Amount of cfDNA and circulating hypermethylated RASSF1A. (A) Level of cfDNA at diagnosis in patients with various pediatric solid tumor entities, compared with healthy adult and pediatric controls. cfDNA was quantified by β-actin (ACTB), in copies/mL plasma or CSF (cerebrospinal fluid). Lines indicate the median. (B) The percentage of hypermethylated RASSF1A of total RASSF1A copies at diagnosis in patients with metastatic neuroblastoma (n = 42), localized neuroblastoma (n = 15), nephroblastoma (n = 11), rhabdomyosarcoma (n = 14), lymphoma (n = 5), and CNS tumors (n = 4). Adult and pediatric controls were used to establish a threshold for positivity. In 41 of 42 patients with metastatic neuroblastoma and 6 of 15 patients with localized neuroblastoma, hypermethylated RASSF1A was detected. In all 11 patients with nephroblastoma, 8 of 14 patients with rhabdomyosarcoma, 4 of 5 patients with lymphoma, and 1 of 4 patients with CNS tumor, hypermethylated RASSF1A was detected. Two plasma samples of patients with benign renal tumors (a Cystic Partially Differentiated Nephroblastoma and a bilateral differentiated nephroblastomatosis) were negative for hypermethylated RASSF1A. cfDNA, cell-free DNA; CFS, cerebrospinal fluid.