Benchmarking taxonomic classifiers with Illumina and Nanopore sequence data for clinical metagenomic diagnostic applications

Culture-independent metagenomic detection of microbial species has the potential to provide rapid and precise real-time diagnostic results. However, it is potentially limited by sequencing and classification errors. We use simulated and real-world data to benchmark rates of species misclassification using 100 reference genomes for each of ten common bloodstream pathogens and six frequent blood culture contaminants (n=1600). Simulating both with and without sequencing error for both the Illumina and Oxford Nanopore platforms, we evaluated commonly used classification tools including Kraken2, Bracken, and Centrifuge, utilising mini (8GB) and standard (30-50GB) databases. Bracken with the standard database performed best, the median percentage of reads across both sequencing platforms identified correctly to the species level was 98.46% (IQR 93.0:99.3) [range 57.1:100]. For Kraken2 with a mini database, a commonly used combination, median species-level identification was 79.3% (IQR 39.1:88.8) [range 11.2:100]. Classification performance varied by species, with E. coli being more challenging to classify correctly (59.4% to 96.4% reads with correct species, varying by tool used). By filtering out shorter Nanopore reads (<3500bp) we found performance similar or superior to Illumina sequencing, despite higher sequencing error rates. Misclassification was more common when the misclassified species had a higher average nucleotide identity to the true species. Our findings highlight taxonomic misclassification of sequencing data occurs and varies by sequencing and analysis workflow. This “bioinformatic contamination” should be accounted for in metagenomic pipelines to ensure accurate results that can support clinical decision making.

Validity and Acceptability of Wearable Devices for Monitoring Step-Count and Activity Minutes Among People With Multiple Sclerosis

Multiple wearable devices that purport to measure physical activity are widely available to consumers. While they may support increases in physical activity among people with multiple sclerosis (MS) by providing feedback on their performance, there is little information about the validity and acceptability of these devices. Providing devices that are perceived as inaccurate and difficult to use may have negative consequences for people with MS, rather than supporting participation in physical activity. The aim of this study was, therefore, to assess the validity and acceptability of commercially available devices for monitoring step-count and activity time among people with MS. Nineteen ambulatory adults with MS [mean (SD) age 52.1 (11.9) years] participated in the study. Step-count was assessed using five commercially available devices (Fitbit Alta, Fitbit Zip, Garmin Vivofit 4, Yamax Digi Walker SW200, and Letscom monitor) and an activPAL3μ while completing nine everyday activities. Step-count was also manually counted. Time in light activity, moderate-to-vigorous activity, and total activity were measured during activities using an Actigraph GT3X accelerometer. Of the 19 participants who completed the validity study, fifteen of these people also wore the five commercially available devices for three consecutive days each, and participated in a semi-structured interview regarding their perception of the acceptability of the monitors. Mean percentage error for step-count ranged from 12.1% for the Yamax SW200 to −112.3% for the Letscom. Mean step-count as manually determined differed to mean step-count measured by the Fitbit Alta (p = 0.002), Garmin vivofit 4 (p &lt; 0.001), Letscom (p &lt; 0.001) and the research standard device, the activPAL3μ (p &lt; 0.001). However, 95% limits of agreement were smallest for the activPAL3μ and largest for the Fitbit Alta. Median percentage error for activity minutes was 52.9% for the Letscom and 100% for the Garmin Vivofit 4 and Fitbit Alta compared to minutes in total activity. Three inductive themes were generated from participant accounts: Interaction with device; The way the device looks and feels; Functionality. In conclusion, commercially available devices demonstrated poor criterion validity when measuring step-count and activity time in people with MS. This negatively affected the acceptability of devices, with perceived inaccuracies causing distrust and frustration. Additional considerations when designing devices for people with MS include an appropriately sized and lit display and ease of attaching and charging devices.

