Novel Circulating Hypermethylated RASSF1A ddPCR for Liquid Biopsies in Patients With Pediatric Solid Tumors

Abstract

PURPOSE Liquid biopsies can be used to investigate tumor-derived DNA, circulating in the cell-free DNA (cfDNA) pool in blood. We aimed to develop a droplet digital polymerase chain reaction (ddPCR) assay detecting hypermethylation of tumor suppressor gene RASSF1A as a simple standard test to detect various pediatric tumor types in small volume blood samples and to evaluate this test for monitoring treatment response of patients with high-risk neuroblastoma. METHODS We developed a ddPCR assay to sensitively detect tumor-derived hypermethylated RASSF1A DNA in liquid biopsies. We tested this assay in plasma of 96 patients with neuroblastoma, renal tumors, rhabdomyosarcoma, or Hodgkin lymphoma at diagnosis and in cerebrospinal fluid of four patients with brain tumors. We evaluated the presence of hypermethylated RASSF1A in plasma samples during treatment and follow-up in 47 patients with neuroblastoma treated according to high-risk protocol and correlated results with blood mRNA–based and bone marrow mRNA–based minimal residual disease detection and clinical outcomes. RESULTS The total cfDNA level was significantly higher in patients with metastatic neuroblastoma and nephroblastoma compared with healthy adult and pediatric controls. Hypermethylated RASSF1A was present in 41 of 42 patients with metastatic neuroblastoma and in all patients with nephroblastoma, with the median percentage of 69% and 21% of total RASSF1A, respectively. Hypermethylated RASSF1A levels decreased during therapy and recurred at relapse. CONCLUSION Our findings demonstrate the value of ddPCR-based detection of hypermethylated RASSF1A as a circulating molecular tumor marker in neuroblastoma. Our preliminary investigation of RASSF1A hypermethylation detection in circulating cfDNA of other pediatric tumor entities demonstrates potential as a pan-tumor marker, but requires investigation in larger cohorts to evaluate its use and limitations.

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

“…The RASSF1A- M ddPCR assay was performed using double digestion with the methylation-sensitive restriction enzymes Hhal and Bsh1236I BstUI; Thermo Fisher Scientific, Waltham, MA) using a thermocycler T100 and QX200 reader (Bio-Rad, Hercules, CA) as described previously. 14 The sequences and concentrations of the primers and probes, cycling conditions, and analyses were performed as described previously, with the threshold for RASSF1A -M positivity per sample set at ≥ 14 copies/ml and ≥ 4 RASSF1A -M–positive droplets, as determined in 18 healthy pediatric and 22 adult control plasmas. 14 The percentage of RASSF1A- M was calculated relative to total RASSF1A .…”

“… 10 , 31 Recently, we developed a methylation-specific enzyme-based approach involving ddPCR to detect RASSF1A- M in several pediatric solid tumors, including rhabdomyosarcoma. 14 …”

Skin Cancer Research Goes Digital: Looking for Biomarkers within the Droplets

“…Among the omics technologies that gained momentum in the last years, droplet digital polymerase chain reaction (ddPCR) offered the possibility of accurately detecting and quantifying low-abundance nucleic acids in various biological samples, having important applications in cancer subtyping [ 25 ], prognosis [ 26 ], and minimal residual disease monitoring [ 27 ]. Suitable for both archived and liquid biopsy (LB) samples, ddPCR can be used for numerous omics measurements, including absolute allele quantification [ 28 ], CNVs analysis [ 29 ], rare mutations [ 30 ], DNA methylation detection [ 31 ], transcriptomic evaluations (mRNA, miRNA) [ 32 ] and genomic rearrangements [ 33 ]. Therefore, ddPCR forms a suitable platform to be used in personalized medicine in oncology.…”

Extracellular Vesicles: A New Source of Biomarkers in Pediatric Solid Tumors? A Systematic Review

“…The use of cell-free DNA from plasma has been extensively studied for different tumor entities using various molecular techniques. The presence of the methylated tumor suppressor gene RASSF1A can be detected in plasma for several types of pediatric solid tumors, and can be used to monitor therapy response ( 25 , 26 ). For neuroblastoma, tumor-specific aberrations in the MYCN and ALK genes (mutations and copy number alterations) can be monitored during the course of the disease ( 27 , 28 ).…”

“…Copy number profiling can be performed on cell-free DNA to detect a tumor-derived signal, and this can be combined with the copy number profile from the primary tumor, offering a more comprehensive overview of the genetic landscape of the tumor and its metastatic lesions ( 29 ). However, since plasma mostly contains non-tumor cell-free DNA, the signal-to-noise reduction can be challenging, especially considering that not all tumors shed large amounts of cell-free DNA ( 25 , 30 ) Another option that has been explored, is detection of circulating tumor cells in blood, or bone marrow, using tumor-specific targets. This has been shown to be of clinical value in neuroblastoma and rhabdomyosarcoma ( 31 – 33 ).…”