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It is increasingly understood that cancers can be recognized by the immune system and that inflammation relates to response. Combining stereotactic radiotherapy (SBRT) to increase release of cancer cell antigens with an anti-CTLA-4 and PD-L1 inhibitor may lead to increased response rates. Due to tumor heterogeneity it may be important to irradiate the primary tumor (addressing trunk mutations), instead of its metastasis (branch mutations). No solid predictive biomarker for response and/or immunotherapy related adverse events (TRAEs) is available. Electronic nose (eNose) technology measures the complete mixture of volatile organic compounds in exhaled breath and can detect lung cancer based on pattern recognition of the breath profile. We hypothesize that eNose is able to predict TRAEs and response in patients with NSCLC. Methods: In 3 sequential cohorts, immunotherapy regimes combined with SBRT were studied in stage IIIB/IV NSCLC patients progressing on chemotherapy. All patients were irradiated on the primary tumor (1x20 Gy on 9cc) 1 week after the 1st dose of immunotherapy. The 1st cohort (n=3) received durvalumab. The 2nd and 3rd cohort (both n=6) received a combination of durvalumab and tremelimumab followed by durvalumab monotherapy. Duplicate eNose measurements were performed by using the SpiroNose that contains 7 metal oxide semiconductor sensors. TRAEs were categorized using NCI CTCAE version 4.3. Descriptive statistics were used to summarize baseline characteristics and TRAEs. The relationship between breath profiles and response and TRAEs was analyzed with advanced signal processing, ambient correction and Mann-Whitney U test. Linear discriminant and receiver operating characteristics analysis followed. Pearson correlation and regression assessed duration of response. Findings: Fifteen patients were included as described above. Baseline characteristics of the groups were comparable. Median progression free survival was 2 months, overall survival 10 months (immature). No statistical difference was found in (duration of) response. There was 1 low grade TRAE (CTC 1-2) in cohort 1 and 10 in cohort 2/3. High grade TRAEs (CTC 3) were only present in cohort 2/3 (n=3) and 1 patient discontinued treatment. There was one dose limiting toxicity. At baseline, 12/15 patients performed eNose measurements; 7 with TRAEs and 5 without. The TRAE group had a significant higher sensor 7 signal compared to those without TRAE (p=.042). The cross-validated accuracy for detecting TRAE was 67%. The ROC-AUC was .857 [.638-1]. Interpretation: eNose breath profiles at baseline may predict which patients will develop TRAEs. The small sample size increases the risk of overfitting, therefore another cohort of 34 immuno-monotherapy patients is being analyzed (15 with and 19 without TRAEs, pending). We demonstrated no new safety data. Disclosure: This study was sponsored by a research grant from AstraZeneca. Citation Format: M Benthe Muntinghe-Wagenaar, Hanneke Kievit, Lucie B. Hijmering-Kappelle, Birgitta I. Hiddinga, J Fred Ubbels, Robin Wijsman, Mechteld F. Brasz, Gitte Slingers, Rianne de Vries, Milou M. Schuurbiers, Harry J. Groen, Huib A. Kerstjens, Anthonie J. van der Wekken, Michel M. van den Heuvel, T Jeroen Hiltermann. A phase 1 study to detect adverse events after SBRT and immunotherapy by electronic nose in advanced NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 666.

Rationale Selected ion flow tube mass spectrometry (SIFT-MS) is versatile, rapidly provides result output and determines a wide range of volatiles, making it suitable for biomedical applications. When direct sampling into the SIFT-MS instrument is impractical, combining thermal desorption (TD) and SIFT-MS might offer a solution as it allows sample storage on sorbent tubes for later analysis. This work compares off-line TD SIFT-MS and real-time SIFT-MS for the quantification of selected breath volatiles. Methods Ten healthy non-smoking individuals provided 60 breath samples per method. For off-line analysis, breath was collected onto sorbent tubes via a breath sampler provided with filtered inspiratory air. After TD, samples were re-collected in Tedlar bags which were then connected to the SIFT-MS instrument. For real-time analysis, breath was sampled directly into the instrument. In both cases the analytical method included a total of 155 product ions, and 14 selected volatiles were quantified. The agreement between the methods was assessed using Pearson correlation coefficients and Bland-Altman plots. Results Overall, correlations between real-time and off-line analysis were moderate to very strong (r = 0.43-0.92) depending on the volatile of interest, except for 2,3-butanedione and styrene. The difference between real-time and off-line measured breath concentrations (average bias) ranged between -14.57 and 20.48 ppbv. For acetone and isoprene, it was 251.53 and 31.9 ppbv, respectively. Conclusions Real-time SIFT-MS and off-line TD SIFT-MS for quantification of selected breath volatiles did not show optimal agreement. Analyzing a multitude of analytes in breath via direct exhalation into a SIFT-MS instrument for real-time analysis is challenging. On the other hand, off-line analysis using a breath collection device also has its issues such as possible sample losses due to selective absorption depending on the sorbent used or during desorption and transfer to the instrument. Despite these drawbacks, both methods were moderately well correlated.