Attenuation of Antibody Titers from 3 to 6 Months after the Second Dose of the BNT162b2 Vaccine Depends on Sex, with Age and Smoking Risk Factors for Lower Antibody Titers at 6 Months

Objective: We aimed to determine antibody titers at six months and their percentage change from three to six months after the second dose of the BNT162b2 coronavirus disease 2019 (COVID-19) mRNA vaccine (Pfizer/BioNTech) and to explore clinical variables associated with titers in Japan. Methods: We enrolled 365 healthcare workers (250 women, 115 men) whose three-month antibody titers were analyzed in our previous study and whose blood samples were collected 183 ± 15 days after the second dose. Participant characteristics, collected previously, were used. The relationships of these factors with antibody titers at six months and percentage changes in antibody titers from three to six months were analyzed. Results: Median age was 44 years. Median antibody titer at six months was 539 U/mL. Older participants had significantly lower antibody titers (20s, 752 U/mL; 60s–70s, 365 U/mL). In age-adjusted analysis, smoking was the only factor associated with lower antibody titers. Median percentage change in antibody titers from three to six months was −29.4%. The only factor significantly associated with the percentage change in Ab titers was not age or smoking, but sex (women, −31.6%; men, −25.1%). Conclusion: The most important factors associated with lower antibody titers at six months were age and smoking, as at three months, probably reflecting their effect on peak antibody titers. However, the only factor significantly associated with the attenuation in Ab titers from three to six months was sex, which reduced the sex difference seen during the first three months. Antibody titers may be affected by different factors at different time points.

Interferon Gamma and perforin positive T cells in acquired aplastic anemia: implication in therapeutic response

Acquired aplastic anemia (aAA) is an autoimmune disease, characterized by infiltration of T lymphocytes in the bone marrow with destruction of hematopoietic stem cells by the effector cells. Interferon gamma (IFN-γ) and perforin are important mediators of cell destruction. In this flow cytometry-based study, we have investigated the percentage of intracellular IFN-γ + and perforin + CD5 + T cells in peripheral blood of newly diagnosed aAA patients before and after immunosuppressive therapy (IST). Patients were categorized as per standard disease severity and response to IST. The median percentage of IFN-γ + and perforin + CD5 + T cells was higher in untreated patients compared to healthy controls. The percentage of these cells was also increased in untreated severe and very severe aplastic anemia when compared with non-severe aplastic anemia patients. In patients before and after IST the median percentage of T cells producing IFN-γ and perforin was elevated in non-responders as compared to partial plus complete responders. The higher percentage of IFN-γ + and perforin + CD5 + T cells may be useful as an early diagnostic marker for aberrant activation of immune system and predict poor response to IST in aAA patients, who will benefit from alternative therapy.

Early vascular healing of ultra-thin strut polymer-free sirolimus-eluting stents in acute coronary syndrome: USUI-ACS study

Purpose:Ultra-thin strut polymer-free sirolimus eluting stent (UPF-SES) have two novel characteristics, ultra-thin strut and polymer-free coating, which have the potential to achieve early re-endotherialization. However, a little is known whether early vascular healing of UPF-SES can be achieved in patients with acute coronary syndrome (ACS).The aim of this study was to evaluate the vascular healing after an implantation of UPF-SES in patients with ACS using optical coherence tomography (OCT) at 3 months after the stent implantation. Methods:From September 2020 and January 2021, a total of 31 consecutive patients presenting with ACS who underwent OCT examinations at the initial percutaneous coronary intervention (PCI) and 3-month follow-up were enrolled in the USUI-ACS study. The endpoints of this study were neointimal strut coverage, malapposition, and mean neointimal hyperplasia (NIH) thickness at 3-month follow-up.Results:Over a mean follow-up of 91 days after the initial PCI, the follow-up OCT was examined. The median percentage of covered struts was 98.4% and malapposed struts 0%, and the mean NIH thickness was 60μm.Conclusions:UPF-SES exhibited an excellent early vascular healing at 3-months in patients with ACS.

