RESUMEN
The analysis of cell-free tumor DNA (ctDNA) and proteins in the blood of cancer patients potentiates a new generation of non-invasive diagnostics and treatment monitoring approaches. However, confident detection of these tumor-originating markers is challenging, especially in the context of brain tumors, in which extremely low amounts of these analytes circulate in the patient's plasma. Here, we applied a sensitive single-molecule technology to profile multiple histone modifications on millions of individual nucleosomes from the plasma of Diffuse Midline Glioma (DMG) patients. The system reveals epigenetic patterns that are 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 capture and quantify the tumor-originating oncoproteins, H3-K27M and mutant p53, from the plasma of children diagnosed with DMG. This single-molecule system allows for accurate molecular classification of patients, utilizing less than 1ml of liquid-biopsy material. Furthermore, we show that our simple and rapid detection strategy correlates with MRI measurements and droplet-digital PCR (ddPCR) measurements of ctDNA, highlighting the utility of this approach for non-invasive treatment monitoring of DMG patients. This work underscores the clinical potential of single-molecule-based, multi-parametric assays for DMG diagnosis and treatment monitoring.
RESUMEN
The analysis of cell-free DNA (cfDNA) in plasma provides information on pathological processes in the body. Blood cfDNA is in the form of nucleosomes, which maintain their tissue- and cancer-specific epigenetic state. We developed a single-molecule multiparametric assay to comprehensively profile the epigenetics of plasma-isolated nucleosomes (EPINUC), DNA methylation and cancer-specific protein biomarkers. Our system allows for high-resolution detection of six active and repressive histone modifications and their ratios and combinatorial patterns on millions of individual nucleosomes by single-molecule imaging. In addition, our system provides sensitive and quantitative data on plasma proteins, including detection of non-secreted tumor-specific proteins, such as mutant p53. EPINUC analysis of a cohort of 63 colorectal cancer, 10 pancreatic cancer and 33 healthy plasma samples detected cancer with high accuracy and sensitivity, even at early stages. Finally, combining EPINUC with direct single-molecule DNA sequencing revealed the tissue of origin of colorectal, pancreatic, lung and breast tumors. EPINUC provides multilayered information of potential clinical relevance from limited (<1 ml) liquid biopsy material.
Asunto(s)
Ácidos Nucleicos Libres de Células , Neoplasias , Nucleosomas , Humanos , Biomarcadores de Tumor , Ácidos Nucleicos Libres de Células/metabolismo , Metilación de ADN/genética , Epigénesis Genética/genética , Proteínas de Neoplasias/genética , Neoplasias/diagnóstico , Neoplasias/genética , Nucleosomas/genética , Nucleosomas/metabolismo , Imagen Individual de MoléculaRESUMEN
The COVID-19 pandemic raises the need for diverse diagnostic approaches to rapidly detect different stages of viral infection. The flexible and quantitative nature of single-molecule imaging technology renders it optimal for development of new diagnostic tools. Here we present a proof-of-concept for a single-molecule based, enzyme-free assay for detection of SARS-CoV-2. The unified platform we developed allows direct detection of the viral genetic material from patients' samples, as well as their immune response consisting of IgG and IgM antibodies. Thus, it establishes a platform for diagnostics of COVID-19, which could also be adjusted to diagnose additional pathogens.
Asunto(s)
Prueba de Ácido Nucleico para COVID-19/métodos , Prueba Serológica para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/inmunología , Imagen Individual de Molécula/métodos , Proteínas Virales/genética , Anticuerpos Antivirales/sangre , Secuencia de Bases , COVID-19/sangre , COVID-19/inmunología , COVID-19/virología , Prueba de Ácido Nucleico para COVID-19/normas , Prueba Serológica para COVID-19/normas , Ensayo de Inmunoadsorción Enzimática , Humanos , Sueros Inmunes/química , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Nasofaringe/virología , Poliproteínas/sangre , Poliproteínas/genética , ARN Viral/sangre , ARN Viral/genética , SARS-CoV-2/genética , Sensibilidad y Especificidad , Imagen Individual de Molécula/instrumentación , Proteínas Virales/sangreRESUMEN
The COVID-19 pandemic raises the need for diverse diagnostic approaches to rapidly detect different stages of viral infection. The flexible and quantitative nature of single-molecule imaging technology renders it optimal for development of new diagnostic tools. Here we present a proof-of-concept for a single-molecule based, enzyme-free assay for detection of SARS-CoV-2. The unified platform we developed allows direct detection of the viral genetic material from patients' samples, as well as their immune response consisting of IgG and IgM antibodies. Thus, it establishes a platform for diagnostics of COVID-19, which could also be adjusted to diagnose additional pathogens.
