“With this breakthrough, we are proud to enable a first-in-class test that can address an unmet need for a quick and reliable diagnostic for a complex, challenging-to-identify illness,” said Oxford Biodynamics’ Chief Scientific Officer Alexandre Akoulitchev.
We live in creative times. The number of clever ways researchers can identify and diagnose disease states appears to be growing exponentially. Back in the day we had cytokines, antibodies, and MRIs. It used to be so simple. Now we have “3-dimensional genomic regulatory immuno-genetic profiling”!
Epigenetic changes that occur during infections can turn off “good gene” and turn on “bad genes”. An epigenetic shift makes perfect sense in ME/CFS and other post-infectious diseases, but until now the evidence has not been convincing.
This new technology takes a deep stab at an area of interest for all post-infectious diseases – epigenetics. Epigenetics refers to the changes in gene expression that occur as we encounter pathogens or other stressors. We adapt to these stressors by turning on or off parts of our genomes. Genes that didn’t used to fire strongly now do so. Genes that were firing strongly die down. Given that many people with these diseases can date the start of their illness to an infection, it only stands to reason that an epigenetic shift – and not a good one – may have occurred.
Epigenetic studies, however, have not exactly lit up the ME/CFS world. This one, by using a different technique, has.
This new study, “Development and validation of blood-based diagnostic biomarkers for Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) using EpiSwitch® 3-dimensional genomic regulatory immuno-genetic profiling“, though, takes epigenetic analyses one step deeper.
In the paper, the authors focus nicely on three main factors in ME/CFS: immune dysregulation, dysregulation in the expression of genes involved in the immune response, energy metabolism, neurotransmission, and problems with energy production and metabolism. i.e., energy production. ”
Then they go to the nub of the problem – identifying these problems has not produced much help clinically; i.e., while the scientific findings are generally cohering, they have not helped ME/CFS patients at the doctor’s office.
Why? Because ME/CFS is quite heterogeneous, the findings have not always been reproducible, and the studies have generally been small. None of this is news, of course. The question is why Oxford Biodynamics thinks they can help out
THE GIST
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EpiSwitch® charts how the folds in our chromosomes that occur as a result of epigenetics changes which genes can interact with each other.
We live in creative times. The number of clever ways researchers can identify and diagnose disease states appears to be growing exponentially. Take the latest study employing “3-dimensional genomic regulatory immuno-genetic profiling”!
- This new technology takes a deep stab at an area of interest for all post-infectious diseases – epigenetics. Because pigenetics refers to the changes in gene expression that occur as we encounter pathogens or other stressors, epigenetic changes could play a huge role in ME/CFS. Thus far, though, epigenetic studies have had less than enticing results. This study, which received widespread media attention, is different.
- This study attempted to get at epigenetic changes in ME/CFS through the back door, so to speak. The chromosomes, which hold our genes, change the way they are folded when confronted with pathogens or other stressors. They do this to bring genes that are not normally near each other close together, allowing them to interact. These new interactions are called “loops”
- Usually, these loops produce a boosted immune defense, but sometimes the chromosomes become folded in such a way as to promote adverse effects.
- This new study from Oxford Biodynamics used specialized technology to assess the loops in housebound ME/CFS patients vs healthy controls.
- The results were startling. Their ability to accurately (96%) identify which people had ME/CFS and which people were the healthy controls was remarkable. The fact that they did this using 200 genomic biomarkers suggested that their findings had a strong foundation.
- Using pathway analyses, they identified three main immune issues in ME/CFS: an IL-2 network hub, innate immune activation, and JAK-STAT signaling. All could conceivably make sense with ME/CFS as we know it today.
- The authors identified approximately a dozen drugs that, if the IL-2 finding is replicated, could be applied to patients with high IL-2 levels. Plus, at least 3 JAK/STAT-inhibiting drugs are being trialed in ME/CFS.
- Encouragingly, the authors also noted that the inflammatory pathways found in ME/CFS are similar to those found in better-studied and treated diseases such as multiple sclerosis and rheumatoid arthritis. Validating that could conceivably open the door to dozens of immune drugs that have never been trialed in this disease.
- Considerations regarding this proof of concept study include its small size, its focus on only one slice of ME/CFS (housebound patients), and the use of a specialized procedure that may not be easily or cheaply replicable in other labs. Additionally, larger studies involving more diseases are needed.
- That last point appears to be the sticking point in ME/CFS diagnosis. Creative attempts to accurately diagnose ME/CFS are popping up more and more. What the field lacks are the larger, follow-up studies that could validate them.
