New Kind of Analysis Yields Promise of Diagnostic Test
In advance of the high-powered ME jamboree underway at Stanford this weekend it seems right to take a close look at the paper, published on July 31, on cytokine expression in the disease. The study was the brainchild of Stanford’s premier research clinician Jose Montoya and the logical extension of a years-long exploration of cytokines in ME by numerous doctors and scientists.
Cytokine expression, a marker for inflammation in the body, has been the subject of literally hundreds of ME studies over the years. But the results often have been contradictory and sometimes faulted for being small in size or analytic power.
This cytokine analysis---“Cytokine signature associated with disease severity in chronic fatigue syndrome patients,” which appeared online in the Proceedings of the National Academy of Sciences (PNAS), moves the ME field forward in a way that most have not. It’s not surprising that lay press coverage of it was wall-to-wall. Not only does the study make a case for inflammation being a driver of the multitudinous symptoms experienced by ME patients, its methods suggest a new, more effective way of studying ME and perhaps other diseases.
Stratifying ME patients by severity of disease and comparing them to healthy controls generated remarkably persuasive data in this study. The authors of this paper suggest such stratification may be a more productive way of structuring research trials in ME and possibly other maladies going forward.
Equally important, at least one of its authors, Stanford’s Mark Davis, who is among the top U.S. immunologists and microbiologists, remarked that the study paves the way for the development of a diagnostic test for ME.
That alone was a showstopper, akin to announcing the location of the Holy Grail—or at least the map.
It’s not unusual these days for journal studies to end their reports with the comment that the work described “may” lead to a diagnostic test and treatments. In most cases one suspects such language is “blue sky” talk, more of a shill to generate financial contributions to the lab in question than it is an accurate reflection of reality. But in the case of the Montoya-Davis PNAS study, the notion of a blood test deriving from this work seems more substantial.
But there was so much more to be absorbed from this study and much that could be inferred.
Almost 200 of Jose Montoya’s patients—186, to be precise—were involved, along with nearly 400 healthy controls, pushing the study into the realm of some of the largest legitimate studies of the disease. Since seeing his first patient in 2004, Montoya has worked unfailingly to interest and engage his colleagues in ME and his patience and strategies are clearly coming to fruition.
Montoya approached Davis, who is the co-creator of Stanford’s ten-year-old Human Immune Monitoring Center, to undertake an evaluation of cytokines in this large group. The Center is able to analyze thousands of immune system data points in large populations—like the nearly 600 people involved in this study--providing tremendous statistical significance. The Center provides opportunities to cancer researchers to study human immune markers, including—according to its website—“…changes in lymphocyte signalling pathways, serum cytokines and specific immune responses that target tumor cells.”
The Center can also isolate infectious agents: “…[P]athogens have been implicated in the etiology of many cancers and the Resource offers an array of methodologies for pathogen identification.”
Although the Center plays a helpful role in cancer research, its technologies can be applied in other fields, as well. Mark Davis’s interest and commitment to using the resources of the Stanford Center he co-created suggests he and his colleagues view ME as a disease worthy of significant investments of time, brain power and money. The ME research underway at this Center is among its largest projects.
Mark Davis is also the director of the Stanford Institute for Immunity, Transplantation and Infection.
MILD, MODERATE, SEVERE
Comparing patients to controls as has been the standard for ME studies for decades and the traditional way of structuring controlled studies in general. Prior to stratifying the data into categories of disease severity, Stanford investigators found little difference between patients and controls. This has been a not-uncommon finding in other cytokine studies.
Striking differences emerged, however, when Montoya, Davis et. al. evaluated the data based on three categories--mild, moderate and severe. Stanford collaborators looked at 51 cytokines in controls and in patients. Montoya and his colleagues discovered that 17 of the 51 cytokines studied correlated with ME severity.
A majority, 13 of 17, are known to be pro-inflammatory. Four are known to be immunosuppressive, or anti-inflammatory.
In the words of the scientists, these cytokines had a “statistically significant linear upward trend across the sequence of mild, moderate and severe ME/CFS severity.” The more severe the disease, the more florid the production of cytokines.
Cytokines are proteins expressed by immune system cells. So-called “pro-inflammatory” cytokines, when elevated, indicate that inflammation is underway in the body. According to the Human Immune Monitoring Center’s website, cytokines “regulate infections and fight cancer.” Production of cytokines is also a response to physical trauma.
Increasingly, cytokines are being studied in neurological diseases, including Parkinson’s, Multiple Sclerosis and ALS, by scientists who are investigating whether brain inflammation may be responsible for symptoms.
