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

Background:

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). It is designed for versatile research use - ideal for investigating biomarkers in lung cancer, infectious diseases, or chronic respiratory conditions, with minimal contamination risk and compatible with standard diagnostic assays. Its compact design and ease of use makes it particularly well-suited to clinical and point-of-care environments. Importantly, pilot studies have demonstrated the feasibility of recovering DNA, RNA, and protein from similar volumes of EBC, although the yield and integrity of these components at this low volume remain incompletely characterised (Kharitonov and Barnes, 2006; Phillips et al., 2003).

The aim of this pilot study is to evaluate whether analysable cellular material can be identified within small-volume EBC collected using the VosCryo™ device from patients with advanced lung cancer. This exploratory work 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. The work has the potential to inform larger-scale biomarker studies and, ultimately, contribute to improved outcomes through earlier diagnosis.

Primary objective:

The primary objective is 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.

Secondary objective:

A secondary aim is 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.

Primary Outcome:

The primary outcome is the proportion of participants with lung cancer with analysable material within their exhaled breath condensate.

Secondary Outcome:

The secondary outcome is comparison of exhaled breath condensate from lung cancer patients with participants without lung cancer

Study Design:

This is a pilot study. Potential participants will be approached when attending lung cancer clinics at Weston Park Hospital. Two cohorts of study participants will be recruited:

1. Patients with previously untreated advanced lung cancer
2. An accompanying patient friend or relative with no history of lung cancer

Recruitment:

Potential participants will be approached by a Clinical Trials Assistant/Research Nurse when attending lung cancer clinics at Weston Park Hospital. Participants will be provided with the Participant Information Sheet and given time to read this. There will be no minimum time required for this. Participants will be given the chance to discuss the study and ask any questions, then be consented and registered and the EBC research sample collected. Any consenting participants who are unable to complete the research breath sample will be withdrawn. Reasons for sample collection failure will be collected in a screening log.

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. Recruitment will continue until 12 participants have completed the EBC sample collection in both cohorts.

Inclusion criteria:

• Age ≥16 years old.
• Signed written consent.
• Advanced non-small cell or small cell lung cancer with intra-thoracic disease (pulmonary primary or pulmonary metastases), previously untreated by systemic anti-cancer treatment or radiotherapy. Patients with previously treated early-stage lung cancer are permitted. Patients with advanced disease with extra-thoracic disease previously treated by local therapy (e.g. palliative radiotherapy to a bone metastasis, or post brain metastectomy), which in the opinion of the principal investigator is unlikely to impact the quality of the EBC research sample are eligible; or
• Attending relative or friend with no history of lung cancer or any other primary cancer, except for curatively treated non-melanomatous skin cancer or carcinoma in situ, unless the participant has been free of malignancy and off treatment for a period of at least 2 years prior to study enrolment.
• No intercurrent upper or lower respiratory tract infection.
• Able to complete the EBC sample collection.

Exclusion criteria:

• Participants with lung metastases from non-lung primaries.
• Participants unable to consent or unable to comply with the requirements for

Study procedures:

Study participants will be allocated a study number. All study data and study research samples will be labelled and recorded using the study number.

All study procedures will be performed at baseline. No follow up procedures are required for this study.

Participant characteristics recorded at baseline:

• 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)

Collection of the EBC Research Sample:

The EBC will be collected only once participant informed consent is obtained. The EBC need not be collected on the same visit as consenting but must be collected before starting any anti-cancer treatment.

EBC Research Sample Analysis:

Specimens will be analysed in the School of Bioscience, University of Sheffield, as follows:

1. The device will be subjected to centrifugation at 90 g for 1 min at ambient temperature using a swing-out rotor.
2. The total sample volume will be noted.
3. The supernatant will be removed and stored at -20oC.
4. Pelleted material will be resuspended in phosphate-buffered saline, and an aliquot dispensed onto a poly-L lysine coated coverslip.
5. Material will be fixed using 4% paraformaldehyde for 15 minutes and then analysed using immunocytochemistry and indirect immunofluorescence microscopy.
6. Another aliquot of the pelleted material will be subjected to RNA extraction, cDNA synthesis and qPCR-based analysis of genes expressed in epithelial and immune cell types.
7. Any remaining samples will be stored at -20oC.
8. The results will be recorded for data analysis and statistical purposes.
9. Samples will be stored for the duration of the project (18 months), and may be re-analysed in future prototype versions of this project, and their composition further characterised either by ELISA, fluorescence microscopy or mass spectrometry. After which time, samples will be destroyed by the Chief Investigator.

Statistical Analysis:

12 participants completing the research breath sample will be recruited to each cohort (12 patients with advanced lung cancer, 12 accompanying relatives); i.e. a minimum of 24 participants in total.

Descriptive statistics will be used to summarise and compare the two cohorts.

Study conduct:

The study will be conducted in accordance with the trial protocol, the International Conference on Harmonisation (ICH) for Good Clinical Practice (GCP) and appropriate regulatory requirements. The study will have been approved by the HRA and an NHS Research Ethics Committee prior to commencement.

Recording and reporting of events and incidents:

An Adverse Event (AE) is any untoward medical occurrence in a participant, including occurrences which are not necessarily caused by or related to the intervention.

A serious adverse event (SAE) is any untoward medical occurrence that:

1. results in death;
2. is life-threatening;
3. requires hospitalisation or prolongation of existing hospitalisation;
4. results in persistent or significant disability or incapacity; or
5. consists of a congenital abnormality or birth defect; or
6. is otherwise considered medically significant by the investigator. NOTE: The term "life-threatening" in the definition of "serious" refers to an event in which the participant was at risk of death at the time of the event; it does not refer to an event which hypothetically might have caused death if it were more severe.

The research device is single use and the procedure to collect the EBC research sample does not require any period of extended restricted breathing, hyperventilation or forced breathing. With no increased work of breathing, there are no expected adverse or serious adverse events (SAEs) from study participation.

In the unlikely event of any SAE occurring, these will be managed as per local standard practise. Any SAEs that are related to the trial (i.e., they result from administration of any of the research procedures) and unexpected (i.e., not listed in the protocol as an expected occurrence) will be emailed to the research ethics committee (REC) using the non-CTIMP safety report to REC form. These will be sent within 15 days of the chief investigator becoming aware of the event.

Data Handling:

Consenting participants will be assigned a study number. This number will be used to label the research sample and participant study data. No participant personal identifiers will be stored.

Project Timescales:

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. A final study report will then be generated.

Funding Arrangements:

This project is funded by VosBio (the manufactures of the VosCryo™ device). The University of Sheffield will be the fundholder and any NHS funds will be paid upon submission of invoices as per the study contract.