Understanding Liquid Biopsy for Brain Cancer
Any cancer diagnosis is devastating, but the discovery of a brain tumor comes with a unique set of challenges for both patient and doctor. For the approximately 25,000 individuals diagnosed with a primary tumor of the brain and spinal cord in the US, the 5-year survival is about 36%—one of the lowest across different cancers (1). In this article, we discuss the potential role of liquid biopsy for management of brain cancer, current research in this space, and remaining questions in the field.
Current strategies for brain cancer management
Brain tumors are typically diagnosed by MRI or CT scan, with a tumor resection or biopsy obtained when possible. Information from the tumor tissue is critical for accurate diagnosis, treatment decision-making, and prognosis, because brain tumors can be highly molecularly diverse. However, a surgical biopsy or resection may be dangerous or impossible for some patients because of the tumor location in the brain.
Most brain cancers are treated by maximal surgical resection followed by radiation, with some patients also receiving chemotherapy. During treatment, brain tumors are conventionally assessed by imaging. However, radiographic methods are a highly imperfect means of assessing tumor response: the changes detected are time-delayed and can be difficult to distinguish from pseudoprogression. Consequently, there is an urgent need for clinically validated and minimally invasive biomarkers that can inform diagnosis, treatment decisions, and treatment response in neuro-oncology.
Liquid biopsy is an exciting potential addition of the standard-of-care (SOC) for brain cancer patients, providing a host of new information about the molecular characteristics of the tumor and putative biomarkers to track response to therapy. Figure from Escuerdo et al. (2021), found here.
Strategies for liquid biopsy in brain cancer
Liquid biopsy has emerged as a highly promising non-invasive method for cancer diagnosis, treatment monitoring, and recurrence surveillance. No currently available commercial liquid biopsy assays are FDA-approved for use in brain cancer (2), but many research groups have worked to understand the value of using plasma and cerebrospinal fluid (CSF) in the management of brain tumors.
The first studies of liquid biopsy for brain cancer analyzed patient plasma samples. At first, plasma was thought to be an unreliable source of brain tumor-derived cell-free DNA due to the blood-brain barrier, which blocks egress of tumor cells and tumor cell contents (3–5). One early study from 2014 found that less than 10% of patients with glioma, the most common primary brain cancer in adults, have detectable circulating tumor DNA (ctDNA), as compared to >75% of patients with advanced solid tumors outside the brain (6). However, several groups have since shown that the ctDNA burden in plasma is influenced by multiple factors including the type of brain tumor, tumor burden, and timing of collection relative to treatment (7). Some success has been achieved in diagnosis and longitudinal monitoring of brain cancers by targeting specific mutations in plasma ctDNA (5,8–11), and a first-in-human study was recently published in which focused ultrasound was used to increase the permeability of the blood-brain barrier and the yield of cell-free DNA in the plasma (12). Nevertheless, the relative paucity of ctDNA in blood and the need for targeted assays make this a challenging avenue for further development (5,13).
Cerebrospinal fluid (CSF) is found within the ventricles, around the brain and spinal cord, and between the meninges. Image from National Cancer Institute.
CSF has been identified as a more promising biofluid for liquid biopsy because the ctDNA content is orders of magnitude higher than in plasma and has been shown to be molecularly faithful to the tumor (14,15). CSF is a clear fluid secreted by a tissue called the choroid plexus in the ventricles, or hollow spaces, of the brain. CSF flows into and around the brain and spinal cord and is also found between the meninges (tissues that cover the brain and spinal cord). CSF can be obtained by lumbar puncture– the most common method– or directly from ventricles during brain surgery. Unlike plasma, CSF is typically low in white blood cells and consequently does not contain the high levels of background non-cancerous DNA that complicate accurate detection of ctDNA in the blood.
Recent studies of liquid biopsy in brain cancer
As with other solid tumors, analysis of ctDNA in brain cancer can be performed at a number of points along the patient journey, including at the time of diagnosis, post-surgery for recurrence monitoring and molecular residual disease (MRD), and during systemic treatment to track tumor response and evolution. The field is in the process of understanding how and when liquid biopsies can provide the most clinical utility for brain cancer patients.
A recent report of real-time CSF liquid biopsy to inform patient care was published recently by a team at Memorial Sloan Kettering Cancer Center (16). Miller et al. collected and analyzed CSF samples at the time of diagnosis, at recurrence, over the course of treatment, and during surveillance. Importantly, while 14/15 CSF samples shared mutations with the matched tumor biopsy, 8 samples contained novel mutations likely representing tumor heterogeneity or evolution, some of which were potentially actionable mutations. This work and other similar studies are advancing our understanding of how liquid biopsy can be embedded into the current standard-of-care for optimal patient benefit (17,18). Below, we discuss progress to date for the major stages at which liquid biopsy has been studied in brain cancer.
Liquid biopsy in brain cancer has multiple use cases along the patient journey. The field is still working to understand the most complementary uses of this technology in the context of the current standard-of-care, as shown above. Figure created with Biorender.
Diagnosis
When tissue biopsy is impossible due to location, CSF liquid biopsy via lumbar puncture would be a highly valuable tool for brain cancer diagnosis. In this use case, the assay would need to be highly specific in order to allow a high-confidence molecular diagnosis. Digital droplet PCR (ddPCR) targeting cancer-specific known mutations is currently the most sensitive approach, but targeted NGS panels have also been used to detect ctDNA with good success and also have the advantage of detecting patient-specific mutations that could serve as biomarkers in subsequent testing (13). In addition, routine liquid biopsy collected at the time of tissue biopsy could provide additional molecular insight into the tumor, potentially informing treatment decision (16).