Although the science of this paper is complex given the myriad observations to be made about the disease from the data generated, one simple take home message is that, as the authors wrote, “…severity may be the key variable in subgrouping patients.”
That’s a significant departure from the oft-heard description of ME as a “heterogeneous” disease or even multiple “diseases” that should be sub-grouped for study based on potentially artificial delineations of clusters of symptoms.
“For decades, the ‘case vs. healthy controls’ study design has served well to advance our understanding of many diseases,” Jose Montoya told Stanford’s medical reporter Bruce Goldman. “However,” Montoya added, “it’s possible that for certain pathologies in humans, analysis by disease severity or duration would be likely to provide further insights.”
Just one other study has found abnormal cytokine activity based on duration of illness, by some reports a serendipitous finding. Two years ago, Columbia University scientists Mady Hornig and Ian Lipkin wrote in the Journal of Translational Medicine that they found higher levels of cytokines in people who had been ill for less than three years, and lower levels of cytokines in people who had been ill more than three years. Reportedly, Hornig and Lipkin had been looking for differences between controls and patients and came up with little of significance. They did see a difference, however, when they stratified by years of illness.
“We can only speculate,” they wrote at the time, that “…the exuberant stimulation of cytokine producing cells seen early in the illness leads to an ‘exhaustion’ of the cytokine producing cells thereafter.”
That speculation was not borne out in the new study, although Stanford investigators looked.
“We did not find any cytokine to be significantly different between these two groups,” the authors noted in the paper.
Nevertheless, Stanford researchers do not rule out that disease duration may yet be another important mode of stratification in the future.
Interestingly, the PNAS investigators found that some cytokines were lower in mild patients than they were in controls, even though the same cytokines were higher in severely ill patients. This finding has been reported in studies of other patients with specific infectious diseases, in particular occular toxoplasmosis, the authors noted. Montoya is among the nation’s top toxoplasmosis experts.
“The levels of circulating cytokines in response to inflammatory triggers such as infection have been reported by various groups to be lower than in controls for some cytokines and higher for others,” the authors wrote.
TRANSFORMING GROWTH FACTOR - BETA
One of the cytokines that was significantly correlated with ME severity was transforming growth factor-beta, a primarily immunosupressive cytokine with, confusingly, pro-inflammatory properties in some cases that has been implicated in numerous severe diseases including cancer.
This particular cytokine has been a target of investigation in ME for some time The PNAS authors noted that in five out of eight earlier studies, transforming growth factor-beta has been found to be higher in ME patients compared to controls.
Transforming growth factor-beta long has been associated with myriad serious diseases, most notably cancer. Literally hundreds of papers can be found in the literature on the subject of transforming growth factor-beta and its associations with numerous cancers, particularly lymphoma and leukemia.
Given that, it may not be surprising that the authors noted, “Because TGF-β has been implicated in the development of cancer, elevation of this cytokine in ME/CFS patients older than 65 years of age could contribute to their possibly increased risk of NHL (non-Hodgkin’s lymphoma) and two defined NHL subtypes… following their ME/CFS diagnosis.”
An increase in lymphoma among ME patients was observed originally by clinicians in Nevada during an outbreak there and by a cancer epidemiologist studying an outbreak of ME in a North Carolina orchestra nearly three decades ago. This association has been an ongoing source of interest among some ME researchers, although it's an association the federal government seems less than eager to address. As long ago as in 1986 and 1987, doctors in Incline Village, NV reported an unusual incidence of NHLs in their sick population to staff at the Centers for Disease Control. At the time, Gary Holmes, one of two investigators who had been sent to Incline Village to investigate that outbreak in late 1985, told me, “I was wanting to go back,” when he heard reports of increased lymphoma and abnormal brain scans in that population. Holmes was overruled by his superiors (and four years later, fired).
During our conversation, Holmes alluded to animosity among CDC staff toward the Incline Village doctors and their patients as well as bad publicity about CDC generated by its own investigation as the reason no one from CDC returned to Nevada and a decision made by his superiors to do nothing further. By 1988, the CDC had invented and formalized the name “Chronic Fatigue Syndrome” for a malady that clearly was not limited to Nevada.
Since then, an association between ME and lymphoma has been documented in journal reports, however, though that link is rarely if ever referred to in government publications or in ME definitions. The fact that Montoya and Davis chose to link lymphoma in ME with a cancer-related cytokine is significant, suggesting neither investigator is fearful of government reprisals in the form of grant turn-downs by reviewers who may not believe the association exists or are unfamiliar with early observations.
Non-Hodgkin’s lymphoma is an umbrella term for a large collection of lymphomas and leukemias that are differentiated by cell association and/or appearance. NHLs have surged in the last three decades in tandem with ME. No one knows why NHLs are epidemic, although theories abound, including radiation exposure, pesticide exposure and pathogens, in particular, retroviruses.
