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.

MRNA RT-qPCR is shown to be a very sensitive technique to detect minimal residual disease (MRD) in patients with neuroblastoma. Multiple mRNA markers are known to detect heterogeneous neuroblastoma cells in bone marrow (BM) or blood from patients. However, the limited volumes of BM and blood available can hamper the detection of multiple markers. To make optimal use of these samples, we developed a multiplex RT-qPCR for the detection of MRD in neuroblastoma. GUSB and PHOX2B were tested as single markers. The adrenergic markers TH, GAP43, CHRNA3 and DBH and mesenchymal markers POSTN, PRRX1 and FMO3 were tested in multiplex. Using control blood and BM, we established new thresholds for positivity. Comparison of multiplex and singleplex RT-qPCR results from 21 blood and 24 BM samples from neuroblastoma patients demonstrated a comparable sensitivity. With this multiplex RT-qPCR, we are able to test seven different neuroblastoma mRNA markers, which overcomes tumor heterogeneity and improves sensitivity of MRD detection, even in those samples of low RNA quantity. With resources and time being saved, reduction in sample volume and consumables can assist in the introduction of MRD by RT-qPCR into clinical practice.

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.

Neuroblastoma affects mostly young children, bearing a high morbidity and mortality. Liquid biopsies, e.g., molecular analysis of circulating tumor-derived nucleic acids in blood, offer a minimally invasive diagnostic modality. Cell-free RNA (cfRNA) is released by all cells, especially cancer. It circulates in blood packed in extracellular vesicles (EV) or attached to proteins. We studied the feasibility of analyzing cfRNA and EV, isolated by size exclusion chromatography (SEC), from platelet-poor plasma from healthy controls (n = 40) and neuroblastoma patients with localized (n = 10) and metastatic disease (n = 30). The mRNA content was determined using several multiplex droplet digital PCR (ddPCR) assays for a neuroblastoma-specific gene panel (PHOX2B, TH, CHRNA3) and a cell cycle regulation panel (E2F1, CDC6, ATAD2, H2AFZ, MCM2, DHFR). We applied corrections for the presence of platelets. We demonstrated that neuroblastoma-specific markers were present in plasma from 14/30 patients with metastatic disease and not in healthy controls and patients with localized disease. Most cell cycle markers had a higher expression in patients. The mRNA markers were mostly present in the EV-enriched SEC fractions. In conclusion, cfRNA can be isolated from plasma and EV and analyzed using multiplex ddPCR. cfRNA is an interesting novel liquid biopsy-based target to explore further.

PURPOSE: About 60% of patients with high-risk neuroblastoma relapse. Specific mRNA detection in bone marrow (BM) by reverse transcriptase quantitative PCR (RT-qPCR) is associated with survival outcomes. Peripheral blood (PB) sampling is less invasive. Therefore, we prospectively validated an RT-qPCR panel of neuroblastoma mRNA in PB of patients with high-risk neuroblastoma, treated in NB2004-HR (GPOH) and NBL2009 (DCOG). METHODS: From 312 patients, 634 PB samples were prospectively collected (2009-2017) at diagnosis, after two cycles and end-of-induction therapy. RT-qPCR was performed using our panel of neuroblastoma mRNA markers: PHOX2B , TH , DDC , CHRNA3 , and DBH . Results were compared with paired BM samples. The association between neuroblastoma detection and event-free survival (EFS) and overall survival (OS) was estimated using Kaplan-Meier's methodology and multivariable Cox regression model. RESULTS: Clear correlation between calculated infiltration by neuroblastoma mRNA expression in PB and BM was seen at diagnosis ( r _s 0.70 [95% CI, 0.62 to 0.76]), and heterogeneity was seen after two cycles (0.37 [95% CI, 0.12 to 0.58]) and end of induction (0.61 [95% CI, 0.10 to 0.87]). mRNA expression was significantly lower in PB compared with BM. At diagnosis, PB infiltration ≥1% was a prognostic factor for survival: adjusted hazard ratio (HR) was 2.37 [95% CI, 1.56 to 3.60] for EFS and 2.60 [1.65-4.08] for OS. At the end of induction, PHOX2B positivity in PB samples (n = 11) was associated with poor outcomes: HR 3.06 [1.51-6.20] for EFS and 2.88 [1.36-6.11] for OS. In patients with ≥10% BM infiltration at diagnosis, detection of the mRNA panel in PB samples could significantly distinguish between survival groups; the adjusted HR of PB infiltration ≥1% was 2.09 [1.01-4.30] for OS. CONCLUSION: PB infiltration is associated with EFS and OS at diagnosis; it is also significantly associated with survival outcomes of patients with ≥10% BM infiltration at diagnosis. During follow-up, neuroblastoma mRNA detection in PB can be of added value, when BM analysis is not possible.