Mutations of the Novel Tumor Suppressor Gene SAMHD1 Are Frequent and Correlate with Decreased Protein Expression in Peripheral T-Cell Lymphomas (PTCL)

Introduction: The SAM domain and HD domain 1 (SAMHD1) protein is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase, which depletes the intracellular dNTP substrates and thus protects the host (human) cells from replication of viruses such as HIV. Mutations of SAMHD1 gene have been linked to Aicardi-Goutières syndrome (AGS). In lymphoid malignancies, SAMHD1 gene mutations have been detected in a subset of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) resulting in decreased SAMHD1 mRNA levels and also mantle cell lymphoma (MCL) among B-cell neoplasms as well as in a subset (20%) of T-prolymphocytic leukemia (T-PLL). Therefore, SAMHD1 may play a role in oncogenesis as a tumor suppressor. In addition, SAMHD1 may confer resistance to cytarabine by hydrolysing their active triphosphate metabolites and its high protein levels correlate with poorer clinical outcome in acute myeloid leukemia. The mutation status of SAMHD1 gene and its expression patterns in peripheral T-cell lymphoma types is not known yet. The purpose of this study was to investigate SAMHD1 gene alterations using next generation sequencing and SAMHD1 protein expression in common types of PTCL. Methods: The study group included 81 adult patients with peripheral T-cell lymphomas (PTCL) including 26 patients with ALK+ anaplastic large cell lymphoma (ALCL), 20 ALK- ALCL, 13 angioimmunoblastic T-cell lymphomas (AILT) and 22 PTCL, not otherwise specified (NOS) with pre-treatment, formalin-fixed, paraffin-embedded (FFPE) tumor tissues available for immunohistochemical analysis. Double immunostaining (SAMHD1/CD68) was used to distinguish CD68+ histiocytes from the neoplastic T-cells. The Ventana autostainer and a previously validated monoclonal antibody for SAMHD1 (#A303-691A; Bethyl Laboratories, San Antonio, TX, USA) was utilized. The percentage of SAMHD1-positive cells was calculated by counting at least 500 tumor cells in each case. In a subset of 28 PTCLs, next generation sequencing (NGS) was performed using FFPE tissues and an enriched custom TruSight gene panel of 52 genes relevant to lymphoma biology. In addition, 3 control tissue samples were included in the analysis. The analysis pipeline was based on GATK best practices guidelines and all variants were annotated using Ensembl VEP v94.5. Freedom from progression (FFP) and overall survival (OS), were the clinical endpoints. Survival analyses were performed using the Kaplan-Meier method (log-rank test). Results: The expression level of SAMHD1 (percentage of positive neoplastic T-cells) varied significantly with AILT showing the highest level (median percentage 80%) as compared to ALK+ ALCL that showed the lowest level (median percentage 40%) of SAMHD1 expression (p=0.019, Kruskall-Wallis test). SAMHD1 mutations were detected for the first time in a subset of PTCL including 4/11 (36%) ALK+ ALCL, 1/5 (20%) ALK- ALCL, 3/6 (50%) AILT and 2/5 (40%) PTCL, NOS. The SAMHD1 gene alterations included missense mutations, nonsense (stopcodon) and splice region mutations. Importantly, reduced level (low percentage of positive tumor cells) of SAMHD1 protein expression was significantly associated with the presence of SAMHD1 mutations. More specifically, the median percentage of SAMHD1+ neoplastic T-cells was 80% in the PTCL group with wild-type SAMHD1 gene compared to 30% in the PTCL group with mutated SAMHD1 gene (p=0.01, Mann-Whitney U test), thus suggesting that alterations of SAMHD1 gene may represent a mechanism of SAMHD1 protein downregulation in a subset of PTCL. SAMHD1 expression or gene alterations did not correlate with FFP or OS in any PTCL histologic type, although the number of patients included in each group was not adequate to draw definite conclusions for prognostic significance. Conclusions: SAMHD1 gene mutations are frequently detected in a subset of PTCL and are associated with reduced expression of SAMHD1 protein. These findings reveal a novel mechanism (SAMHD1 mutations) of SAMHD1 downregulation in PTCL, and further support the tumor suppressor function of SAMHD1 gene in lymphomas. Disclosures Rosenquist Brandell: AbbVie: Honoraria; AstraZeneca: Honoraria; Illumina: Honoraria; Janssen: Honoraria; Roche: Honoraria.

