Cell-Free DNA as a Diagnostic and Prognostic Biomarker in Pediatric Rhabdomyosarcoma

Abstract

PURPOSE Total cell-free DNA (cfDNA) and tumor-derived cfDNA (ctDNA) can be used to study tumor-derived genetic aberrations. We analyzed the diagnostic and prognostic potential of cfDNA and ctDNA, obtained from pediatric patients with rhabdomyosarcoma. METHODS cfDNA was isolated from diagnostic plasma samples from 57 patients enrolled in the EpSSG RMS2005 study. To study the diagnostic potential, shallow whole genome sequencing (shWGS) and cell-free reduced representation bisulphite sequencing (cfRRBS) were performed in a subset of samples and all samples were tested using droplet digital polymerase chain reaction to detect methylated RASSF1A ( RASSF1A-M). Correlation with outcome was studied by combining cfDNA RASSF1A-M detection with analysis of our rhabdomyosarcoma-specific RNA panel in paired cellular blood and bone marrow fractions and survival analysis in 56 patients. RESULTS At diagnosis, ctDNA was detected in 16 of 30 and 24 of 26 patients using shallow whole genome sequencing and cfRRBS, respectively. Furthermore, 21 of 25 samples were correctly classified as embryonal by cfRRBS. RASSF1A-M was detected in 21 of 57 patients. The presence of RASSF1A-M was significantly correlated with poor outcome (the 5-year event-free survival [EFS] rate was 46.2% for 21 RASSF1A-M ‒positive patients, compared with 84.9% for 36 RASSF1A-M ‒negative patients [ P < .001]). RASSF1A-M positivity had the highest prognostic effect among patients with metastatic disease. Patients both negative for RASSF1A-M and the rhabdomyosarcoma-specific RNA panel (28 of 56 patients) had excellent outcome (5-year EFS 92.9%), while double-positive patients (11/56) had poor outcome (5-year EFS 13.6%, P < .001). CONCLUSION Analyzing ctDNA at diagnosis using various techniques is feasible in pediatric rhabdomyosarcoma and has potential for clinical use. Measuring RASSF1A-M in plasma at initial diagnosis correlated significantly with outcome, particularly when combined with paired analysis of blood and bone marrow using a rhabdomyosarcoma-specific RNA panel.

Targeted locus amplification to develop robust patient-specific assays for liquid biopsies in pediatric solid tumors

“…Since hypermethylation of RASSF1A has been found in many types of tumors ( 54 – 60 ), this assay offers another approach for liquid biopsy-based disease monitoring. The combination of both RNA and DNA-based platforms for the analysis of liquid biopsies could be complementary, as we have showed previously in a cohort of patients with rhabdomyosarcoma ( 61 ).…”

Case series on clinical applications of liquid biopsy in pediatric solid tumors: towards improved diagnostics and disease monitoring

“…Detection of RASSF1A-M by ddPCR in plasma of patients with highrisk neuroblastoma clearly showed a significantly poorer event-free survival when hypermethylated RASSF1A exceeded 27,681 copies/mL at diagnosis (32). In rhabdomyosarcoma, detectable levels of RASSF1A-M in diagnostic plasma samples clearly correlate with poor outcome, especially in patients with metastatic disease (80). RASSF1A-M detection could therefore be complementary to the rhabdomyosarcoma RT-qPCRpanel (80).…”

“…In rhabdomyosarcoma, detectable levels of RASSF1A-M in diagnostic plasma samples clearly correlate with poor outcome, especially in patients with metastatic disease (80). RASSF1A-M detection could therefore be complementary to the rhabdomyosarcoma RT-qPCRpanel (80).…”

Detection of pretreatment circulating tumor DNA as a biomarker of poor outcome in intermediate-risk rhabdomyosarcoma

“…As we consider incorporating detection of ctDNA into clinical practice in the context of the results of these studies, attention should be paid to the generalizability of the methodologies with efforts toward standardization. As an example, we and others have developed plasma-based liquid biopsy assays for pediatric solid tumors ( 10 , 11 , 25 , 26 ). It remains to be seen if the sensitivity and specificity of the results are comparable across platforms.…”