A major challenge for breath research is the lack of standardization in sampling and analysis. To address this, a test that utilizes a standardized intervention and a defined study protocol has been proposed to explore disparities in breath research across different analytical platforms and to provide benchmark values for comparison. Specifically, the Peppermint Experiment involves the targeted analysis in exhaled breath of volatile constituents of peppermint oil after ingestion of the encapsulated oil. Data from the Peppermint Experiment performed by proton transfer reaction mass spectrometry (PTR-MS) and selected ion flow tube mass spectrometry (SIFT-MS) are presented and discussed herein, including the product ions associated with the key peppermint volatiles, namely limonene, alpha- and beta-pinene, 1,8-cineole, menthol, menthone and menthofuran. The breath washout profiles of these compounds from 65 individuals were collected, comprising datasets from five PTR-MS and two SIFT-MS instruments. The washout profiles of these volatiles were evaluated by comparing the log-fold change over time of the product ion intensities associated with each volatile. Benchmark values were calculated from the lower 95% confidence interval of the linear time-to-washout regression analysis for all datasets combined. Benchmark washout values from PTR-MS analysis were 353 min for the sum of monoterpenes and 1,8-cineole (identical product ions), 173 min for menthol, 330 min for menthofuran, and 218 min for menthone; from SIFT-MS analysis values were 228 min for the sum of monoterpenes, 281 min for the sum of monoterpenes and 1,8-cineole, and 370 min for menthone plus 1,8-cineole. Large inter- and intra-dataset variations were observed, whereby the latter suggests that biological variability plays a key role in how the compounds are absorbed, metabolized and excreted from the body via breath. This variability seems large compared to the influence of sampling and analytical procedures, but further investigations are recommended to clarify the effects of these factors. Marie Skodowska-Curie Actions Innovative Training Network: Ion-Molecule Processes for Analytical Chemistry Technologies (IMPACT) through the European Commission's HORIZON 2020 Programme [674911]; UK's National Physical Laboratory

The performance of the MSD immunoassay, as an allergy blood test, was compared to the skin prick test (SPT; golden standard in clinical practices) and ImmunoCAP™ assay (most commonly used blood test). The MSD immunoassay, detecting sIgE against respiratory allergens, showed high sensitivity and specificity (compared to the ImmunoCAP™ assay or SPT), and low inter‐ and intra‐assay variation with good reproducibility., Summary Allergic sensitization is commonly assessed in patients by performing the skin prick test (SPT) or determining specific immunoglobulin (IgE) levels in blood samples with the ImmunoCAP™ assay, which measures each allergen and sample separately. This paper explores the possibility to investigate respiratory allergies with a high throughput method, the Meso Scale Discovery (MSD) multiplex immunoassay, measuring IgE levels in low volumes of blood. The MSD multiplex immunoassay, developed and optimized with standards and allergens from Radim Diagnostics, was validated against the SPT and the ImmunoCAP assay. For 18 adults (15 respiratory allergy patients and three controls), blood collection and the SPT were performed within the same hour. Pearson correlations and Bland–Altman analysis showed high comparability of the MSD multiplex immunoassay with the SPT and the ImmunoCAP assay, except for house dust mite. The sensitivity of the MSD multiplexed assay was ≥78% for most allergens compared to the SPT and ImmunoCAP assay. Additionally, the specificity of the MSD multiplex immunoassay was ≥ 87% – the majority showing 100% specificity. Only the rye allergen had a low specificity when compared to the SPT, probably due to cross‐reactivity. The reproducibility of the MSD multiplex immunoassay, assessed as intra‐ and interassay reproducibility and biological variability between different sampling moments, showed significantly high correlations (r = 0·943–1) for all tested subjects (apart from subject 13; r = 0·65–0·99). The MSD multiplex immunoassay is a reliable method to detect specific IgE levels against respiratory allergens in a multiplexed and high‐throughput manner, using blood samples as small as from a finger prick.