Peripheral Levels of Monocytic Myeloid-Derived Suppressive Cells at Diagnosis Predict Survivals in AML Patients Eligible for Intensive Chemotherapy

Introduction: Myeloid Derived Suppressive Cells (MDSC) constitute a heterogeneous population of immature myeloid cells characterized by their capacity to suppress innate and adaptive immune responses. As such, they have been proven, in solid tumors, to modulate malignancy by increasing tumor cell survival, angiogenesis, metastasis and tissue invasion. By contrast, reports on the role of MDSC in either acute myeloid (AML) or lymphoid (ALL) leukemias are very limited with unknown established impact on long-term outcomes. Patients and Methods: This monocentric prospective study included all adult patients eligible for first-line intensive chemotherapy for AML or ALL. The main objective was to investigate the presence of peripheral blood monocytic MDSC at diagnosis and after induction in such patients and to correlate their levels to complete remission (CR/CR with incomplete platelet recovery), cytologic relapse, leukemia-free (LFS) and overall (OS) survivals. Monocytic MDSCs were defined as CD15- CD34- CD16- CD14+ CD33+ CD11b+ DR-/low cells and assessed in a lysis-no-wash flow cytometry technique. Data acquisition was performed on a Navios® flow cytometer (Beckman Coulter, Miami, FL). MDSC were expressed as a percentage (%) of total nucleated cells defined as CD45+. MDSC% were compared to those of 21 healthy controls. The study was registered at the French Commission Nationale de l'Informatique et des Libertés as CNIL 2016-038. All patients gave informed consent. Analyses were performed in July 2021. Results: Between October 2017 and March 2021, 73 AML and 14 ALL were enrolled (Table 1). The median MDSC% in controls was 0.24% (range: 0.02-1.21). This % was significantly higher in AML compared to ALL (0.19% (range: 0-0.54) vs 0.14% (range: 0-0.35), p=0,01) and differed significantly from controls in ALL (p=0.0004) but not in AML (p=0.94). MDSC% after chemotherapy induction were available for 61 AML and 13 ALL at medians of 37,5 and 37 days, respectively. At that time, median MDSC% were similar between AML (0.84%, range: 0-28) and ALL (0.97%, range: 0-4.75) patients (p=0.52) but significantly higher than in controls for AML patients (p=0.001; ALL p=0.07). AML: MDSC% were not correlated to any other factors, especially ELN2017 classification (p=0.79). ROC curves for LFS established the threshold of 0,55% of MDSC at diagnosis as the best cut-off for analyses. MDSC% (&lt; vs &gt;0,55%) was not predictive of CR/CRp (86.6% n=39/45 vs 78.5% n=22/28, p=0.56). However, 2-year LFS (67.7+8% vs 30.1+10%, p=0.005) and 2-year OS (71.5+8% vs 30.1+10%, p=0.001). (Figure 1) were significantly higher for patients with low MDSC% (&lt;0.55%). The incidence of cytologic relapse after achieving CR/CRp was significantly lower in these low MDSC% patients (12.8% n=5/39 vs 45.4% n=10/22, p=0.01). The median percentage of MDSC increased significantly between diagnosis (0,19%) and post-induction (0,84%; p=0.001). Median post-induction MDSC% were similar between patients achieving CR/CRp (0.9%, n=53 evaluable) vs others (0.44%, n=8 evaluable, p=0.34). No impact on relapse incidence nor on LFS and OS was observed when comparing patients based on the median post-induction level of MDSC and ROC curves did not identify thresholds able to predict LFS or OS using MDSC% post-induction. Multivariate analysis confirmed the independent prognostic value of MDCS% at diagnosis for AML patients (LFS p=0.026, HR 3.6, 95%CI 1.88-6.91; OS p=0.02-, HR 2.6, 95%CI 1.11-5.95) together with ELN 2017 classification (LFS p=0.0001, HR 2.34, 95%CI 1.10-4.97; OS p=0.01, HR 2.57, 95%CI 1.18-4.11). ALL: MDSC% at diagnosis were not predictive of response as 13/14 patients achieved CR/CRp after induction. The percentage of MDSC increased significantly between diagnosis (0.14%) and post- induction (0.97%; p=0.002). Again, this had no consequence on relapse incidence in CR/CRp patients nor on LFS and OS when comparing patients based on median post induction MDSC%. Discussion: This study evidenced that a higher percentage of peripheral monocytic MDSC at diagnosis predict lower survivals in AML patients because of more relapse. This result has to be confirmed on larger cohorts as it may implicate to propose immune intervention before or in combination with chemotherapy to improve these patients' outcome. Indeed, these cells seem to be an independent biomarker and potentially promising targets for the development of novel therapeutic strategies. Figure 1 Figure 1. Disclosures Moreau: Sanofi: Honoraria; Amgen: Honoraria; Celgene BMS: Honoraria; Janssen: Honoraria; Abbvie: Honoraria; Oncopeptides: Honoraria.

