MR-guided focused ultrasound liquid biopsy enriches circulating biomarkers in patients with brain tumors

Abstract

Background: Liquid biopsy is promising for early detection, monitoring of response, and recurrence of cancer. The blood-brain barrier (BBB) limits the shedding of biomarker, such as cell-free DNA (cfDNA), into the blood from brain tumors, and their detection by conventional assays. Transcranial MR-guided focused ultrasound (MRgFUS) can safely and transiently open the BBB, providing an opportunity for less-invasive access to brain pathology. We hypothesized that MRgFUS can enrich the signal of circulating brain-derived biomarkers to aid in liquid biopsy.

Methods: Nine patients were treated in a prospective single-arm, open-label trial to investigate serial MRgFUS and adjuvant temozolomide combination in patients with glioblastoma (NCT03616860). Blood samples were collected as an exploratory measure within the hours before and after sonication, with control samples from non-brain tumor patients undergoing BBB opening (BBBO) alone (NCT03739905).

Results: Brain regions averaging 7.8 ± 6.0 cm3 (range 0.8-23.1 cm3) were successfully treated within 111 ± 39 minutes without any serious adverse events. We found MRgFUS acutely enhanced plasma cfDNA (2.6 ± 1.2-fold, P < .01, Wilcoxon signed-rank test), neuron-derived extracellular vesicles (3.2 ± 1.9-fold, P < .01), and brain-specific protein S100b (1.4 ± 0.2-fold, P < .01). Further comparison of the cfDNA methylation profiles suggests a signature that is disease- and post-BBBO-specific, in keeping with our hypothesis. We also found cfDNA-mutant copies of isocitrate dehydrogenase 1 (IDH1) increased, although this was in only one patient known to harbor the tumor mutation.

Conclusions: This first-in-human proof-of-concept study shows MRgFUS enriches the signal of circulating brain-derived biomarkers, demonstrating the potential of the technology to support liquid biopsy for the brain.

Keywords: blood-brain barrier; brain tumor; focused ultrasound; glioblastoma; liquid biopsy.

Fig. 1

Opening of the blood-brain barrier in large brain regions is achieved with transcranial MR-guided focused ultrasound. (A) Schematic diagram of MRgFUS device and study design with BBBO procedures overlapping adjuvant chemotherapy regimen in patients with glioblastoma after surgical resection and concurrent chemoradiation. Blood samples are collected immediately before and after sonications on the same day of the procedure. (B) Representative images of the lesions (yellow circles) on baseline MRI for each patient. (C) Ultrasound can be targeted specifically to an operator-defined contour. The green polygon represents one of many target contours (others not shown). The color map indicates the cavitation dose detected by device upon ultrasound delivery. In (D), the top panel shows contrast extravasation in the areas of increased BBB permeability after MRgFUS for P5 (yellow arrow). The bottom panel shows partial restoration of the BBB integrity approximately 24 hours later, in comparison to the top panel and baseline image of the tumor in (B). (E) Intensity difference maps further show increased BBB permeability in P5 within large regions spatially distributed in the tumor and tumor margins. Heterogeneous changes in contrast extravasation are partially due to the underlying tumor. Abbreviations: BBB, blood-brain barrier; BBBO, BBB opening; MRgFUS, MR-guided focused ultrasound.

Fig. 2

Transient blood-brain barrier opening enriches signal of circulating biomarkers. (A) Plasma cfDNA concentration was significantly enhanced after noninvasive MRgFUS BBBO. Each data point represents the mean cfDNA concentration for each patient. (B) The fold change in plasma cfDNA concentration is graphed over the course of adjuvant chemotherapy. Each gray line represents a single patient. The red line indicates the group mean at each cycle. (C) The concentration of single nucleosome length cfDNA was also increased after BBBO. (D) The fold change in plasma cfDNA concentration at each cycle is plotted against the volume of BBBO, demonstrating a positive Spearman’s correlation between the two variables. Each point represents the data at one cycle. (E) L1CAM and NCAM double-positive extracellular vesicles are increased after BBBO. (F) Plasma S100b levels are significantly increased after BBBO. In (C), (E), and (F), measurements were taken pre- and post-BBBO at only one cycle per patient, given the availability of samples. Abbreviations: BBBO, BBB opening; cfDNA, cell-free DNA; MRgFUS, MR-guided focused ultrasound; ndEV, neuron-derived extracellular vesicle.

Fig. 3

Post-BBBO cfDNA has distinctive disease-specific signature. (A) Principal component analysis shows a separation of the methylation signature of pre- and post-BBBO cfDNA. (B) Results of a functional enrichment analysis of the differentially hypomethylated probe set based on the Human Protein Atlas. (C) Results of a principal component analysis of methylation data with cfDNA from non-brain tumor patients’ post-BBBO as controls suggest disease-specific signatures. Abbreviations: BBBO, BBB opening, cfDNA, cell-free DNA; GBM, glioblastoma.

Fig. 4

A MRgFUS-based platform for both enhanced drug delivery and liquid biopsy. Concept diagram of a MRgFUS-based platform for both enhancing drug delivery and liquid biopsy to deliver personalized medicine for patients with brain tumors. MRgFUS can be combined with repeated doses of anti-tumor therapy (1), each time providing an opportunity to collect blood samples containing analytes derived from the pathology in the sonicated areas (2). After preprocessing (3) and extraction for specific markers (4), commercially available techniques (5, eg, droplet digital PCR). Biological changes in the pathology might inform a change in anti-tumor therapy (6). Abbreviation: MRgFUS, MR-guided focused ultrasound.