- The small 2019 nanoneedle produced the most stunningly accurate results yet (100% accuracy) but failed to get further funding. Recently, though, ME Research UK and the ME Association have begun funding more nanoneedle work.
- The single-cell Raman microspectroscopy + AI study stood out with its high accuracy rate 91%, its inclusion of both multiple sclerosis patients and healthy controls, and its remarkable ability to accurately distinguish between mild, moderate, and severe ME/CFS cases. The Open Medicine Foundation and ME Association are funding more work into Raman spectroscopy.
- Some metabolomic and proteomic studies have also achieved high accuracy rates.
- As noted earlier, though, what this field lacks is the next step: large, comprehensive trials that contain both healthy controls and several different disease states. Hopefully, Oxford Biodynamics and/or the nanoneedle group and/or the Raman spectroscopy group, or another group, can get their diagnostic work across the finish line, and we will finally have a biological diagnostic marker for ME/CFS.
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Looking for Loops
Epigenetics occurs when parts of the genome which regulate gene expression (promoters, enhancers, insulators) get turned on. Because these epigenetic changes can get passed down during cell division, the changes can be more or less permanent (until the next epigenetic change occurs).
It turns out that the chromosomes in which our genes occur are amazingly flexible. When faced with an infection, the chromosomes in our immune cells, for instance, fold themselves in such a way as to turn on certain genes. They do this by folding themselves to bring genes which are ordinarily distant from each other, close together. When this happens a “loop” is formed.
These loops bring regulator genes (promoters, enhancers, etc.) next to active genes in order to turn them on or off. Most of the time, this is helpful, but viruses and inflammation can hijack this process and turn on genes that produce inflammation and immune activation.
The EpiSwitch® technology, developed by Oxford BioDynamics, basically identifies genes that, by virtue of the unique folding patterns found, have become able to talk to each other in diseases. Once they uncover these loops, they look for genes within a specific distance that these loops may be turning on or off; i.e., they’re identifying disease-specific epigenetic wiring patterns.
Using EpiSwitch® technology, Oxford Biodynamics has identified epigenetic signatures for amyotrophic lateral sclerosis, rheumatoid arthritis, and urothelial cancer. The authors reported that EpiSwitch®-based commercial tests are now available to diagnose prostate cancer with 94% accuracy (PSE test) (!) and the response to immune checkpoint inhibitors across 14 cancers with 85% accuracy (CiRT test)
EpiSwitch charts how the folds in our chromosomes, resulting from epigenetic changes, determine which genes can interact with each other.
The authors reported that Oxford BioDynamics has also produced a blood-based test that can predict COVID-19 severity, and have uncovered genomic markers that identify potential treatments for the biological pathways associated with them.
In this proof-of-concept study, they tested their procedure on an even bigger challenge – ME/CFS. The study included 47 people with severe ME/CFS, and 61 age-matched healthy controls. The goal was to see if the chromosomes in ME/CFS had become folded in a way that turned on pathogenic gene combinations that produced or contributed to ME/CFS.
Oxford Biodynamics used a more rigorous approach in this study compared to their past disease studies. Because we didn’t know as much about ME/CFS instead of targeting their analysis on specific loops they searched through the entire genome of the ME/CFS patients. Because of this they were more apt to uncover more novel “loops” or DNA folding patterns.
The Study
The study assessed chromosome folding patterns in 47 patients with severe ME/CFS (housebound) and n = 61 age-matched healthy controls.
This study found significant amount of aberrant chromosome folding has occurred in ME/CFS. Using 200 genomic biomarkers, the researchers were able to identify ME/CFS patients with a sensitivity of 92% (correctly identify 92/100), and specificity of 98% (identified virtually everyone who was healthy as healthy) had an overall accuracy of 96% (when putting ME/CFS and healthy controls together it was accurate 96% of the time).
Right now, the Epswich test is one of the most accurate diagnostic tests ever reported for ME/CFS, and has received plenty of coverage in the media.
The fact that the study used approximately 200 broad-based biomarkers suggested that a) ME/CFS is polygenic; i.e. many genes contribute to it and b) the results rested on a broad biological foundation. The accuracy also suggests that, for all the discussions of subsets in ME/CFS, core biological features involving the immune system, in particular, persist throughout ME/CFS. Once again, (how many times does this need to happen?) the idea that ME/CFS is some sort of wastebasket disease was demolished.