Sampling of volatile organic compounds (VOCs) has shown promise for detection of a range of diseases but results have proved hard to replicate due to a lack of standardization. In this work we introduce the 'Peppermint Initiative'. The initiative seeks to disseminate a standardized experiment that allows comparison of breath sampling and data analysis methods. Further, it seeks to share a set of benchmark values for the measurement of VOCs in breath. Pilot data are presented to illustrate the standardized approach to the interpretation of results obtained from the Peppermint experiment. This pilot study was conducted to determine the washout profile of peppermint compounds in breath, identify appropriate sampling time points, and formalise the data analysis. Five and ten participants were recruited to undertake a standardized intervention by ingesting a peppermint oil capsule that engenders a predictable and controlled change in the VOC profile in exhaled breath. After collecting a pre-ingestion breath sample, five further samples are taken at 2, 4, 6, 8, and 10 h after ingestion. Samples were analysed using ion mobility spectrometry coupled to multi-capillary column and thermal desorption gas chromatography mass spectrometry. A regression analysis of the washout data was used to determine sampling times for the final peppermint protocol, and the time for the compound measurement to return to baseline levels was selected as a benchmark value. A measure of the quality of the data generated from a given technique is proposed by comparing data fidelity. This study protocol has been used for all subsequent measurements by the Peppermint Consortium (16 partners from seven countries). So far 1200 breath samples from 200 participants using a range of sampling and analytical techniques have been collected. The data from the consortium will be disseminated in subsequent technical notes focussing on results from individual platforms. The Marie Sklodowska-Curie Actions Innovative Training Network: Ion-Molecule Processes for Analytical Chemistry Technologies (IMPACT) (www.impact-h2020itn.com), which has supported BH, MM and GP as Early Stage Researchers through the European Commission's HORIZON 2020 Programme under Grant Agreement Number 674911. The EMBER consortium for support in the dissemination of the Peppermint Initiative; researchers involved in the application of the protocol to specific techniques (to be published in due course) Abigail Joyce, Amisha Manek, Bellagambi Francesca Giuseppa, Bonsignori Alessio, Ghimenti Silvia and Biagini Denise, Liesl Carr, Luke Bryant and Rebecca Cordell. MW and SJF are supported by the NIHR Manchester Biomedical Research Centre. The group from Li`ege University is supported by the FNRS through the EOS and the FRIA initiatives. Ruszkiewicz, DM (, orresponding author), Loughborough Univ, Ctr Analyt Sci, Dept Chem, Loughborough, Leics, England ; Univ Birmingham, Sch Phys & Astron, Mol Phys Grp, Birmingham, W Midlands, England. D.M.Ruszkiewicz2@lboro.ac.uk

Volatile organic compounds in breath can reflect host and pathogen metabolism and might be used to diagnose pneumonia. We hypothesized that rats with Streptococcus pneumoniae ( SP) or Pseudomonas aeruginosa ( PA) pneumonia can be discriminated from uninfected controls by thermal desorption-gas chromatography-mass-spectrometry (TD-GC-MS) and selected ion flow tube-mass spectrometry (SIFT-MS) of exhaled breath. Male adult rats ( n = 50) received an intratracheal inoculation of 1) 200 µl saline, or 2) 1 × 107 colony-forming units of SP or 3) 1 × 107 CFU of PA. Twenty-four hours later the rats were anaesthetized, tracheotomized, and mechanically ventilated. Exhaled breath was analyzed via TD-GC-MS and SIFT-MS. Area under the receiver operating characteristic curves (AUROCCs) and correct classification rate (CCRs) were calculated after leave-one-out cross-validation of sparse partial least squares-discriminant analysis. Analysis of GC-MS data showed an AUROCC (95% confidence interval) of 0.85 (0.73–0.96) and CCR of 94.6% for infected versus noninfected animals, AUROCC of 0.98 (0.94–1) and CCR of 99.9% for SP versus PA, 0.92 (0.83–1.00), CCR of 98.1% for SP versus controls and 0.97 (0.92–1.00), and CCR of 99.9% for PA versus controls. For these comparisons the SIFT-MS data showed AUROCCs of 0.54, 0.89, 0.63, and 0.79, respectively. Exhaled breath analysis discriminated between respiratory infection and no infection but with even better accuracy between specific pathogens. Future clinical studies should not only focus on the presence of respiratory infection but also on the discrimination between specific pathogens.

