A Pilot Study of the VosCryoTM Breathalyser in Lung Cancer (CryoPilot)

Lung cancer remains one of the leading causes of cancer-related mortality worldwide, with late-stage diagnosis contributing substantially to poor prognosis (Bray et al., 2024). Despite advancements in imaging and molecular diagnostics, early detection strategies remain inadequate for population-wide screening, particularly in resource-limited settings. There is therefore an urgent need for novel, non-invasive, and cost-effective diagnostic approaches suitable for use in at-risk populations, such as individuals with a family history of lung cancer or those with a history of significant exposure to tobacco smoke.

Exhaled breath condensate (EBC) offers a promising, non-invasive source of biomarkers for respiratory diseases including lung cancer (Carpagnano et al., 2005; Palomba et al., 2023). EBC is a biological fluid formed by the cooling of exhaled breath and contains aerosolised droplets from the respiratory tract, including proteins, nucleic acids, volatile organic compounds, and, occasionally, cellular material (Horvath et al., 2005; Mutlu et al., 2001). These components may reflect pathological processes occurring in the lower airways and alveoli, thus offering a window into the disease state without the risks associated with bronchoscopy or biopsy.

Recent technological developments have enabled the reproducible collection of small volumes of EBC in a clinically practical timeframe. The VosCryo™ device, is a commercially available disposable device that collects approximately 100-200 µL of condensate in about one minute. The VosCryo™ which we will use, is a fast, non-invasive, and reliable EBC collector, optimized for small-volume sampling (50-200 µL/min).

Primarily the study aims to determine whether small-volume EBC obtained via VosCryo™ from late-stage lung cancer patients contains quantifiable and analysable cellular material, including epithelial cells, leukocytes, or exfoliated tumour cells. This will be determined from the proportion of participants with lung cancer with analysable material within their exhaled breath condensate. The study also aims to compare the presence and quality of such material with that obtained from healthy relatives (acting as a low-risk control group), to assess whether EBC-based analysis may differentiate between cancer and non-cancer states, and therefore will recruit from two cohorts - patients with late stage lung cancer and relatives of late-stage cancer patients with no evidence/low risk of cancer. This exploratory pilot study will inform the viability of future development of EBC as a low-cost, non-invasive diagnostic in patients with advanced and early-stage lung cancer, and as a potential screening tool for use in at-risk populations, and may lead to larger scale studies.

This will be a single-centre pilot study run at Weston Park Cancer Centre in Sheffield, United Kingdom, with patients and relatives recruited by clinical/medical oncologists and clinical trials assistants during their routine oncology clinic appointments. Patients attending the lung cancer clinic without an accompanying relative can be approached for study participation. Consenting patients with an accompanying relative who declines study participation can be included, as can consenting relatives with a patient who declines.

With patient consent, the study will collect a range of data at the single baseline visit for descriptive statistical analysis, including:

• Sex.
• Age.
• Smoking status - never smoker, ex-smoker (not smoked >12 months), recent ex-smoker (smoked ≤12 months), current smoker (smoked within past 14 days), vapes.
• TNM tumour stage 9th edition (where appropriate) from most recent staging scans performed as part of NHS standard of care.
• Tumour histology (where appropriate) including histological subtype, immunohistochemistry profile, mutation profile and PD-L1 status from results reported as part of NHS standard of care investigations.
• History of any concurrent pulmonary disease (e.g. emphysema, interstitial lung disease)

EBC Research Samples will be analysed using immunocytochemistry, indirect RNA extraction, cDNA synthesis and qPCR-based analysis of genes expressed in epithelial and immune cell types. The samples will also be stored for the duration of the project (18 months), and may be re-analysed in future projects using techniques such as ELISA, fluorescence microscopy or mass spectrometry.

It is estimated that participant recruitment and sample collection will be completed over a 6-month period, with subsequent sample and data analysis completing over an additional 12-month period.