A Comprehensive Circulating Tumor DNA Assay for Detection of Translocation and Copy-Number Changes in Pediatric Sarcomas

Abstract

Most circulating tumor DNA (ctDNA) assays are designed to detect recurrent mutations.Pediatric sarcomas share few recurrent mutations but rather are characterized by translocations and copy number changes. We applied CAncer Personalized Profiling by deep Sequencing (CAPP-Seq) for detection of translocations found in the most common pediatric sarcomas. We also applied ichorCNA to the combined off-target reads from our hybrid capture to simultaneously detect copy number alterations. We analyzed 64 prospectively collected plasma samples from 17 pediatric sarcoma patients.Translocations were detected in the pre-treatment plasma of 13 patients and were confirmed by tumor sequencing in 12 patients. Two of these patients had evidence of complex chromosomal rearrangements in their ctDNA. We also detected copy number changes in the pre-treatment plasma of 7 patients. We found that ctDNA levels correlated with metastatic status and clinical response. Furthermore, we detected rising ctDNA levels before relapse was clinically apparent, demonstrating the high sensitivity of our assay. This assay can be utilized for simultaneous detection of translocations and copy number alterations in the plasma of pediatric sarcoma patients. While we describe our experience in pediatric sarcomas, this approach can be applied to other tumors that are driven by structural variants.

Circulating tumor DNA – A potential aid in the management of chordomas

“…The risk of event and death increased in patients diagnosed with EWS or osteosarcoma as the levels of ctDNA increased (42). These findings are supported by Shah and colleagues whose work highlights that EWS and osteosarcoma patients with high ctDNA levels have a significantly increased risk of disease-related death and that a rise in ctDNA levels is predictive of patient relapse (43).…”

“…In addition to serving as a valuable predictor of patient survival and highlighting patient response to treatment, ctDNA can reveal complex chromosomal rearrangements. In the study by Shah and colleagues, the research team was able to identify a novel EWSR1-PKNOX2 translocation in a EWS patient’s plasma at the time of their relapse ( 43 ). This translocation has not been previously described in the literature.…”

Molecular Characterization of Circulating Tumor DNA in Pediatric Rhabdomyosarcoma: A Feasibility Study

“…The sensitivity for detection of ctDNA in diagnostic patient plasma samples (78% and 100% for ddPCR and WES, respectively) was on par with that of previous studies in pediatric sarcomas. 9 , 10 , 14 - 17 Interestingly, all three frontline patients in whom baseline ctDNA could not be detected by ddPCR had tumors in a favorable anatomic site (genitourinary tract, excluding the bladder and prostate) and were fusion-negative, which are both positive survival indicators in RMS. 2 Two of the three patients had their tumors resected before collecting baseline blood samples, which explains why no ctDNA could be found in them.…”

“…5,6 The detection of mutations is of particular importance, as ctDNA studies of RMS have focused on identifying gene fusions with little evidence for detection of ctDNA in fusion-negative patients. [8][9][10]14 In this study, we have also developed a custom sequencing panel, suitable for formalin-fixed paraffin-embedded tissue, to define the unique PAX3/7-FOXO1 DNA breakpoints. This is more practical for clinical implementation than a requirement for fresh-frozen material.…”

“…Quantitative polymerase chain reaction (qPCR), targeted sequencing panels, and whole-genome sequencing have previously been used to detect the PAX3-FOXO1 gene fusion in ctDNA from a limited number of patients with aRMS. [8][9][10] However, more evidence is needed (particularly for eRMS or fusion-negative patients) to support the clinical utility of ctDNA in this tumor type.…”