A pathway analysis found that the top 200 genomic markers existed within a densely connected immune network strongly enriched for (biased towards) immune and inflammatory signaling and cellular stress responses. Both the idea of inflammation and stressed-out cells, of course, makes sense in ME/CFS.
IL-2 Stands Out
One cytokine – called IL-2 – appeared to play a key role.
Within that network, several hubs appeared. The top hub focused on the IL-2 cytokine, which appeared to influence several other immune nodes. IL-10 and other interleukins also showed up strongly, as did T-cell regulation – another immune cell which has shown up recently (aka T-cell exhaustion) in ME/CFS.
Il-2 was the major focus, though. The authors reported that IL-2 can cross the blood-brain barrier, and, by influencing microglial activity, result in central sensitization and cognitive symptoms, and neuroinflammation. Interesting animal studies suggest that IL-2 administration can produce fatigue-like behaviours and cognitive problems.
IL-2 levels have been variable in ME/CFS, but as we’ve seen, the level of a cytokine is less important than the role the cytokine plays in the immune networks. One study suggested that in the lab, IL-2 can restore NK cell functioning in ME/CFS cells. Low levels of the soluble IL-2 receptor were also associated with a poor response to IVIG in ME/CFS.
IL-2 plays a key role in the survival and expansion of T-regulatory (Treg) cells. Levels of these cells, which rein in the immune response, tend to be high in ME/CFS, presumably because they’re trying to tamp down an overactive (and exhausted) immune system.
Low-dose IL-2 is used in several autoimmune/inflammatory diseases to boost T-regulatory cells and get autoimmune/inflammatory processes under control. The authors focused more, though, on dampening IL-2 levels in ME/CFS patients to stop chronic T-cell activation and ultimately the T-cell exhaustion.
Innate Immune System Activated – Several hubs (TNF / NF-κB axis, Toll-like receptor signaling) were associated with activation of the innate immune system, which increasingly appears to be a prominent feature of ME/CFS. Nath and others have proposed that problems in the adaptive, later-acting immune system may be causing the early, more inflammatory innate immune system to become hyperactivated.
JAK/STAT Pathways Highlighted– Finding evidence of activated JAK/STAT immune pathways was a welcome sign in many ways. For one, JAK/STAT pathway plays a fundamental role in a wide range of diseases including autoimmune and inflammatory diseases. It’s getting a lot of study, drugs are available, and one (Baricitinib) is being trialed in long COVID.
Problems in the JAK/STAT signaling pathway increase the expression of inflammatory genes in several diseases.
The JAK/STAT pathway translates signals from cytokines that land on the outside of the cell into gene expression in the cells. Aberrant JAK/STAT functioning chronically activates immune cells, leading to inflammation and damage. JAK/STAT inhibitors calm this pathway down, taking immune cells off the ledge. If ongoing immune activation is exhausting T, NK, and other immune cells, JAK/STAT inhibitors could help return the system to normal.
The pathway map suggests, if I understand the authors’ report, that altered cytokine signaling produces innate immune hypersensitivity. That, plus a pathogenic JAK/STAT transcriptional program that promotes immune activation and inflammation, could be producing the fatigue, cognitive symptoms, and autonomic dysfunction described in ME/CFS. It’s a nice model!
ME/CFS Not Alone?
In what can only be good news, the authors reported that similar immune pathways have been found in multiple sclerosis, rheumatoid arthritis, and other chronic inflammatory disorders. They stated that the “epigenetic alterations” found in ME/CFS aligned “closely with known pathways involved in chronic inflammation and immune dysregulation”.
That’s good news for anyone looking for sooner rather than later treatment options. The best outcome would be for the immune and other findings in ME/CFS to line up closely enough with other disorders that the treatments which work in those disorders could be tried. These are the kinds of findings, which, if validated, promise to bring ME/CFS “out from the cold”, and into the broad mainstream of disease.
This is why it’s so darn important to identify the molecular pathways at work in ME/CFS. Lots of studies do identify molecular pathways and we are seeing overlaps but we still apparently don’t have the consensus needed to launch major drug trials that attempt to alter them.
It would seem that we’re at or near the place where patients with alterations in specific molecular pathways could be identified and treated but we haven’t seen those kinds of personalized studies yet.
Possible IL-2 Treatment Regimen
If the authors of this paper are right, the path could be much simpler: identify the subset of ME/CFS patients with high IL-2 levels and try them on IL-2-reducing drugs.
