Liquid Biopsy in Non-Invasive Parkinson’s Disease Testing

A team led by Purdue University researchers and the Purdue spinoff business Tymora Analytical Operations has developed a technology – liquid biopsy, that may detect Parkinson’s disease in urine samples.

The technique allows researchers to assess if LRRK2 (leucine-rich repeat kinase 2) proteins and their downstream pathways, which have been related to Parkinson’s disease, are altered in Parkinson’s patients’ samples. This liquid biopsy technique could potentially lead to widespread non-invasive testing for other neurological diseases, as well as cancer.

“We believe this is a logical and rational approach to move forward for diagnosing Parkinson’s disease,” said W. Andy Tao, professor of biochemistry at Purdue. “Diagnosis for this type of neurodegenerative disease is difficult.” Cognitive and movement testing can take a year or more to confirm the diagnosis, so molecular tests for early diagnosis and intervention can help people with Parkinson’s faster, he explained.

Tao and his colleagues at Purdue, Tymora, The Michael J. Fox Foundation for Parkinson’s Research, and Columbia University reported their findings in the journal Communications Medicine.

“It’s going to be a big new area in diagnostic development,” predicted co-author Anton Iliuk, Tymora’s president and chief technology officer, “especially for neurodegenerative diseases and cancer.”

Parkinson’s disease alone is projected to afflict 1% of the over-60 population. Up to a million Americans are affected by the condition, with 90,000 new instances identified each year.

The paper’s co-authors include Marco Hadisurya, a doctoral student in biochemistry; Kananart Kuwaranancharoen, a doctoral student in electrical and computer engineering; Xiaofeng Wu, who received his doctorate in chemistry at Purdue in 2022; Li Li, Tymora Analytical Operations; Zheng-Chi Lee, West Lafayette Junior/Senior High School; Roy Alcalay, MD, Columbia University; and Shalini Padmanabhan from The Michael J. Fox Foundation.

Padmanabhan proposed a collaboration after reading some of Iliuk and Tao’s work on this liquid biopsy, EVtrap (Extracellular Vesicles total recovery and purification) approach for urinary analysis.

“When I reviewed the data from their previous publication,” Padmanabhan said, “it was interesting to note the expression of an important Parkinson’s disease-linked protein, LRRK2. This piqued my interest since this approach provided us with an opportunity to determine if LRRK2 proteins or the downstream pathways they impact are actually altered in urinary samples from Parkinson’s patients who harbor a mutation in the gene.”

Tao headed a team that developed a blood test that could potentially identify breast cancer in 2017. Tao and his colleagues examined samples from breast cancer patients with a healthy control group in that study.

“We identified phosphorylated proteins, which are a typical hallmark of cancers,” Tao said. The researchers also discovered extracellular vesicles, which are little containers that cells use as a molecular delivery method. The discovery indicated that a blood sample containing phosphoproteins might be used as a possible diagnostic for early cancer diagnosis or disease progression monitoring.

Using the Liquid Biopsy technique of Tymora’s EVtrap, the team was able to rapidly separate vesicles from urine samples.

“We have used the method for a number of indications, primarily focusing on different cancers for biomarker discovery and validation,” said Iliuk, who received his doctorate in biochemistry at Purdue in 2011. Tymora Analytical, which specializes in technology and services for detecting illness biomarkers in biofluids, was founded by Iliuk and Tao.

“This kind of analysis opens a new frontier in non-invasive diagnostics development. It’s showing that biomarkers previously thought to be undetectable have become uncovered and do a really good job of differentiating disease from non-disease state,” Iliuk said. “It’s not obvious that urine would be a source of brain-based chemicals or signatures, but it is. These EVs can penetrate the blood-brain barrier quite easily.”

They are concentrated or filtered into urine after being exported from the brain into the bloodstream. However, collecting such biomarkers from the brain through spinal tap is a rather intrusive operation.

“Especially for early diagnosis that is not the preferred sampling method,” Tao said. Urine samples contain proteins that could be disease markers, but many perform housekeeping functions that are unrelated to disease.

“Extracellular vesicles provide a way to focus on disease markers because they are released by certain types of cells,” he said.

Tracking the biological pathway of LRRK2 is one of many approaches to study its impact, which can be done by examining urine, blood, and cerebrospinal fluid. The EVtrap technology made it simple to track changes in urine, which is collected for a variety of clinical research.

Columbia University’s LRRK2 Biobanking Study has a vast bank of urine samples available for significant Parkinson’s disease research. Alcalay, a Columbia University co-author who contributed several of the samples, also assisted in correlating EVtrap findings with clinical data. The scientists investigated samples from people with and without the LRRK2 gene mutation, as well as patients with and without the condition, for the Communication Medicine study.

Padmanabhan noted, “This study also highlighted that changes in urinary proteins could serve as a proxy for changes in protein signatures that occur in brain diseases like Parkinson’s disease.”

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