The positive results validate Owlstone Medical’s EVOC® probe strategy for the development of breath-based diagnostic and prognostic tests for Nonalcoholic Fatty Liver Disease (NAFLD) and associated Nonalcoholic steatohepatitis (NASH). The results have been published in Clinical and Translational Gastroenterology.
In the study, exhaled breath from 32 cirrhosis patients and 12 cirrhotic hepatocellular-carcinoma patients was compared with 40 controls. Breath samples were analyzed by Owlstone Medical’s Breath Biopsy platform. The results (sensitivity of 73%, specificity of 77%) clearly demonstrated that limonene levels are associated with alterations in liver function. Limonene therefore holds great promise as a marker of liver metabolic capacity, particularly when considering that the study results reflected only normal dietary intake of limonene as a single biomarker rather than being deployed as an optimized EVOC probe, which can be expected to yield far better performance.
Driven by the global epidemics of obesity and diabetes, NAFLD/NASH is becoming increasingly common with a worldwide prevalence estimated at 25% for NAFLD, and with many of these cases (1.5–6.45% global incidence) progressing to NASH1. This is placing a significant and growing demand on healthcare systems globally. In the United States, estimated annual direct medical and societal costs for NASH and NAFLD amount to $292B per year and in Europe total approximately €235B per year2.
[[{"fid":"288848","view_mode":"default","fields":{"format":"default","alignment":"","field_file_image_alt_text[und][0][value]":"NAFLD map","field_file_image_title_text[und][0][value]":"NAFLD map"},"link_text":false,"type":"media","field_deltas":{"1":{"format":"default","alignment":"","field_file_image_alt_text[und][0][value]":"NAFLD map","field_file_image_title_text[und][0][value]":"NAFLD map"}},"attributes":{"alt":"NAFLD map","title":"NAFLD map","class":"media-element file-default","data-delta":"1"}}]]
The key to preventing deaths and reducing the economic impact of liver disease is earlier detection and intervention. Unfortunately however, 75% of diagnoses occur at late stage3 due to a lack of suitable and specific diagnostic tools that can be deployed in a screening setting.
Owlstone Medical is currently developing non-invasive quantitative breath tests that will use EVOC Probes4, including limonene, a GRAS (Generally Recognized as Safe) compound, along with inflammatory exhaled biomarkers, to measure liver enzyme activity and provide information on the health of the liver and for NAFLD/NASH diagnosis and prognosis. These tests will initially be offered in research settings including to pharma companies developing NAFLD/NASH drugs, with launch as clinical tests to follow.
Dr Chris A Mayhew, Professor of Molecular Physics, University of Birmingham, and Director of the Institute for Breath Research, at the University of Innsbruck, Austria, commented: “The results from this study, and its implications for liver disease diagnostics are very promising. When a probe with higher levels of limonene optimized for this application is used, along with other markers to measure liver function, the potential for this approach to non-invasively stage liver disease and to measure the progression or recovery from disease is excellent.”
Dr Victoria Snowdon, Consultant, Transplant Hepatology, Cambridge University Hospitals noted: “The burden of liver disease is high and continues to grow, and so assessment of liver function is essential for determining prognosis and establishing effective treatment. The findings of this study suggest that a breath test could non-invasively help us determine liver function, helping to monitor patients with liver disease from an early stage and optimize treatment. We are pleased to have contributed to these findings through the PAN study and would like to thank all our patients at Addenbrooke’s who took part. Without their help we would be unable to develop new and improved ways of diagnosing and treating disease.”
Billy Boyle, co-founder and CEO at Owlstone Medical, said: “This study represents a major milestone for Owlstone Medical, as it is the first proof of the value of our EVOC probe approach for the development of breath tests in areas of high clinical need. Much like how PET scans work, this involves the introduction of a compound to the body that elicits a strong and clearly readable signal over background. We have identified a number of additional highly promising compounds that we are working to validate as EVOC probes for liver disease and we believe our EVOC probe approach can be applied to other disease areas, including lung cancer, where earlier and improved diagnosis is clearly needed to save lives.”
The study operated as part of Owlstone Medical’s PAN study5. Patients were recruited from the clinical research facility at Cambridge University Hospitals or through the Cambridge BioResource. Cirrhotic hepatocellular-carcinoma patients had an established diagnosis prior to entry into the study.