Burkitt’s lymphoma, an aggressive cancer formerly limited to what is known as Africa’s “lymphoma belt,” a continent-spanning swath of equatorial Africa, has been directly linked to Epstein-Barr infection. Researchers have proposed malaria as a co-factor. The once-exceedingly rare Burkitt’s lymphoma began to be seen in the U.S. with the advent of the AIDS and ME epidemics of the 1980s.
As an aside, in at least one AIDS-related Non-Hodgkin's Lymphoma study published in 2012, the authors found elevated cytokines and chemokines in patients with these diseases. They concluded, "These findings support the current hypothesis that A-NHL (AIDS-related Non-Hodgkin's Lymphoma) develops in response to persistent immune stimulation and inflammation. A network of inflammatory mediators was identified demonstrating correlations with disease progression and overexpression in A-NHL patients." (J Clin Oncol)
Approximately 85 to 90 percent of all lymphomas originate in B-cells; one percent originate in NK cells and the rest in T-cells. Burkitt’s is a B-cell lymphoma.
Another cytokine associated with disease severity in ME patients is leptin, known primarily as a “satiety” indictor that signals the brain that the stomach is full. Leptin increases during infections. A 2006 study about leptin suggested that, “…leptin deficient mice are resistant to a wide range of autoimmune diseases and display features of immune deficiency. Subsequent work has confirmed that leptin has a pleiotrophic role (multiple effects) on the immune response and can rightly be considered, both structurally and functionally, as a proinflammatory cytokine.” Leptin is also considered a hormone.
Secreted by fat cells, this cytokine-hormone is of particular interest because leptin is more abundant—in one study (http://ajcn.nutrition.org/content/85/4/1121.full) three times more abundant—in women’s blood than in men’s. Women also have higher ratios of fat, as much as ten percent or more, though they typically eat fewer calories.
That abundance of leptin may help unlock the puzzle of why the preponderance of ME sufferers are women. The latter have been estimated to comprise 80 percent or more of all sufferers, making female gender the number one risk factor for acquiring ME among adults. In the new Stanford study the patients and controls reflected that reality; almost 77 percent of the patients and controls were women. Anecdotal reports suggest that the ratio is less extreme among pediatric patients.
A few expert ME clinicians have observed that women with ME tend to be sicker and less likely to recover than men. Investigators at Nova University in Ft. Lauderdale’s Institute for Neuro Immune Disorders are recruiting currently for a study of men with ME.
Leptin has intrigued Montoya for some time. Four years ago researcher Jarred Younger, who was then at Stanford but today is at the University of Alabama at Brimingham, and Montoya decided to study leptin in ten ME patients defined by Fukuda criteria and ten healthy controls. All twenty participants were women. That study, like the recent PNAS study, attempted to analyze the link between cytokines and severity of illness, too.
Given that severity can rise and fall in the matter of a day, Younger and Montoya used a longitudinal study design to accommodate for fluctuations. They measured blood levels of leptin and assessed severity of symptoms every day for 25 consecutive days in patients and controls. In their 2013 paper in the Journal of Translational Medicine the authors wrote “…fatigue severity was significantly correlated with leptin levels in six of the participants with CFS.”
Cytokines have long been a focus of scientific inquiry in ME given the myriad self-reported symptoms implicating inflammation and often dramatically elevated cytokines measured by doctors who order specific cytokine lab tests. A recent PubMed search using the words “cytokine” and “chronic fatigue syndrome” turned up a total of 432 papers that either included a focus on cytokine expression in the disease or, more often, made cytokine expression a sole focus of inquiry.
Typically, however, the numbers of ME patients in those studies were small, at least in comparison to the recent Stanford study. Also, no matter how much data has been generated to suggest that ME is an inflammatory disease, academic or government scientists who do not believe the disease is important or does not even exist frequently have dismissed them as irrelevant citing inconsistent results and small sample sizes. The earlier cytokine investigations have been viewed at best as pilot studies requiring repetition and confirmation, or worse, in the words of one observer, “will-o-wisp.” In a field where money is scarce, confirmatory studies have been a challenge.
One of the beauties of this study, in addition to the decision to look at patients by disease severity, which found the disparities described, is that the investigators had the ability to utilize the powerful analytic tools available to them in Stanford’s Human Immune Monitoring Center.
Montoya, the paper’s first author, spoke to just one journalist, Stanford University's own medical reporter Goldman. Montoya took the opportunity to express in powerful ways the fact that ME is a serious disease that if there was any doubt about that fact, this study proves it. His comment that ME “…could turn a life of productive activity into one of dependency and desolation” was widely re-published in mainstream media reports on the paper.
