NEW YORK – Nonprofit research organization Brain Chemistry Labs has identified a set of eight microRNAs that may speed up the diagnosis of amyotrophic lateral sclerosis (ALS) and help to differentiate it from other neurodegenerative disorders.
The Jackson Hole, Wyoming-based institution published a confirmatory study of their earlier work identifying this biomarker on Thursday in the journal Brain Communications and is now in discussions with potential commercialization partners to bring the test to market in the next 18 to 24 months.
ALS, also known as Lou Gehrig's disease, is a rare and fatal neurodegenerative illness. Over the course of the disease, motor neurons deteriorate, resulting in the progressive loss of muscle control. Respiratory failure caused by the loss of control over the muscles responsible for breathing is the leading cause of death in ALS, and few treatments specific to the disorder exist.
The highly variable clinical presentation of ALS complicates diagnosis and drives the need for more accurate biomarkers, particularly fluid-based ones, as symptoms can make neuroimaging uncomfortable for patients and sometimes cannot distinguish between ALS and other neuropathies, such as primary lateral sclerosis and Parkinson's disease.
To search for such biomarkers, a team of scientists with Brain Chemistry Labs turned their attention to exosomes, small lipid-bound particles released into circulation by cells.
Sandra Banack, senior scientist at Brain Chemistry Labs and the first author of the study, explained that exosomes carry protein markers on their surface that are specific to their cells of origin, in this case, brain tissue. "Material that comes from neurological tissue has a biomarker," she said, "and we use those markers to see which of these exosomes are coming from the brain."
Rachael Dunlop, a senior research fellow at the institute and a coauthor of the study, added that the phospholipid bilayer surrounding exosomes plays a critical function in making them attractive targets for liquid biopsies.
"MicroRNA is highly susceptible to degradation from [circulating] nucleases," she said. "The fact that it's contained in this phospholipid bilayer protects it, not just in situ but once it comes out of the patient. It doesn't break down in transit, or because someone left the plasma on the bench and went for a cup of tea or something."
In their retrospective study, Banack, Dunlop, and their colleagues used immunoaffinity purification to extract exosomes found in blood samples from 119 individuals diagnosed with amyotrophic lateral sclerosis, 42 with primary lateral sclerosis, and 20 with Parkinson's disease, along with 150 healthy controls.
The eight ALS-specific miRNAs that the Brain Chemistry Labs team identified came from two prior publications. In the first, published in Open Biology, two 20-person cohorts of 10 ALS patients and 10 healthy controls each led to the discovery of 101 neural-associated miRNAs that differed significantly between cases and controls.
"Critically, they were all patients who had been diagnosed by the same neurologist and collected under a very specific protocol," Dunlop said.
In the second study, published in the Journal of the Neurological Sciences, the researchers obtained 100 samples from the US National ALS Biorepository, comprising 50 samples from people diagnosed with ALS and 50 healthy controls.
"[That] biorepository has a wide range of samples," Dunlop said, including ones that were collected via different protocols, were stored differently, and were from patients diagnosed by different neurologists.
Dunlop and her colleagues used this dataset to narrow down the set of candidate biomarkers from the earlier set of 101 miRNAs to 34 that were the most differentially expressed between individuals diagnosed with ALS and healthy controls. The Brain Chemistry Labs team further whittled these down to eight miRNAs that most consistently separated ALS samples from control samples with the strongest statistical significance, which they then used in the latest study.
Dunlop said that in the current Brain Communications study, "we've now been able to incorporate neurological diseases that are similar [to ALS] in their symptoms, in the early stages of disease."
The comparison to other neurological disorders is vital for ALS biomarker development, she explained, because diagnosis currently relies heavily on observing how symptoms progress.
"This is not ideal for people with ALS," Dunlop said, "considering the median life expectancy [post-diagnosis] is about three years. They don't really have time to wait for progression."
Banack and Dunlop tested three different computational approaches –– random forest split, random forest separate cohorts, and logistic regression –– to estimate the accuracy of their miRNA biomarker.
"They have very different approaches as to how they use the data to classify whether a patient has or does not have ALS," Banack said.
Across all methods, sensitivity ranged between 96 percent and 100 percent, and specificity was consistently 97 percent. While Banack said that these numbers are likely to change as the biomarker set is further developed into a diagnostic assay, she said the data presented in their current study should give a sense of the range that can be expected.
"And as sample size increases, we're hoping that we can nail that down even further," she said.
Sulev Koks, head of epidemiology research at Australia's Murdoch University who specializes in neurodegenerative disorders, praised the study for its methodology and clarity, adding that its focus on miRNA is one of its key strengths, as total RNA analysis can be much more challenging.
He also applauded the use of exosomes as a means of isolating brain-derived miRNA.
"The problem with the pathologies affecting the brain is that the brain is non-accessible tissue," he said. "We cannot take a biopsy from the brain."
This has created a "massive gap" in obtaining biosamples from individuals with diseases of the brain, Koks explained, with numerous research efforts underway to figure out how to extract relevant biomarkers from peripheral tissues and blood.
"Using exosomes makes the analysis more specific and increases accuracy," he said. "I think that this is really where the field is moving."
Koks noted that some technological challenges remain to be solved to translate the study's results into the clinical setting, such as developing simpler methods to purify exosomes, which would facilitate its implementation into everyday practice.
"But this can be useful for clinical trials and [as] an additional tool for practitioners," he said.
Brain Chemistry Labs intends to develop its miRNA ALS signature into a diagnostic test, with the help of an industry partner.
"We are speaking with diagnostic companies but have not yet selected one to become our partner," Banack said.