Liquid Biopsy – Priming Agents Transiently Reduce the Clearance of Cell-Free DNA to Improve Liquid Biopsies

Martin-Alonso et al., 2024, Science 383, 274

During the course of my Master’s degree in Cancer Biology at Imperial College London, UK, one of the projects I have been working on was titled “Searching for oncogenic mutations in circulating tumor DNA (ctDNA) from gestational trophoblastic tumors (GTT)”. The rationale behind this project was to identify oncogenic driver mutations in cfDNA and tumour DNA samples of patients with gestational and non-gestational trophoblastic tumors.

Whilst I was working in my current lab, I came across circulating tumor DNA in the context of pediatric high-grade gliomas. Our lab as a whole have largely been involved in establishing patient-derived primary tumor samples from patient biopsies. When we collect biopsies, we have also been collecting patient blood samples too. Whilst we have always wanted to effectively detect ctDNA from patient’s plasma samples, we were only able to detect a minimal amount of ctDNA from cerebrospinal fluid (CSF) instead.

I therefore went into literature to look for alternative methods and came across this journal which I really wanted to introduce to you and also wanted to encorporate into future studies in pHGG settings, if possible. The paper introduced a technology which I believe no-one has thought of or at least put it into action before.

In this paper, Martin-Alonso and her colleagues states that it is important to realise that currently, the efforts to improve the sensitivity for detecting ctDNA have been largely shifted towards ex-vivo sequencing and analysis method whilst the intrinsic challenge lies in the in-vivo scarcity of ctDNA. Therefore, the group hypothesized that transiently attenuating ctDNA clearance in-vivo prior to blood draw will increase the level of cfDNA in circulation and therefore will increase the amount of ctDNA recovered from a blood draw.

They developed two different intravenous priming agents given 1 to 2 hours prior to blood draw which increases the recovery of ctDNA by >10 fold. The two orthogonal priming agents are:

1. Competing nanoparticle (SPE liposome)
2. DNA-binding priming agent (monoclonal antibodies, IgG2a mAb (3519), variant aST3)

These agents each intervene in-vivo natural mechanisms for clearing cfDNA:

1. Uptake by liver-resistant cells of the mononuclear-phagocyte system (MPS)
2. Degradation by circulating nucleases.

This is highly relevant in terms of pediatric brain tumors. To enable precision medicine for pHGG patients, the underlying epigenetic modifications are characterised through methylation profiling of tumor biopsy samples. However, due to highly invasive nature of procedure and limitations in reaching critical tumor locations (e.g. Pons for DIPG), routine sampling is avoided. Also, due to the highly-diffused nature of the disease, it is often really difficult to preempt the emerging tumor via MRI. Therefore, a non-invasive alternative strategy of identifying mutations from tumor in cell-free DNA (cfDNA) from patient’s plasma, or circulating tumor DNA, has emerged as a promising biomarker for advanced solid tumors.

Even though previous benchmarking showed that somatic copy number alterations (SCNAs) were detectable using 0.1x whole genome sequencing of cfDNA in solid tumours, minimal research has been done in the context of pHGGs. The main reason behind this is the obstacle posed by the blood-brain barrier (BBB) which possibly limits the release of tumour-derived DNA into the bloodstream, making it more difficult to detect and analyse SCNAs (although SCNAs better represent metastatic tumors). However, despite the highly transient nature of ctDNA with half-life of less than two hours, there are studies claiming that ddPCR allows monitoring of single nucleotide variants (SNVs) associated with histone H3 mutations in both blood and cerebrospinal fluid (CSF) of pHGGs whilst some studies counter-argue these findings with negative results for cfDNA detection.

In line with this above statement, I will share another journal with you which investigates ctDNA of pHGG patients’ blood and CSF samples – written by our previous PhD student – please follow along to this POST!

For your reference, the below is the paper (Martin-Alonso et al.) and my notes on the paper for anyone interested!

The study very well describes the ethical and biological considerations they have taken into account which are important when proposing newly developed therapeutic interventions. Generating empty liposomes with cholesterol (50 mol%), such as lipids sometimes used in the FDA-approved liposomal formulations (e.g. [1,2-dipalmitoylsn-glycero-3-phosphoethanolamine-N-(succinyl) (SPE), 1,2-distearoyl-sn-glyccero-3-phospho-(1′-rac-glycerol)(DSPG), or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)]) and designing the average hydrodynamic diameter of the liposomes to match the size of the fenestrae of murine liver capillaries (230-260 nm) are some exampes. However, since these modulations are based on murine models, I am excited to keep up with their future research to see how the team would eventually apply their priming agents in the context of a human.