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Beyond Oncology – Liquid Biopsy In Neurological Disorders

This feature is written using content from our upcoming Liquid Biopsy report, due to be released late January 2024.

Liquid biopsy is a hot topic in the cancer world. But what are the other applications of this tech, and how much wider is the scope?

One clear example is the early detection and surveillance of neurological disorders, which massively benefit from the fast and non-invasive nature of liquid biopsy assays1. As populations worldwide begin to age and the incidence of neurological disease rises, identifying biomarkers to diagnose and inform treatment is becoming an increasingly urgent concern.

Research into CSF biomarkers for neurological disease has been progressing steadily over the last decade. Interestingly, despite CSF analysis via lumbar puncture being a common procedure for neurological disorder diagnosis, the term “liquid biopsy” has only just begun to be commonly used in the field – perhaps thanks to the increased development and interest in blood-based liquid biopsy techniques in oncological care. The rapid advancement of liquid biopsy techniques has made blood-based analysis for neurological disease a promising goal, as it is less-invasive and more repeatable than CSF sampling.

What are neurological disorders?

The term “neurological disorder” covers a range of conditions and diseases, all of which affect the central or peripheral nervous system. The central nervous system (CNS) is comprised of the brain and spinal cord; the core components for receiving, processing and responding to sensory information in the form of action potentials. Conversely, the peripheral nervous system (PNS) refers to the network of nerves and ganglia that are present throughout the body, responsible for relaying signals between the limbs and organs to the CNS. Neurodegenerative diseases are some of the most well-known examples of neurological disorders. These diseases are characterised by neuron death in the CNS and PNS, resulting in progressive cognitive impairment, loss of speech and mobility, and deficits in other basic functions2. Across all the major neurodegenerative diseases (such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and amyotrophic lateral sclerosis), disease pathogenesis is caused by mitochondrial dysfunction, inflammation and oxidative stress that impacts neuronal health and survival. Importantly, the major risk factor in the development of neurodegenerative conditions is age, with 1 in 10 adults over the age of 65 being diagnosed with Alzheimer’s disease3.

How could liquid biopsy be used to understand Alzheimer’s disease?

Given its worldwide incidence, Alzheimer’s disease poses a major threat to public health. Yet, despite intense research efforts, it remains difficult to diagnose and treat, due to its complex pathology and late onset of symptoms. To this end, it’s thought that up to 60% of a patient’s neurons are already dead by the time they begin to present with visible symptoms4. This severely limits the therapeutic options available to the patient.

Caused by the loss of neurons and synapses (small structures responsible for the propagation of electrical impulses between neurons) within the cerebral cortex, Alzheimer’s disease is characterised by two histological abnormalities – the presence of Aβ plaques and neurofibrillary tangles within the brain. These classic hallmarks of disease are often used for post-mortem diagnosis. Unsurprisingly, Alzheimer’s biomarkers are a major area of research. One particular area of interest is nucleic acid biomarkers, where several types of non-coding RNA and DNA have been illustrated to be detectable in various biological fluids. Given their high stability and abundance, non-coding RNAs such as microRNAs (miRNAs) and circular RNAs (circRNAs) have recently come into the limelight for early AD detection5. However, more traditional DNA biomarkers such as cfDNA also hold promise, with a recent study pairing methylation analysis and nanopore sequencing to profile neuron-derived cfDNA biomarkers in the blood plasma, identifying Alzheimer’s patients with 100% accuracy6.

Unsurprisingly, liquid biopsy approaches that detect Aβ-42 and Aβ-40 biomarkers in the blood have dominated the field in recent years. To date, several liquid biopsy assays have been developed for Alzheimer’s disease diagnosis. For example, the Lumipulse G β-Amyloid Ratio (1-42/1-40) assay (FUJIREBIO) recently came onto the market as an CSF-based in vitro diagnostic tool. The assay, which received FDA marketing approval in 20227, measures the ratio of β-amyloid 1-42 and β-amyloid 1-40 within CSF samples, with a high ratio indicating the possible development of Aβ plaques. Similarly, this year Roche received FDA clearance for two Alzheimer’s CSF assays: Elecsys beta-Amyloid (1-42) CSF II (Abeta42) and Elecsys Total-Tau CSF assays (tTau). Like the Lumipulse assay, these tests also measure specific biomarker rations (pTau181/Abeta42 ratio or tTau/Abeta42 ratio) that are reflective of Alzheimer disease pathology. Additionally, Roche also have a blood-based liquid biopsy test for Alzheimer’s disease in development, the Elecsys® Amyloid Plasma Panel, which is being developed in collaboration with Eli Lilly52. Other blood-based assays have also been gaining traction over recent years. The Simoa® phospho-Tau 181 (pTau-181) blood test (Quanterix) is a notable example that has received FDA breakthrough designation for Alzheimer’s disease diagnostic evaluation8. This assay measures the pTau-181 concentration in human serum and plasma at high sensitivity, with phosphorylated tau protein concentrations being associated with cognitive decline and neurodegenerative disease9.

How can liquid biopsy help us understand Parkinson’s disease?

After Alzheimer’s disease, Parkinson’s disease is the second most common neurodegenerative condition worldwide10. Also characterised by neuronal death (in the midbrain), it’s a condition that causes mobility issues, such as tremor and rigidity, with dementia being a common development in the later stages of disease progression. Notably, the brains of Parkinson’s patients contain aggregates of a pre-synaptic protein called alpha-synuclein (aSyn), forming Lewy Bodies. This prominent hallmark of the disease is commonly the target of liquid biopsy analysis. In particular, one landmark study in 2020 analysing plasma samples from both Parkinson’s patients and healthy patients aimed to identify whether aSyn was a clinically relevant biomarker for early disease detection. Through the analysis of aSyn extracted from plasma-derived exosomes, the researchers determined that aSyn levels were significantly higher in patients with early-stage PD compared with the healthy controls11. Furthermore, increasing aSyn concentrations were also associated with higher risk for motor symptom progression, illustrating that aSyn is a helpful biomarker for Parkinson’s disease progression.