Association between BCL2, BCL2 E17, MCL1 and BAX Protein Expression, Bone Marrow Microenvironment Histological Features, Clinical Presentation, Therapeutic Outcome, and the Overall Survival in Newly Diagnosed Multiple Myeloma

We evaluated the correlation between immunohistochemical BCL2, BCL2 E17, MCL1 (Bcl2-related protein family) and BAX (effector proapoptotic protein) expression and intensity of staining in plasma cells (PC) in MM patients (pts) who were eligible for autologous stem cell transplantation (ASCT) with DD4, CD8, and FOXP3 positive Tregs lymphocytes in bone marrow (BM) microenvironment, morphologic features of PC, clinical parameters, and outcomes. Immunohistochemical staining of BCL2, BCL2 E17, MCL1, BAX, CD4, CD8, FOXP3 and Ki67 was performed in BM biopsy of 36 newly diagnosed MM patients who were eligible for ASCT between 2012-2017 at University Hospital Merkur. Pearson's and Spearmans's coefficients of correlation, t-tests, one-way independent sample ANOVAs, mixed ANOVAs, moderation analyses, chi-square tests of independence and Cox's proportional hazards were used to analyze the data. Median age of pts was 57 years (95% CI 52-60), 15 pts were female with equal distribution in rISS I-III (12 pts). Twelve (33%) pts had high risk cytogenetics: del 13q34 (8 pts; 22%), del 17p (5 pts, 13,8%), t(4; 14) (4 pts; 11.1%); t(16;14) (4 pts; 11.1%); 4 pts (11.1%) had t(11;14). The median percentage of PC (PC ptc) in BM was 60 (95% CI 45 - 80); 25 pts (69.4%) had diffuse type of infiltration (TI) in BM; and 23 pts (63.8%) had high grade of PC differentiation (grade 2/3). The median percentage of Ki67 in PC was 3% of PC (95% CI 2 - 4, range 1 - 80). The median percentage of BCL2 positive PC in BM was 50 (95% CI 25 - 60) with high intensity of staining in 23 pts (63.8 %); BCL2 E17 positive PC in BM was 60 (95% CI 60 - 70) with high intensity of staining in 33 pts (91.6%); MCL1 positive PC in BM was 40 (95% CI 20 - 60) with high intensity of staining in 21 pts (58.3%); and BAX positive PC in BM was 17.5 (95% CI 6 - 40) with high intensity of staining in 18 pts (50%). Median CD4 positive T-cells was 8.5 (95% CI 4 - 18); CD8 T-cells 24 (95% CI 18 to 37); and FOXP3 Tregs 1 (95% CI 1 - 2, range 1 - 31). Nineteen pts (52.7 %) underwent tandem ASCT, 34 pts (94.4%) received VCD induction; 2 pts received MP induction; and 1 pts received VD induction. Overall response rate (≥partial response) to induction therapy was 94.4%; ≥ very good partial response rate was 80.5%. Overall response rate (≥partial response) to ASCT was 86.1%; complete response rate was 50%. Median overall survival time was 35.40 months (95% CI 26.61-165.39). Expression of BCL2, BCL2 E17, MCL1 and BAX proteins was significant and positively, moderately to strongly associated (p &lt; 0.05), except for BCL2E17 and BAX association (r (36) = 0.319, p = 0.058). BCL2 and MCL-1 expression was more correlated when PC were well differentiated, grade 1 (p = .016). Higher PC pct was correlated with higher intensity of BLC2 staining (t (34) = 2.51, p = 0.017). Patients with Ki67 &gt;10% had a higher PC pct in BM (t (34) = 2.04, p = 0.049). Higher grade of PC in BM correlated with higher PC pct in BM (t (34) = 2.63, p = 0.012). A higher MCL1 expression was found in MM with high-risk cytogenetic changes (t (34) = 2.09, p = 0.045). Cytoplasmic granular MCL1 staining had greater MLC 1 staining intensity than the diffuse TI (χ 2 (1) = 6.60, p = 0.017). Low BCL2, BCL2E17, MCL1 and BAX expression was found in MM stage rISS1 compared to rISS3 (p &lt; 0.05), and low BCL2 and MCL-1 expression in MM stage rISS1 compared to ISS2 (p &lt; 0.05). Patients with non-diffuse TI had worse response to ASCT compared to their response to induction therapy than patients with diffuse TI (χ 2 (2) = 6.39, p = 0.041). Although we observed high number of CD4 and CD8 T-cell in BM microenvironment and aberrant CD4:CD8 ratio in favor to CD8 positive T-cell, it had no impact on other variables in study. We found significant correlation between BCL2E17 expression and response to ASCT (r s = -0.417, p = 0.011), with higher protein expression being associated with worse outcomes. BCL2 contributed to worse survivability (χ 2 (1) = 7.18, p = 0.007), while BCL2 E17, MCL1 and BAX expression, as well as the number of CD4 and CD8 T-cells, did not predict survival (p &gt; 0.05). Results showed aberrant expression of the BCL2 family members that may plays a role in application strategies for MM therapy. Bcl-2 inhibits Bax but we found aberrant BAX expression in MM that may be result of pre-condition or hormesis like responses in stressful micro-environments that serve to increase apoptotic resistance. Further investigation of the role Bcl2- proteins family in the biology of MM and its impact on clinical and therapeutic outcomes is warranted. Disclosures Jaksic: Roche, Oktal-Pharma/Celtrion, Sandoz: Consultancy, Honoraria.

