Single-molecule systems for the detection and monitoring of plasma-circulating nucleosomes and oncoproteins in diffuse midline glioma

The analysis of cell-free tumor DNA (ctDNA) and proteins in the blood of patients with cancer potentiates a new generation of non-invasive diagnostic approaches. However, confident detection of tumor-originating markers is challenging, especially in the context of brain tumors, where these analytes in plasma are extremely scarce. Here, we apply a sensitive single-molecule technology to profile multiple histone modifications on individual nucleosomes from the plasma of patients with diffuse midline glioma (DMG). The system reveals epigenetic patterns unique to DMG, significantly differentiating this group of patients from healthy subjects or individuals diagnosed with other cancer types. We further develop a method to directly quantify the tumor-originating oncoproteins, lysine 27 to methionine substitution in histone H3 (H3-K27M) and mutant p53, from <1 mL of plasma, allowing for the accurate molecular classification of patients with DMG. We show that our strategy correlates with MRI and droplet-digital PCR (ddPCR) measurements of ctDNA, highlighting the clinical potential of single-molecule-based, multi-parametric assays for DMG diagnosis and treatment monitoring. • Liquid biopsy single-molecule systems for the diagnosis of diffuse midline gliomas • Patients with glioma show distinct epigenetic patterns on plasma-circulating nucleosomes • Capture and quantification of oncoproteins originating directly from the tumor • Rapid and non-invasive monitoring of treatment response correlating with MRI Erez et al. adapt single-molecule technologies to decode histone modification patterns on plasma-circulating nucleosomes from the blood of patients with pediatric high-grade glioma. They further develop systems to directly detect the tumor-originating oncoproteins H3-K27M and mutant p53, allowing for molecular diagnosis and treatment monitoring of this aggressive cancer.