In this interview, we chat to Professor Andy Tao at Purdue University, to learn how he’s discovering new biomarkers in the fight against Parkinson’s disease.

“My name is Andy Tao. I’m a Professor at Purdue University, and I’m also a Cancer Centre member here, and the Faculty Director for the Purdue Life Science mass spec facility. My lab is focused on the development of proteomics technologies. My first 10 years at Purdue, I was working on protein modifications using cell models, occasionally using animal models by collaborating with others. About 10 years ago, we started to look into extracellular vesicles. They gave us the opportunity to look at disease diagnosis using clinical samples. What has surprised us is that we’re able to see a lot of protein phosphorylation and interesting disease molecules that can be identified from these vesicles. From that point onwards, we have been doing research in this field and developing technologies to isolate extracellular vesicles, and we have multiple collaborations investigating different diseases using this approach.”

FLG: What makes extracellular vesicles a good candidate for biomarker discovery?

“This is a very good question. Biomarker discovery is always very hard, especially as my lab is focusing on protein-based analysis. Protein analysis is more difficult to do because the typical biomarkers are based on blood or urine samples, and these samples are dominated by these highly abundant proteins. In that case, your analysis will be limited by this high dynamic range. Secondly, there are some enzymes in circulation and a lot of interesting proteins could get degraded or hydrolyzed – such as cancer-relevant phosphoproteins. These will be hydrolyzed by these alkaline phosphatases in the blood acid, which makes it tremendously difficult to analyse. Extracellular vesicles make a perfect surrogate. By isolating them you avoid contamination from highly abundant proteins and prevent interference. Also, the extracellular vesicles protect the cargo, including proteins, from enzymes in the blood. So, you can preserve the original protein phosphorylation status. Using extracellular vesicles, you can collect these proteins more easily, which, for us, is a great opportunity for diagnosis.”

FLG: Why is there a need to find biomarkers for Parkinson’s disease?

Parkinson’s disease and other neurodegenerative diseases are very hard to diagnose. Compared to cancer it’s more difficult, because it’s very hard to get a sample – unless you get a part of brain, which is certainly not good for early diagnosis. Secondly, usually the clinical approach to diagnosis so far has been based on observations and asking questions, it’s not straightforward and we also want to follow up and see the progression and then eventually make a decision. So, this is not an easy disease to diagnose. There is an urgent need for a better and more accurate diagnosis method.

If you want to know more about the use of liquid biopsy to detect neurological disorders, you can download our Liquid Biopsy report when it releases in late January 2024. Featuring expert collaborators including Klaus Pantel, Michael Hubank and Lauren Leiman, we dive into the potential uses of liquid biopsy in neurological care, the biomarkers that have clinical impact, and how proteomics and AI are being used to detect novel biomarkers and identify patterns in liquid biopsy data that the human eye may miss.

References:

1. Malhotra, S., Miras, M. C. M., Pappolla, A., Montalban, X. & Comabella, M. Liquid biopsy in neurological diseases. Cells 12, (2023).

2. Lamptey, R. N. L. et al. A review of the common neurodegenerative disorders: Current therapeutic approaches and the potential role of nanotherapeutics. Int. J. Mol. Sci. 23, (2022).

3. Hou, Y. et al. Ageing as a risk factor for neurodegenerative disease. Nat. Rev. Neurol. 15, 565–581 (2019).

4. Breijyeh, Z. & Karaman, R. Comprehensive review on Alzheimer’s disease: Causes and treatment. Molecules 25, (2020).

5. Abidin, S. Z., Mat Pauzi, N. A., Mansor, N. I., Mohd Isa, N. I. & Hamid, A. A. A new perspective on Alzheimer’s disease: microRNAs and circular RNAs. Front. Genet. 14, 1231486 (2023).

6. Pollard, C., Aston, K., Emery, B. R., Hill, J. & Jenkins, T. Detection of neuron-derived cfDNA in blood plasma: a new diagnostic approach for neurodegenerative conditions. Front. Neurol. 14, (2023).

7. Office of the Commissioner. FDA permits marketing for new test to improve diagnosis of Alzheimer’s disease. U.S. Food and Drug Administration https://www. fda.gov/news-events/press-announcements/fda-permits-marketing-new-test[1]improve-diagnosis-alzheimers-disease (2022).

8. Quanterix granted Breakthrough Device Designation from U.S. FDA for blood[1]based pTau-181 assay for Alzheimer’s disease. Quanterix https://www.quanterix. com/press-releases/quanterix-granted-breakthrough-device-designation-from-u-s-fda-for-blood-based-ptau-181-assay-for-alzheimers-disease/ (2021).

9. Chong, J. R. et al. Blood-based high sensitivity measurements of beta-amyloid and phosphorylated tau as biomarkers of Alzheimer’s disease: a focused review on recent advances. J. Neurol. Neurosurg. Psychiatry 92, 1231–1241 (2021).

10. Statistics. Parkinson’s Foundation https://www.parkinson.org/understanding-parkinsons/statistics.

11. Niu, M. et al. A longitudinal study on α-synuclein in plasma neuronal exosomes as a biomarker for Parkinson’s disease development and progression. Eur. J. Neurol. 27, 967–974 (2020).