The electronic nose: emerging biomarkers in lung cancer diagnostics

Diagnostic biomarkers

A biomarker, when available, could be useful in three important roles. First, it could aid in establishing a lung cancer diagnosis, especially as part of a thoracic cancer screening programme with a relatively high percentage of false-positive findings. It could also aid in selecting high-risk patients for a CT screening programme. In lung cancer, no such biomarkers have been validated for clinical use but in other fields, such biomarkers have been of interest (e.g.prostate-specific antigen and carcinoembryonic antigen). For lung cancer, the ECLS and bio-MILD trials are currently running to assess blood tumour markers.

Furthermore, the gold standard to diagnose thoracic cancers is a pathology report indicating malignant cells. However, quite often (e.g. in severe COPD patients in whom a biopsy cannot be safely obtained), it is not possible to gain enough tissue to establish a diagnosis, and the multidisciplinary team makes decisions based on radiology and clinical details. A diagnostic biomarker would be valuable to help support these teams in their decisions on probability of malignancy.

Predictive biomarkers

The second important role of a biomarker is to predict a future treatment response. The detection of epidermal growth factor receptor (EGFR) mutations and ALK or other driver mutations are regarded as successful biomarkers in non-small cell lung cancer (NSCLC) [6, 7]. Another biomarker used to predict treatment responses is the programmed death ligand 1 (PD-L1) tumour proportion score. PD-L1 is associated with checkpoint inhibitor efficacy in stage IV, and potentially also in stage III, NSCLC. In clinical trials for mesothelioma, small cell lung cancer and other stages of NSCLC, PD-L1 is also gaining more clinical attention. However, PD-L1 assays are far from perfect, with a low sensitivity and specificity [2]. A significant amount of research activity is aimed at developing new strategies and biomarkers to replace or improve PD-L1. There is a need to improve the sensitivity and specificity of this biomarker, with potential for improvements from new or additional biomarkers.

Biomarkers for monitoring

Thirdly, a biomarker could aid in the serial monitoring of treatment effect, or to distinguish between disease progression and toxicity from treatments such as radiation or immunotherapy. Such a biomarker could be effectively used in parallel to information gained from CT or positron emission tomography scans. In lung cancer, no such biomarkers are available. However, this role for biomarkers is currently being used in other cancer treatments by, for example, prostate-specific antigen and carcinoembryonic antigen.

Ideally, a diagnostic biomarker that is designed to optimise efficacy and safety of low-dose CT screening for lung cancer and subsequent invasive diagnostics should have a high sensitivity and specificity, should be noninvasively obtained, and should be easy to use and inexpensive. Exhaled breath analysis could fit this profile in the detection of thoracic malignancies. In this narrative review, we provide an overview of the currently available data on electronic nose (e-nose) as a potential diagnostic, predictive or monitoring biomarker for lung cancer treatment.

An e-nose can detect volatile organic compounds (VOC). It is used for both medical and nonmedical purposes. The e-nose for medical purposes is most commonly used to measure VOCs in exhaled breath from patients but has also been used in the assessment of several biological samples including faeces, biopsies, saliva and skin [8–10].

What are VOCs?

VOCs were identified in the 1970s [11]. Since then, breath analysis has boomed into a high-throughput breathomics research field with >3000 different VOCs discovered in human breath [12, 13]. Most particles in the air are biogenic and emitted through external processes such as the environment and atmospheric pollution [10]. However, due to metabolic processes within the human body, VOCs can be emitted or VOC patterns can be altered [13]. These processes can be physiological but can be also induced by or altered due to disease. Therefore, it is believed that these VOCs cause a specific “smell” or “breathprint” for different diseases. When a concentration of VOCs is measured directly or measured after being captured and stored (e.g. via collection bags or canisters), different breathprints or patterns can be detected [14].

VOCs in other diseases

VOC and breathprint detection with e-noses have previously been shown to be effective in respiratory diseases [15]. For COPD, the technique not only allows a COPD diagnosis but can also detect the origin of COPD exacerbations, and can be used in the differential diagnosis of COPD and asthma [15–17]. In asthma, it is used to detect the disease and to determine its phenotype. VOCs are used in cystic fibrosis and in the detection of tuberculosis [18]. Furthermore, nonrespiratory diseases can be detected using an e-nose (e.g.Barrett's oesophagus and inflammatory bowel disease) [8, 19]. For other cancer types, positive e-nose studies have been published, including head and neck, bladder, and colon cancer [20].

