From Blood to Breath

Asthma is a very common and well-known disease, but unfortunately, it’s also a disease that can be very difficult to diagnose. At the moment, we use lung function tests that rely on measuring how fast we can blow air out of our lungs – but these tests are often unable to make a definitive diagnosis, and it’s difficult for children, and even some adults, to perform them. That’s not all that complicates the matter; we now also know that asthma is very heterogeneous. It has a number of subtypes, including allergic and non-allergic disease and high and low eosinophil presence. It’s not always easy to determine what type a particular patient has, and that means that we can’t always decide which treatments might make a difference – not everyone benefits from inhaled corticosteroids, the standard medication, and we don’t always understand why. As a result, there is a great need to find tests that can both diagnose asthma and characterize the disease further.

Ideally, for patients who present with respiratory symptoms, we’d be able to conduct a simple test – perhaps with a blood or saliva sample – to get the information we need. Until now, there have been no such tests. Recently, though, we’ve discovered a collection of microRNAs (miRNAs) present in human body fluids that can give us better insight into asthma, both in terms of absolute diagnosis and individual phenotype. 

Asthma answers 

Our research project kicked off when we discovered that RNA species were present in exhaled breath condensates – basically, cooled breath vapor harboring particles that originate in the lungs. After making that discovery, we cloned the exhaled RNA and found that miRNAs were enriched in lung fluid. We then went on to show that a number of miRNAs were differentially expressed in patients with asthma, in comparison with healthy control subjects. It turned out that those miRNAs were differentially expressed in the blood of asthmatics compared to non- asthmatic subjects, suggesting that blood could serve as a source of miRNAs capable of describing inflammation in the lungs. 

Initially, we identified a panel of 30 miRNAs that were different in the blood of asthma patients compared with healthy controls (1). Many of these had similar expression patterns, though, so we were able to come up with a final panel of seven that could differentiate between asthma, healthy controls, and subjects with allergic rhinitis (an upper respiratory disease; asthma, in contrast, is a lower respiratory disease). The ability to narrow our options down to a limited panel is a plus because it means that we can make a simpler, cheaper diagnostic test.

How do the expression profiles of various asthma subtypes differ? We performed a cluster analysis on the original 30 differentially expressed miRNAs in asthma, and we found that they clustered primarily into two groups. One group of asthmatics had a high level of eosinophils, whereas the other cluster had low eosinophils. Emerging research shows that patients with non-eosinophilic asthma may not respond to typical asthma therapies, so our hope is that we can start to predict whether or not patients are likely to respond to treatment by measuring their miRNA profiles. We think that, as we expand the numbers of subjects we study, we’ll be able to uncover even more asthma subtypes based on miRNA profiles – sort of a “fingerprint.” This would be a huge stride toward personalizing asthma therapy! 

We’re beginning to understand that the miRNAs we’ve identified may play important roles not just in asthma, but in allergic and inflammatory diseases. Some of the miRNAs (miR-155, miR-570, and miR-1248, for example) have pro- inflammatory roles and may be necessary for the development of asthma. Others, like Let7a and miR-146a, seem to be anti-inflammatory. So, in addition to the diagnostic potential of these miRNAs, they may have therapeutic potential as well. So far, we’ve found that miR- 146a works in parallel to glucocorticoid medications, and that when the two are combined, they have additive effects. We currently have some difficulty treating patients who fail to respond properly to glucocorticoids, so adding miR-146a to their treatment regimens may help increase their sensitivity to the drugs. 

Partnering with pathology 

It’s interesting to note that such tests are being referred to as “liquid biopsies.” One day, we may be able to find out more about our patients’ lung pathology and airway inflammations simply by examining the miRNAs in their blood – or even other fluids like exhaled breath condensates. We’re collaborating with the Milton S. Hershey Medical Center’s pathology department on our current work, as they may be able to apply the assays we develop to other diseases. Tests like that would really open up our ability not only to diagnose diseases, but also to learn about their pathology. And because the current diagnostic workflow involves simple, PCR- based tests, we anticipate that our new assays could be readily integrated. 

Right now, we’re in the process of acquiring larger numbers of subjects to validate our initial results and see how good the miRNA test is at diagnosing asthma in an unknown population. As the technology we use to measure miRNA levels in the blood is sensitive, reproducible, and cheap, we hope that our recent findings can soon be translated into a diagnostic test – hopefully within the next five years. We’d eventually like to develop a chip-based assay that would allow physicians to take and analyze saliva or fingerstick blood samples at their patients’ bedsides.

Towards personalization 

Moving forward, we have three main goals. First, we’d like to reduce the size of the miRNA testing technology so that it can fit on a chip for analysis by a cell phone. Next, we’re trying to understand the biology of the miRNAs; we think that some of them are crucial to the development of asthmatic inflammation, so we’re working on understanding what they do. Third, we’re moving toward using this technology to really personalize asthma treatment. We will determine whether or not we can use miRNA profiles to predict treatment responses. Putting all three of these goals together, in a decade or less, we’d like to see this happen: 

• A patient with wheezing enters the office. 
• A fingerstick blood sample is analyzed by a chip the size of a credit card, plugged into a cell phone. 
• The software not only confirms an asthma diagnosis, but also tells you what form of asthma is present, and may even make a recommendation about which medication to initiate. 

The ability to diagnose asthma quickly, conveniently and noninvasively – perhaps even on a patient’s first visit to the clinic – would offer significant advantages over current tests. Patients unable to perform respiratory tests could still receive a diagnosis and begin treatment rapidly, and characterizing their disease at the same time has the potential to prevent overtreatment in situations where steroids won’t be effective, and to eliminate the period of uncertainty as patient and physician wait to see whether or not the drugs will help. And we might even gain a better understanding of the molecular pathogenesis of asthma and allergic rhinitis, knowledge that may allow us to identify new therapeutic targets and continue to improve patient care.