sequencing to allow for detection and quantification of circulating tumor DNA.
We have isolated cfDNA from pediatric sarcoma patients and found that their cfDNA levels are higher than levels found in adult oncology patients and healthy individuals, likely due to a large fraction of contributing ctDNA. We have applied our selector to pretreatment plasma samples from 5 EWS patients, 2 OS patients, 4 RMS patients, and 1 SS patient. We detected translocations in 10/12 of these samples. Tumor was available for 7/12 of these patients, and we were able to confirm our plasma results by whole-genome sequencing of the tumor, as a validation of our findings. Additionally, we have applied our selector to serial plasma samples collected over the course of treatment and found that ctDNA levels correlate with clinical status. We have detected translocations at allelic frequencies &lt;0.01%, demonstrating that our method is ultrasensitive and could be used to detect minimal residual disease. Our work demonstrates that CAPP-Seq can serve as an ultrasensitive, broadly applicable tool for circulating tumor translocation detection and offers promise as a method for noninvasive diagnosis and disease monitoring.
Citation Format: Avanthi Tayi Shah, Tej D. Azad, Jake J. Chabon, Marcus Breese, Bogdan Tanasa, Aviv Spillinger, Stanley G. Leung, Maximilian Diehn, Ash A. Alizadeh, E. Alejandro Sweet-Cordero. Quantitating circulating tumor DNA in translocation-positive sarcoma patients using CAPP-Seq [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B49.