Biology subjects

Deubzer, H. E.

Publications and source records attributed to Deubzer, H. E..

4 recordsLinked to original sources

bioRxiv2026

CRISPR-dPCR: A unified multiplex digital PCR framework for quantitative characterization of targeted knock-in

Accurate characterization of CRISPR knock-in requires distinguishing site-specific integration from total donor abundance and donor-derived background while measuring editing at the target site. Here, we present CRISPR-dPCR, a modular multiplex digital PCR framework combining a locked nucleic acid-enhanced cut-site assay with junction-based detection and two orthogonal short-amplicon strategies: Poisson linkage and homology-arm subtraction. We evaluated assay modules across genomic loci, neuroblastoma models and primary human T cells, with selected assays compared on two dPCR platforms. In genotype-defined clones and mixtures with low residual donor, homology-arm subtraction most closely recovered expected knock-in abundance. Linkage provided highly specific evidence of target-cargo association without junction-spanning amplification, whereas junction assays supplied boundary-specific confirmation but were more sensitive to amplicon length and multiplexing. Five-channel panels integrated cut-site editing, donor abundance, copy-number normalization and site-specific knock-in in a single reaction. Residual donor DNA limited quantitative interpretation, particularly in early post-editing samples. Modular extensions quantified defined unintended outcomes, including chromosomal translocations. CRISPR-dPCR integrates complementary measurements into a scalable analytical framework for genome-editing research, cell and gene therapy product development and applications in longitudinal clinical monitoring of targeted integration and transgene persistence.

molecular biology↗

bioRxiv2026

Circulating extracellular vesicles display immunotherapeutic targets and complement circulating tumor DNA in high-risk neuroblastoma

Extracellular vesicles (EVs) offer a minimally invasive window into tumor biology through surface molecules reflecting their cellular origin. Here, we investigated whether neuroblastoma-derived EVs retain tumor-associated surface markers and provide complementary information to established biomarkers in children with neuroblastoma. EVs from neuroblastoma cell lines were characterized using orthogonal biochemical, morphological and biophysical approaches, followed by analysis of GD2, L1CAM and NCAM1 surface expression. EV-associated markers were subsequently evaluated in blood plasma-derived EVs from children with neuroblastoma and pediatric controls and compared with clinical and circulating cell-free DNA (cfDNA) parameters. Neuroblastoma EVs displayed GD2, L1CAM and NCAM1 in patterns reflecting donor-cell surface expression, supported by a positive correlation between cellular and EV marker abundance (Spearman {rho}=0.74, P<0.0001). Genetic disruption of GD2 biosynthesis resulted in corresponding loss of EV-associated GD2, further supporting its cellular origin. In plasma, GD2-, L1CAM- and NCAM1-positive EVs were enriched in patients with high-risk neuroblastoma and correlated with LDH level, soft tissue disease burden (GD2, L1CAM) and cfDNA concentration (GD2, NCAM1). Longitudinal analyses demonstrated dynamic and partially discordant changes in EV- and cfDNA-markers. These findings identify tumor-associated EV surface phenotypes as complementary circulating biomarkers and support their further evaluation for minimally invasive monitoring of neuroblastoma evolution.

cancer biology↗

bioRxiv2026

Neuroblastoma-derived small extracellular vesicles retaincellular identity and adrenergic/mesenchymal state signatures

Neuroblastoma is a heterogeneous pediatric malignancy characterized by distinct genomic subgroups and cell-state plasticity between adrenergic (ADRN) and mesenchymal (MES) phenotypes. While small extracellular vesicles (sEVs) mediate pre-metastatic niche priming, the degree to which sEV proteomes reflect parental genomic and phenotypic identity remains unresolved. Here, we isolated sEVs from six neuroblastoma cell models (IMR-5/75, BE(2)-C, GI-ME-N, CLB-GA, LAN-6, and SK-N-FI) representing three genomic subgroups (MYCN-amplified, TERT-rearranged, and unaltered) using OptiPrep density gradient centrifugation followed by size exclusion chromatography and ultrafiltration. High-resolution mass spectrometry of sEV proteomes revealed that sEVs carry highly reproducible protein signatures that tightly correlate with the parental genomic subgroups. Analyzing paired cell/sEV samples, we demonstrate cell-to-EV concordance of lineage-specific markers: ADRN sEVs selectively enriched L1CAM and dopamine {beta}-hydroxylase (DBH), whereas MES sEVs enriched SERPINE1 and type III collagen (COL3A1). These findings indicate that neuroblastoma-derived sEVs systematically preserve both the genomic landscape and the lineage transdifferentiation states of their cells of origin. This study establishes a robust molecular foundation for utilizing sEV-based proteomics as a surrogate to track cellular plasticity and for studying sEV-mediated microenvironmental remodeling.

cancer biology↗

bioRxiv2026

Quantitative and sensitive neuroblastoma minimal residual disease detection using extrachromosomal DNA (ecDNA) breakpoints

Sensitive detection of minimal residual disease (MRD) remains a major unmet need in high-risk neuroblastoma. MYCN amplification, a hallmark of high-risk disease, typically occurs on extrachromosomal DNA (ecDNA), but the potential of ecDNA-associated genomic rearrangements for individualized MRD monitoring has not been fully exploited. Here, we applied neuroblastoma-specific hybrid capture-based panel sequencing to identify patient-unique breakpoints within MYCN amplicons, and used Circle-seq and Nanopore sequencing to resolve the extrachromosomal amplicon structure in representative samples. Analysis of 8 neuroblastoma cell lines and 22 primary tumors identified 69 tumor-specific breakpoints. Those selected for assay development were validated by breakpoint-specific PCR and Sanger sequencing. Breakpoints detected in primary tumors remained detectable at relapse, supporting their stability as MRD markers. Breakpoint-specific real-time quantitative PCR and droplet digital PCR detected these junctions in bone marrow aspirates with high specificity and reached sensitivities down to a tumor DNA fraction of 10^-6. We applied this approach to 53 serial bone marrow aspirates from 14 patients with high-risk neuroblastoma to monitor MRD dynamics, resolving treatment response and molecular persistence. In six samples, breakpoint-positive DNA was detected in bone marrow that was negative by conventional cytology and immunocytology, highlighting the added value of molecular monitoring. Together, these findings establish ecDNA breakpoint-based detection as a strategy for MRD assessment in neuroblastoma, that is, in principle, applicable to any ecDNA-amplified oncogene.

cancer biology↗