Access recorded webinar, Measuring Exhaled Limonene in Cirrhosis Patients
References:
- Meta-analysis of literature, performed by Owlstone Medical
- https://aasldpubs.onlinelibrary.wiley.com/doi/full/10.1002/hep.28785
- https://www.britishlivertrust.org.uk/
- https://www.owlstonemedical.com/about/news/2019/jun/18/owlstone-medical-announces-novel-approach/
- https://www.owlstonemedical.com/about/news/2019/jan/3/clinical-trial-launches-develop-breath-test-multip/
About NASH and NAFLD and the potential of breath diagnostics
Non-alcoholic fatty liver disease (NAFLD) refers to a group of conditions where there is accumulation of excess fat in the liver of people who drink little or no alcohol. Although fueled by increasing obesity rates, NAFLD is not exclusively present in those who are overweight, and indeed encompasses a spectrum of disease arising from a range of underlying causes. Risk factors for developing NAFLD are obesity, type 2 diabetes, dyslipidaemia, metabolic syndrome, polycystic ovary syndrome, gender (male), age, and ethnicity (due to genetics).
While NAFLD is often asymptomatic, it is the first step on a process where a portion of the population with NAFLD may have their disease progress to non-alcoholic steatohepatitis (NASH). When NASH is present, physiological damage is taking place. Ongoing liver scarring can lead to cirrhosis, liver fibrosis, liver cancer, hepatic encephalopathy and a range of other severe and debilitating conditions. Further, NASH worsens the cardiovascular condition of patients, and is related to higher risk of death caused by cardiovascular events.
The symptoms of NAFLD and NASH are often silent or non-specific (most commonly fatigue, mild upper abdominal pain), making them difficult to diagnose. Currently, blood-based liver function tests are widely used but are non-specific and are used to identify potential risk for a broad range of indications, while the gold standard for definitive diagnosis of liver disease is liver biopsy, which is costly, invasive, and not suited for screening. This is one of the greatest challenges in addressing these forms of liver disease, particularly distressing as up to NASH, the process is reversible, but from cirrhosis treatment shifts to delay of progression and addressing symptoms rather than curing.
Breath is an area that holds great potential however. Studies have suggested that metabolic dysfunction in liver disease is reflected in the composition of exhaled breath, with limonene, a naturally occurring chemical that is found in citrus foods, being particularly promising. Limonene was identified as a biomarker for liver disease in a paper by Mayhew (Owlstone Medical Scientific Advisory Board member) et al. at the University of Birmingham in 20151, and was studied more recently by Plevris et al.2, who provided validation of the potential of limonene to be used to stratify liver disease. Key to the work by Mayhew et al. is that pre- and post- liver transplant breath samples were investigated, which unambiguously allowed the researchers to assign limonene as a distinct biomarker for liver disease.
1. https://www.sciencedirect.com/science/article/pii/S2352396415300797
2. https://www.sciencedirect.com/science/article/pii/S2589555920300719
About EVOC® Probes
Owlstone Medical’s Exogenous Volatile Organic Compound (EVOC) Probes are compounds which undergo metabolism in the body and are excreted via breath, offering a readout of metabolism by enzymes and organs. Where possible, EVOC probes are designed utilizing GRAS (Generally Recognized as Safe) compounds, which are largely food additives. This approach enables high levels of exogenous compounds to be administered, such that fold changes in levels are much more visible with substantially improved signal-to-noise ratios over those that come from endogenous sources. The concept is similar to PET scans or glucose challenge studies where a probe is administered to the body to elicit a clear and unambiguous response. This can be a powerful approach, best deployed in applications where the underlying biology is well understood, operating through known metabolic pathways so that measurement of their breakdown or the triggering of another biochemical marker provides direct insight into the functioning of that pathway. A key paper on EVOC Probes was recently published in the Journal of Breath Research [https://doi.org/10.1088/1752-7163/ab1789].
What is Breath Biopsy®?
Breath Biopsy represents an entirely new way to measure the chemical makeup of breath by measuring volatile organic compounds (VOCs), which are gaseous molecules that can be sampled quickly and non-invasively from breath and enabling whole-body sampling. These compounds are produced as the end product of metabolic processes within the body, meaning underlying changes in metabolic activity can produce particular patterns of VOCs characteristic of specific diseases. Breath Biopsy also includes sampling of microscopic aerosol particles originating from the lungs and airways. Both VOCs and breath aerosol represent rich sources of biological information.
VOCs originating from all parts of the body are captured in breath, making Breath Biopsy applicable to a wide range of diseases including cancer, inflammatory disease, infectious disease, metabolic disease, cardiovascular disease and respiratory disease. The nature of Breath Biopsy, and VOC and aerosol biomarkers, make them perfectly suited to addressing two of the major challenges of healthcare today: early detection of disease and precision medicine.
Breath collection is carried out using Owlstone Medical’s ReCIVA® Breath Sampler, which ensures reliable, reproducible collection of VOCs. Subjects breathe a controlled supply of air, and samples of their exhaled breath are captured and stabilized on Breath Biopsy Cartridges, which can then be shipped for analysis with Owlstone Medical’s Breath Biopsy analytical platform, using mass spectrometry or FAIMS to determine their VOC profile. Advanced data analytic techniques can then be applied in order to pinpoint the VOCs of interest.