Montoya has been studying ME since seeing his first patient in 2004. He has, during the intervening years, worked in a strategic, focused way to involve his colleagues at Stanford in ME research and largely been successful. In this way, he’s expanded the study of the disease at Stanford, involving ever-larger numbers of collaborators in several areas of expertise, ensuring the widest degree of participation by creating what he calls a “big umbrella.”
He has also given years of thought to the origins of the disease and its pathophysiology. He has searched for ways to prove its seriousness to the rest of the medical cosmos in a logical, step-wise fashion. Although he says most ME research, including his own, is still just “touching the periphery” of the disease—describing abnormalities instead of the cause—his ultimate goal is to reach the center: the cause.
(For a comprehensive look at Montoya’s history of discovery and treatment strategies in ME, his ideas, as well as his near-single-handed conversion of his institution from one of the most hostile to ME to one of the most productive centers of ME research, please see my spring-time four-part interview with Montoya, accessible in Eye View Exclusives. In addition, a new Q&A with Montoya about the implications of this paper is now available at Eye View and makes good reading in concert with this report.
Montoya's PNAS collaborator Mark Davis participated in last weekend’s Open Medicine Foundation’s conference on ME at Stanford and his comments during the public presentations on Saturday, August 12 were in theory about the possibility of ME being an auto-immune disease. Nevertheless, Davis ultimately noted that the “…evidence for an autoimmune antibody is not strong yet,” during a question and answer session that followed his presentation.
During both his presentation and the Q&A session that followed, he relayed some interesting findings related to pathogen identification in the disease, unrelated to cytokines, about which he showed more enthusiasm than he did for autoimmunity theories.
Davis noted that thus far he and Montoya had observed significant “T cell clonal expansion” in ME sufferers, in particular in CD8 T-cells. Clonal expansion suggests that there is, as Davis explained, “…an antigen even that’s causing (the T cells) to divide. We’ve gone on to look for the antigen. We have just one hit—does this mean there is one pathogen?”
He stopped short of identifying the antigen or the identity of the “one hit”—presumably a pathogen.
An hypothesis promoted by many scientists over the years, first introduced by NIH investigator Stephen Straus in 1986, is that any infection can cause ME and whatever the pathogen is, it soon is “gone,” never to be found after having launched the immune system into the incurable chaos that is ME. Others, in contrast, have sought to identify a pathogen they believe continues to be present throughout the disease--reflected by ME’s aberrant immunology--and is responsible for the diseases' hypometabolic abnormalities, as well as its brain, heart and other organ damage.
Davis conjectured that evidence for dramatic cytokine activity suggested the disease could be caused by “…a pathogen or some sort of autoimmunity…What’s most exciting—if we knew more about these T cells…it could lead us directly to the pathogen. That’s our hope.”
His research with Montoya into the T-cell clonal expansion finding is ongoing.
In the Q&A session, Davis added that “It could go quickly if we can use the T-cell we have.” He asked rhetorically, “Is there is a drug for that pathogen? Can we develop a vaccine for that pathogen?”
According to PNAS, the Stanford cytokine study is among the 50 most read articles to appear in PNAS thus far this year.
Even given the significant numbers of patients and healthy controls in the study just published, other scientists are calling for replication of results. One can only hope that there are other major centers with the data analysis Stanford possesses to undertake a study of similar analytic power, combined with doctors who have intimate knowledge of the clinical aspects of the disease to help investigators design it and interpret results.
Not unexpectedly, two British psychiatrists weighed in on the study. Their opinions were sought out by the Science Media Center, a public relations entity notorious for its unfailing support of the discredited UK “PACE” study authors.
One, Alan Carson, said the data in the study was likely correct, especially with regard to TGF-beta. “There have been reports on this for the previous two decades and a meta analysis of 38 papers in 2015 demonstrated it as a consistent finding (Blundell et al 2015).”
He concluded, “This study therefore confirms what was already known but doesn’t take the field forward,” missing the fact that the Stanford study moves the field forward by demonstrating that the degree of severity and cytokine expression are intimately linked.
The other psychiatrist, Anthony Cleare, inexplicably complained, “…there is no indication that the course of illness is linked to any measures of inflammation,” when, in fact, that is exactly what the study proved.
Gordon Broderick, Ben Katz and long-time ME expert Anthony Komaroff reviewed the paper for PNAS. Broderick and Katz have published together and independently on the subject of cytokine expression in ME. In 2012 they collaborated with Suzanne Vernon and others in a study of cytokines in “post-mononucleosis chronic fatigue” in the Journal of Translational Medicine.