The development of a breath test for the early detection of lung cancer


Lung Cancer

Lung cancer is one of the most common forms of cancer in the world with 2.2 million new cases in 2020 and is a leading cause of death (1.8 million globally in 2020), with 5-year survival at 25% in the United States and just 15% in the United Kingdom (1–3). The best way to increase the current poor survival probabilities for patients with lung cancer is to detect the disease early when it can be treated with curative intent by surgery or radiotherapy: diagnosis at an early stage increases the 5-year survival rate to 56%, compared to just 5% for the late stage (4). In the United States there are guidelines in place for using Low-dose CT (LDCT), and a similar program is being launched in the U.K. to screen for lung cancer. This use of LDCT for lung cancer screening has been shown to detect more cancers at an early stage, however, uptake is less than 6% due to inconvenience, radiation dose, test performance, and in some cases, cost.

Figure 1 – Statistics about lung cancer.

Therefore, there is a significant need for alternate screening tools for lung cancer. Breath analysis has emerged as a highly promising, non-invasive, potential solution. An effective breath test for lung cancer could further increase the efficacy, reach, and accessibility of lung cancer screening.

Breath contains volatile organic compounds (VOCs) that can inform on underlying physiology, therefore could offer a much-needed alternative approach to overcome the current barriers to lung cancer screening (5). For a lung cancer test for screening and early detection to be successful, it must be able to detect signals coming from even small early-stage tumors, alongside potential multiple co-morbidities. Through careful evaluation of the metabolic pathways underpinning the production of the volatile compounds linked to the presence of cancer, the EVOC® probe approach was suggested to be able to improve the signal-to-noise ratio of breath tests and has shown success for a candidate test to diagnose liver disease (6,7). 

vocs and cancer
Figure 2 – Exhaled breath contains volatile metabolites that can be used as biomarkers.
The D5-EthGlu Probe

A Probe-Based Breath Test for Lung Cancer

An innovative and alternative approach for breath-based testing involves the utilization of exogenously produced volatile compounds as probes. Owlstone Medical has developed Exogenous Volatile Organic Compound (EVOC®) Probes. These are compounds that can be introduced into the body (through ingestion or inhalation) to explore how the probe is absorbed, metabolized, and excreted through dynamic analysis of the changes in breath VOC composition in response. This approach can target specific pathways of interest and amplify the signal from these pathways for better breath test performance. This concept draws similarities to how labeling urea with 13C is currently employed as a metabolic probe in clinical settings to evaluate H. pylori infection within the gastrointestinal tract.

In the EVOLUTION trial, the diagnostic potential of D5-ethyl-βD-glucuronide (also known as D5-EthGlu or OWL-EVO1) as an exogenous volatile organic compound (EVOC®) probe for detection of lung cancer is currently being evaluated (8,9).  

Figure 3 – The proposed workflow of using the D5-ethyl-βD-glucuronide probe to screen for lung cancer. (1) A baseline breath sample is collected, and then the probe is inhaled into the lungs. (2) The breath is then collected at regular time points after inhalation of the probe to monitor the levels of the probe product D5-ethanol. (3) The results will then be compared to a reference range to distinguish whether the patient has a breath test result aligning with potential presence of lung cancer.


Under normal conditions, the enzyme β-glucuronidase is present within cells in the lysosome and is responsible for the catalytic hydrolysis of β-d-glucuronides. Research in humans and mice has pointed towards β-glucuronidase being differentially expressed in the tumor microenvironment (includes tumor cells and the extracellular space with surrounding cells, blood vessels and tissues) (10,11) of many types of lung cancer, including squamous, adeno, and adeno-squamous non-small-cell lung cancers (12). Interestingly, high levels of β-glucuronidase in the tumor microenvironment this has also been linked to poorer prognostic outcomes​ (13,14)​. This approach utilizing β-glucuronidase has previously been used and tested in vivo for targeted cancer therapy (15,16). Therefore, the presence of β-glucuronidase outside of the cellular environment could serve as a biomarker for the early detection of lung cancer and is a promising target for an EVOC probe approach.

Figure 4 – A schematic indicating how β-glucuronidase in the extracellular space around a tumor in the lungs can metabolize the D5-ethyl-βD-glucuronide probe into D5-ethanol.

To exploit this mechanism, the probe D5-ethyl-βD-glucuronide was created. This probe can be metabolized by the β-glucuronidase enzyme to produce D5-ethanol. In someone without lung cancer, the D5-ethyl-βD-glucuronide probe will come into contact with the cells in their lungs. There is not normally β-glucuronidase enzyme present outside of the cells (extracellular), and therefore the probe should not be metabolized into D5-ethanol. If there is cancer in the lungs, the D5-ethyl-βD-glucuronide probe will come into contact with extracellular β-glucuronidase around the tumor and be metabolized into D5-ethanol which can subsequently be exhaled in the breath. Therefore, presence of D5-ethanol in the breath in response to administration of the probe could provide a reliable indicator for lung cancer, and potentially boost the signal to detect even small tumors. Potential also exists for such a breath test to be used to differentiate benign from malignant lung nodules identified through incidental findings or LDCT screening.

EVOLUTION Trial progress

Results from Phase 1

Results from Phase 1 of the EVOLUTION trial demonstrated that the molecular pathway targeted by the D5-ethyl-βD-glucuronide probe could indeed produce D5-ethanol, and provided crucial safety data, and evidence to support further test development. The EVOLUTION trial is currently at Phase 2, and is assessing the diagnostic performance of the test – enabling optimization of the probe through refinement of the breath testing protocol. Supporting this, the study has been designed to allow differentiation between individuals with lung cancer and relevant contrast groups (e.g. COPD) representative of the clinical populations in which the test is intended to be used.


Catch up on our recent news and data involving the EVOLUTION trial:

POSTER: Proof-of-mechanism for a diagnostic probe generating D5-ethanol as an on-breath reporter molecule for lung cancer – Evolution phase 1

POSTER: Using exogenous volatile organic compound (EVOC) probes to target tumour-associated aldo-keto reductase activity: a potential tool to detect lung cancer

NEWS: Owlstone Medical Recruits First Patient in Phase 2 Clinical Trial for the Early Detection of Lung Cancer through Breath Biopsy®

BBC NEWS: Royal Papworth Hospital: First-of-its-kind lung cancer test trialled

NEWS: Owlstone Medical Presents Data Demonstrating Progress in Development of Breath Biopsy® Test for the Early Detection of Lung Cancer



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