Highly specific enrichment of rare nucleic acid fractions using Thermus thermophilus argonaute with applications in cancer diagnostics.

Detection of disease-associated, cell-free nucleic acids in body fluids enables early diagnostics, genotyping and personalized therapy, but is challenged by the low concentrations of clinically significant nucleic acids and their sequence homology with abundant wild-type nucleic acids. We describe a novel approach, dubbed NAVIGATER, for increasing the fractions of Nucleic Acids of clinical interest Via DNA-Guided Argonaute from Thermus thermophilus (TtAgo). TtAgo cleaves specifically guide-complementary DNA and RNA with single nucleotide precision, greatly increasing the fractions of rare alleles and, enhancing the sensitivity of downstream detection methods such as ddPCR, sequencing, and clamped enzymatic amplification. We demonstrated 60-fold enrichment of the cancer biomarker KRAS G12D and ∼100-fold increased sensitivity of Peptide Nucleic Acid (PNA) and Xenonucleic Acid (XNA) clamp PCR, enabling detection of low-frequency (<0.01%) mutant alleles (∼1 copy) in blood samples of pancreatic cancer patients. NAVIGATER surpasses Cas9-based assays (e.g. DASH, Depletion of Abundant Sequences by Hybridization), identifying more mutation-positive samples when combined with XNA-PCR. Moreover, TtAgo does not require targets to contain any specific protospacer-adjacent motifs (PAM); is a multi-turnover enzyme; cleaves ssDNA, dsDNA and RNA targets in a single assay; and operates at elevated temperatures, providing high selectivity and compatibility with polymerases.

Clinical Utility of Plasma Cell-Free DNA in Adult Patients with Newly Diagnosed Glioblastoma: A Pilot Prospective Study.

PURPOSE: The clinical utility of plasma cell-free DNA (cfDNA) has not been assessed prospectively in patients with glioblastoma (GBM). We aimed to determine the prognostic impact of plasma cfDNA in GBM, as well as its role as a surrogate of tumor burden and substrate for next-generation sequencing (NGS). EXPERIMENTAL DESIGN: We conducted a prospective cohort study of 42 patients with newly diagnosed GBM. Plasma cfDNA was quantified at baseline prior to initial tumor resection and longitudinally during chemoradiotherapy. Plasma cfDNA was assessed for its association with progression-free survival (PFS) and overall survival (OS), correlated with radiographic tumor burden, and subjected to a targeted NGS panel. RESULTS: Prior to initial surgery, GBM patients had higher plasma cfDNA concentration than age-matched healthy controls (mean 13.4 vs. 6.7 ng/mL, P < 0.001). Plasma cfDNA concentration was correlated with radiographic tumor burden on patients' first post-radiation magnetic resonance imaging scan (ρ = 0.77, P = 0.003) and tended to rise prior to or concurrently with radiographic tumor progression. Preoperative plasma cfDNA concentration above the mean (>13.4 ng/mL) was associated with inferior PFS (median 4.9 vs. 9.5 months, P = 0.038). Detection of ≥1 somatic mutation in plasma cfDNA occurred in 55% of patients and was associated with nonstatistically significant decreases in PFS (median 6.0 vs. 8.7 months, P = 0.093) and OS (median 5.5 vs. 9.2 months, P = 0.053). CONCLUSIONS: Plasma cfDNA may be an effective prognostic tool and surrogate of tumor burden in newly diagnosed GBM. Detection of somatic alterations in plasma is feasible when samples are obtained prior to initial surgical resection.

Imaging and histopathologic correlates of plasma cell-free DNA concentration and circulating tumor DNA in adult patients with newly diagnosed glioblastoma.

BACKGROUND: Plasma cell-free DNA (cfDNA) concentration is lower in glioblastoma (GBM) compared to other solid tumors, which can lead to low circulating tumor DNA (ctDNA) detection. In this study, we investigated the relationship between multimodality magnetic resonance imaging (MRI) and histopathologic features with plasma cfDNA concentration and ctDNA detection in patients with treatment-naive GBM. METHODS: We analyzed plasma cfDNA concentration, MRI scans, and tumor histopathology from 42 adult patients with newly diagnosed GBM. Linear regression analysis was used to examine the relationship of plasma cfDNA concentration before surgery to imaging and histopathologic characteristics. In a subset of patients, imaging and histopathologic metrics were also compared between patients with and without a detected tumor somatic mutation. RESULTS: Tumor volume with elevated (>1.5 times contralateral white matter) rate transfer constant (K ep, a surrogate of blood-brain barrier [BBB] permeability) was independently associated with plasma cfDNA concentration (P = .001). Histopathologic characteristics independently associated with plasma cfDNA concentration included CD68+ macrophage density (P = .01) and size of tumor vessels (P = .01). Patients with higher (grade ≥3) perivascular CD68+ macrophage density had lower volume transfer constant (K trans, P = .01) compared to those with lower perivascular CD68+ macrophage density. Detection of at least 1 somatic mutation in plasma cfDNA was associated with significantly lower perivascular CD68+ macrophages (P = .01). CONCLUSIONS: Metrics of BBB disruption and quantity and distribution of tumor-associated macrophages are associated with plasma cfDNA concentration and ctDNA detection in GBM patients. These findings represent an important step in understanding the factors that determine plasma cfDNA concentration and ctDNA detection.