VOCs in lung cancer

At the foundation of breath pattern research in cancer lies a trial by McCulloch et al. [21]. This trial showed that trained dogs were able to detect lung cancer patients in a group of volunteers including healthy subjects. In the 1980s, specific VOCs were demonstrated for the first time in patients with lung cancer [22]. Research then focused on identifying specific VOCs. Different individual VOCs assessed were propranolol, isoprene, acetone, pentane, hexanal and benzene [23]. However, at the time, this proved to be inadequately accurate, expensive and time consuming [24].

As such, interest declined. As technology progressed and superior sensors were developed, interest re-emerged, and new tests were performed on tissues and cell lines in the laboratory [9, 25]. The sensors currently being used focus on pattern recognition. These patterns need to be “learned” first by the machine using artificial intelligence in a manner analogous to the training of dogs used in the original McCulloch study [21]. With this principle, it has now been possible to differentiate lung cancer from healthy subjects and from COPD patients [14, 26–28]. Currently, issues preventing the technique from being widespread in clinical practice include stability of the VOCs, and stability and interchangeability of the devices [29]. If these issues can be resolved, we anticipate that e-noses may find their way to routine practice.

Progress has also been made in mesothelioma. The first tests were published in 2012 [30, 31], showing that molecular pattern recognition of exhaled breath could distinguish mesothelioma patients from healthy controls. More recently, this was confirmed by a study combining breath analysis by gas chromatography–mass spectrometry and an e-nose [32, 33].

Gas chromatography

This technique allows the separation of different types of molecules. The air sample is combined with a carrier gas and then moves against a stationary component with a reaction as result. Different substances provide a different responses, with simple chromatography as an obvious example [34, 35].

Spectrometry

Spectrometers are most often combined with gas chromatography. These are used as devices for the identification of specific chemicals. After ionisation of the compounds, the ions from the molecules are separated according to mass-to-charge ratio. This separation normally occurs in a vacuum with a magnetic field. The technique is cumbersome, expensive and difficult to transport, and to date, no point-of-care system with this technique is widely used [34, 36].

Colorimetry

Colorimetric devices work with sensors with chemically responsive dyes. These dyes can be adapted based on the targeted VOC. Multiple dyes can be used in one sensor, allowing patterns of VOCs to be detected [36].

Surface acoustic wave

Acoustic sensors work by exposing their sensors to gases. The gases then change an already emitted acoustic wave due to reactions with the sensor surface. These waves are then analysed for VOC or pattern identification [34].

Conductometry

This technique works with sensors (e.g. metal oxide or polymeric sensors) that consist of different metals that allow for various interactions with volatile compounds. Exhaled air is guided over these sensors, allowing redox reactions to occur, resulting in conductivity changes of the sensors [16, 34, 37].

Diagnostic e-nose biomarkers

When we assess the potential roles of biomarkers in thoracic disease, most VOC-based research has been dedicated to the detection of cancer. Todate,e-noses have been able to detect cancer in different settings and have been tested in vitro. They can distinguish lung cancer patients from healthy patients, both in volunteers and in those suspected of having cancer [38]. Such studies most commonly assess VOCs emitted by cells in a laboratory setting, with some in vivo studies, but mostly in pilot form.

These studies have demonstrated the detection of lung cancer with a sensitivity ranging from 71% to 96% and specificity from 33 to 100% (table 1). Several attempts have been made to validate these results but have mostly only been conducted using internal validation. There is a lack of standardised sampling and analysis method, impeding interstudy comparison and clinical implementation [29, 43, 44].

Predictive e-nose biomarkers

For the role of a biomarker in predicting future treatment responses, research is very limited. In a small pilot study, the e-nose has been able to differentiate between EGFR-mutated and wild-type EGFR NSCLC; however, more research to assess this is necessary [45]. A recently published study tested an e-nose for the prediction of response to anti-PD-1 therapy in patients with NSCLC with the area under the curve confirmed in the validation set to be 0.85 [46].

Monitoring e-nose biomarkers

For this specific role, at this stage, we have found no published data. However, efforts to study this specific role for the e-nose are being developed.