Recurrence and MRD
In the solid tumor space, much work has been done developing assays to detect MRD as an indicator of possible cancer recurrence. In the neuro-oncology space, research into liquid biopsy for MRD detection is in early stages. One recent study by Liu et al. measured tumor-associated copy-number variation in CSF as an MRD surrogate in pediatric medulloblastoma patients and showed that patients with persistent MRD had a significantly higher risk of progression (19). Molecular MRD detection preceded radiographic evidence of progression in half of patients who relapsed, underscoring the potential value of liquid biopsy in allowing impactful intervention at a lower tumor burden. A major continuing challenge with MRD detection in brain cancer is the observed high ctDNA burden in some patients (indicative of positive MRD) who do not relapse—more research is needed on the most informative timepoints for MRD liquid biopsy in the context of different tumor types and treatments (17).
Treatment Monitoring
Given the current limitations of MRI scans for brain cancer monitoring, there is great interest in using liquid biopsy to assess tumor response in conjunction with radiographic imaging. Several studies have shown the utility of using serial liquid biopsy measurements to inform therapeutic decision-making (4). One study used a targeted mutation panel to screen longitudinal samples of CSF and plasma from 48 patients with pediatric glioma and found a significant decrease in ctDNA burden associated with response to radiotherapy in 83% of patients (10). Similar findings were reported by Cantor et al. in their analysis of plasma and CSF liquid biopsy collected by lumbar puncture during a phase I glioma clinical trial (20). Patients with a decrease in CSF variant allele fraction (VAF) after initial treatment had a significantly longer progression-free survival, and VAF spikes of >25% increase over baseline were found to precede tumor progression in 8/16 cases. The next step for the field will be designing and commercializing the sensitive, cancer-specific mutation panels needed to show utility in a real-time clinical trial setting.
Open questions in liquid biopsy for brain cancer
Many of the fundamental aspects of ctDNA biology are not fully understood. For example, the factors that influence ctDNA release, abundance, and kinetics, both in CSF and plasma, require further study. It is clear there is significant inter-patient variation in the baseline abundance of ctDNA in both biofluids, which must be better understood and accounted for as liquid biopsy evolves in this field (4,13). Similarly, the effects of different types of treatment on the release of ctDNA is a remaining unknown, and serves as a significant confounding variable in attempting to correlate tumor burden with ctDNA characteristics during therapy (4,13). And finally, although CSF liquid biopsy is showing great promise, the fact remains that undergoing a lumbar puncture (or a series of lumbar punctures) is a much more onerous process for the patient than a blood draw. Continued work on increasing the sensitivity and specificity of plasma-based assays is needed to open the door for this type of biopsy in brain cancer.
The Blood Profiling Atlas in Cancer (BLOODPAC) Consortium recently initiated a subject-matter-expert working group on liquid biopsy for brain tumors with the goal of pooling knowledge across major clinical, academic, and industry groups to accelerate progress in the field. The group is working on identifying key preanalytical variables relevant for cerebrospinal fluid (CSF) ctDNA genomic profiling, with the goal of increasing the quality of the data available and standardizing protocols for CSF acquisition, storage, and processing. The working group is also beginning the process of establishing a CSF dataset within the BLOODPAC Data Commons, one of the largest repositories of liquid biopsy data, to allow researchers to perform meta-analyses and drive continued discovery and development.
Works Cited
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3. De Mattos-Arruda, L. et al. Cerebrospinal fluid-derived circulating tumour DNA better represents the genomic alterations of brain tumours than plasma. Nature Communications 2015 6:1 6, 1–6 (2015).
4. Wadden, J., Ravi, K., John, V., Babila, C. M. & Koschmann, C. Cell-Free Tumor DNA (cf-tDNA) Liquid Biopsy: Current Methods and Use in Brain Tumor Immunotherapy. Front Immunol 13, 1505 (2022).
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12. Focused Ultrasound–Enhanced Blood Test for Brain Tumors: World-First Results Published - Focused Ultrasound Foundation. https://www.fusfoundation.org/posts/focused-ultrasound-enhanced-blood-test-for-brain-tumors-world-first-results-published/.
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14. Escudero, L., Martínez-Ricarte, F. & Seoane, J. ctDNA-Based Liquid Biopsy of Cerebrospinal Fluid in Brain Cancer. Cancers (Basel) 13, (2021).
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16. Miller, A. M. et al. Next-generation sequencing of cerebrospinal fluid for clinical molecular diagnostics in pediatric, adolescent and young adult brain tumor patients. Neuro Oncol 24, 1763 (2022).
17. Miller, A. M. et al. Tracking Tumor Evolution in Glioma through Liquid Biopsies of Cerebrospinal Fluid. Nature 565, 654 (2019).
18. Orzan, F. et al. Liquid Biopsy of Cerebrospinal Fluid Enables Selective Profiling of Glioma Molecular Subtypes at First Clinical Presentation. Clinical Cancer Research 29, OF1–OF15 (2023).
19. Liu, A. P. Y. et al. Serial assessment of measurable residual disease in medulloblastoma liquid biopsies. Cancer Cell 39, 1519-1530.e4 (2021).
20. Cantor, E. et al. Serial H3K27M cell-free tumor DNA (cf-tDNA) tracking predicts ONC201 treatment response and progression in diffuse midline glioma. Neuro Oncol 24, 1366–1374 (2022).