The authors identified at least 12 drugs that can reduce IL-2-producing CD4+ cells while increasing IL-10 levels. (Il-10 tamps down immune activation). The fact that numerous drugs exist indicates how rich a space this could be for ME/CFS if the IL-2 connection is validated.
The drugs included Rituximab, Copaxone, Rapamycin, Tacrolimus, mycophenolate mofetil, azathioprine, low-dose methotrexate, corticosteroids, dimethyl fumarate, hydroxychloroquine and Vitamin D3 (Calcitriol)
Although the large Rituximab ME/CFS trial failed, some participants showed results, raising the question of whether the maintenance dose was sufficiently high. A Japanese study underway will help to answer that question. The Rituximab study was not targeted in the same way as a study focused on patients with high IL-2 levels.
The idea that epigenetic modifications could be driving ME/CFS makes perfect sense. With their new technology, Oxford Biodynamics appears to be making good on that notion. We can’t celebrate yet, though. Much work remains to be done before we can say we finally have a diagnostic test for this disease.
Oxford Biodynamics is off to a good start but many questions remain to be answered before the Epswich test can be considered a diagnostic test for ME/CFS.
Considerations
- The best diagnostic biomarker would be cheap and readily available. Because chromosome folding assessments require specialized labs, one question is whether this tool could ever be widely used as a diagnostic measure. Chris Ponting believes this test would probably cost over $1000. One of the significant advantages of the nanoneedle was that it offered the potential for a cost-effective test that could be easily conducted in a doctor’s office.
- Because all the ME/CFS patients were severe, i.e., housebound, these findings might not reflect those in less ill patients. Chris Ponting pointed out that because gender can make a big difference, genders need to be assessed separately. Activity levels need to be addressed as well; i.e., the comparative group needs to be sedentary. Age needs to be matched more closely.
- This was a proof-of-concept study. It certainly worked, but as the authors (and others) have noted, bigger and more comprehensive studies need to be done, including assessing the test against similar but different disease groups.
- We have several potential diagnostic tests, and more will surely be forthcoming. Finding a potential diagnostic test is becoming increasingly easier. It’s the next step – validating it – and then getting it into a doctor’s office – that has proved more challenging
Diagnosing ME/CFS: Creativity Abounds; Persistence Not So Much
Researchers are investigating ever more ways to diagnose ME/CFS. The small 2019 nanoneedle produced the most stunningly accurate results yet (100% accuracy) but lacked external validation.
Threading the Needle: Nanoneedle Scores Big in First ME/CFS Test
The single-cell Raman microspectroscopy + AI stood out with its high accuracy rate 91%, its inclusion of both multiple sclerosis patients and healthy controls, and its remarkable ability to accurately distinguish between mild, moderate, and severe ME/CFS cases.
A Potential Blood Test for Chronic Fatigue Syndrome (ME/CFS)?
The .95 (AUC) figure in Germain’s small 2021 plasma proteome study suggested that proteins may be able to cull out people with ME/CFS accurately.
Interestingly, some of the highest diagnostic accuracy rates have come from metabolomic studies. Naviaux’s small targeted plasma metabolomics (Naviaux et al., PNAS 2016) came up with an overall accuracy of .96 (AUC) for females and 0.94 (AUC) for males. Similarly, Nagy-Szakal’s 2018 metabolomics model had an accuracy rate of .960 (AUC) with ML (Nagy-Szakal et al., 2018).
The Core Problem in Chronic Fatigue Syndrome Identified? Naviaux’s Metabolomics Study Breaks Fresh Ground
The good news is that researchers appear to be getting better and better at finding ways to accurately diagnose ME/CFS. That’s good news for a disease that’s still struggling for validation and funding.
Possible diagnostic biomarkers exist. The next step is getting the studies together that can validate them.
Despite the creativity shown by ME/CFS researchers, the next steps to validate these diagnostic biomarkers using larger cohorts and more disease groups have not yet been taken; i.e., it all comes down to that perpetual problem – securing funding. The nanoneedle is a telling, if somewhat tragic, example. The NIH funded the creation of the needle, but then, after the needle proved effective at distinguishing ME/CFS patients, it failed to fund it further.
Thankfully, the ME Research UK and the ME Association are funding more nanoneedle work, and the Open Medicine Foundation and ME Association are funding more work into Raman spectroscopy. Hopefully, Oxford Biodynamics will be able to expand its ME/CFS findings.
The Nanoneedle for ME/CFS is Back!
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