Supplementing hand washing with proper use of alcoholic hand rub in a special neonatal care unit in a large academic public health institute at Jabalpur, Madhya Pradesh, India

ObjectiveThe purpose was to increase use of alcoholic hand rub (AHR) in specialised newborn care unit (SNCU) to improve hand hygiene in order to reduce neonatal sepsis and mortality at Netaji Subhash Chandra Bose Medical College and Hospital, Jabalpur.DesignA prospective interventional and observational study.MethodologyWe formed a quality improvement (QI) team in our SNCU consisting of doctors, nurses, auxiliary staff and parents (a floating member) to improve proper use of AHR. To identify the barriers to the problem, we used fishbone analysis tool. The barriers which were not allowing the health providers to use AHR properly identified were amount of AHR in millilitres to be used per day per baby, how much and when the amount of AHR to be indented from the main store and what is the proper site to place the bottle. We used plan–do–study–act cycles to test and adapt solutions to these problems. Within 5–6 weeks of starting our project, AHR use increased from 44 mL to 92 mL per baby per day and this is sustained around 100 mL per baby per day for over 2 years now.ResultsSignificant decrease in neonatal mortality was observed (reduced from median of 41.0 between August 2016 and April 2018 to 24.0 between May 2018 and December 2019). The neonates discharged alive improved from 41.2 to 52.3 as a median percentage value. The percentage of babies who were referred out and went Left Against Medical Advice (LAMA) deceased too.ConclusionMultiple factors can lead to neonatal deaths, but the important factors are always contextual to facilities. QI methodology provides health workers with the skills to identify the major factors contributing to mortality and develop strategies to deal with them. Improving processes of care can lead to improved hand hygiene and saves lives.

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

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.