MAESTRO-Pool Enables Highly Parallel and Specific Mutation-Enrichment Sequencing for Minimal Residual Disease Detection in Cohort Studies.

BACKGROUND: Tracing patient-specific tumor mutations in cell-free DNA (cfDNA) for minimal residual disease (MRD) detection is promising but challenging. Assaying more mutations and cfDNA stands to improve MRD detection but requires highly accurate, efficient sequencing methods and proper calibration to prevent false detection with bespoke tests. METHODS: MAESTRO (Minor Allele Enriched Sequencing Through Recognition Oligonucleotides) uses mutation-specific oligonucleotide probes to enrich cfDNA libraries for tumor mutations and enable their accurate detection with minimal sequencing. A new approach, MAESTRO-Pool, which entails pooling MAESTRO probes for all patients and applying these to all samples from all patients, was used to screen for 22 333 tumor mutations from 9 melanoma patients in 98 plasma samples. This enabled quantification of MRD detection in patient-matched samples and false detection in unmatched samples from other patients. To detect MRD, a new dynamic MRD caller was used that computes a probability for MRD detection based on the number of mutations and cfDNA molecules sequenced, thereby calibrating for variations in each bespoke test. RESULTS: MAESTRO-Pool enabled sensitive detection of MRD down to 0.78 parts per million (ppm), reflecting a 10- to 100-fold improvement over existing tests. Of the 8 MRD positive samples with ultra-low tumor fractions <10 ppm, 7 were either in upward-trend preceding recurrence or downward-trend aligning with response. Of 784 patient-unmatched tests, only one was found as MRD positive (tumor fraction = 2.7 ppm), suggesting high specificity. CONCLUSIONS: MAESTRO-Pool enables massively parallel, tumor-informed MRD testing with concurrent benchmarking of bespoke MRD tests. Meanwhile, our new MRD caller enables more mutations and cfDNA molecules to be tested without compromising specificity. These improve the ability for detecting traces of MRD from blood.

Priming agents transiently reduce the clearance of cell-free DNA to improve liquid biopsies.

Liquid biopsies enable early detection and monitoring of diseases such as cancer, but their sensitivity remains limited by the scarcity of analytes such as cell-free DNA (cfDNA) in blood. Improvements to sensitivity have primarily relied on enhancing sequencing technology ex vivo. We sought to transiently augment the level of circulating tumor DNA (ctDNA) in a blood draw by attenuating its clearance in vivo. We report two intravenous priming agents given 1 to 2 hours before a blood draw to recover more ctDNA. Our priming agents consist of nanoparticles that act on the cells responsible for cfDNA clearance and DNA-binding antibodies that protect cfDNA. In tumor-bearing mice, they greatly increase the recovery of ctDNA and improve the sensitivity for detecting small tumors.