Volatile organic compounds (VOCs) in breath can reflect host and pathogen metabolism and might be used to diagnose pneumonia (Oort J Breath Res 2018). We hypothesized that rats with Streptococcus pneumoniae (SP) or Pseudomonas aeruginosa (PA) pneumonia can be discriminated from uninfected controls by gas-chromatography mass-spectrometry (GC-MS) of exhaled breath. Male adult rats (N = 50) received an intra-tracheal inoculation of 1) 200µL saline, 2) 1x107 colony forming units (cfu) of SP or 3) 1x107 cfu of PA. 24 hours later the rats were anaesthetized, tracheotomized and mechanically ventilated. Exhaled breath was analyzed via GC-MS. Area under the receiver operating characteristic curve (AUROCC) was calculated after leave-one-out cross-validation of sparse partial least squares discriminant analysis (sPLS-DA). The AUROCC was 0.85 (95%-CI: 0.73-0.96) for infected vs. non-infected animals. The AUROCC was 0.98 (95%-CI: 0.94-1) for SP vs. PA, 0.92 (95%-CI: 0.83-1) for SP vs. controls and 0.97 (95%-CI: 0.92-1) for PA vs. controls. Exhaled breath analysis discriminated with good accuracy between respiratory infection and no infection, but with much better accuracy between the specific pathogens. Clinical studies should focus more on the discrimination between specific pathogens, instead of the general presence of respiratory infection.

Exhaled volatile organic compounds (VOCs) could potentially serve as non-invasive biomarkers for the diagnosis of respiratory infections. This study aims to discriminate rats infected with Pseudomonas aeruginosa (PA) or Streptococcus pneumoniae (SP) from non-infected rats. Exhaled breath was collected from 50 male rats 24h after intra-tracheal inoculation of 200µL saline, or 1x107 colony forming units of SP or PA. VOCs in exhaled breath trapped on sorbent tubes were analysed with Selected Ion Flow Tube Mass Spectrometry (SIFT-MS) after thermal desorption. The pattern of reaction ions with mass/charge ratio 15 to 200 was analysed using sparse partial least squares discriminant analysis. Area under the receiver operating characteristic curve (AUROCC) calculated after leave-one-out cross-validation of the multivariate discriminant analysis was 0.73 (95%CI: 0.57-0.89) for infected vs. non-infected animals. For SP vs. PA the AUROCC was 0.97 (95%CI: 0.93-1) while for SP vs. controls and PA vs. controls it was 0.67 (95%CI: 0.47-0.86) and 0.72 (95%CI: 0.53-0.91), respectively. Exhaled breath analysis discriminated with higher accuracy between animals infected with specific respiratory pathogens compared to infected vs. non-infected animals. Similar findings were observed for VOCs analysed with Gas Chromatography Mass Spectrometry (see submitted ERS abstract from P. M. van Oort et al.). Our results suggest that breath analysis in infected patients may be helpful in discriminating different pathogens. Furthermore, SIFT-MS has potential as a point-of-care tool to assess respiratory infections in clinical practice.

Introduction: Categorizing noisy breathing sounds is of major clinical relevance to identify the most likely underlying cause. However frequently mislabeling this sound increases the risk of wrong treatment. No standardized objective method is available for diagnosis. Analysis of exhaled breath volatile metabolites has the potential to fulfill this need. This study aimed to validate Selected Ion Flow Tube Mass Spectrometry (SIFT-MS) for analysis of breath volatiles. Method: Quality parameters of the SIFT-MS such as quantification limit and reproducibility were determined for selected voltiles linked to lung diseases. These were hexanol, pentanal, decanal, pentane, dodecane, p-xylene, decene, acetophenone, hexanoic acid, valeric acid, acetic acid, dimethyl disulphide and isoprene. Measurement uncertainty was calculated from absolute bias and reproducibility. A commercially available breath sampler ReCIVA®) with integrated sorbent tube trapping was optimized for use in infants (2-24 months). Results: Two of the 16 compounds resulted in a measurement uncertainty above 50% (theoratically acceptable). All others had an acceptable measurement uncertainty ranging from 12.28 to 38.78%. The quantification limit ranged from 1.48 to 14.12ppb with an exception of 70.71ppb for pentane. The mouth piece of the breath sampler was adjusted for infants and the flow volume of the clean air supply for inhalation was lowered from 40L/min (for adults) to 10L/min. Conclusion: SIFT-MS was suitable for analysis of exhaled breath volatiles. For some breath volatiles a preconcentration using sorbent tubes will be needed to assess them in the lower ppb level. This could be essential in the diagnosis and classification of noisy breathing infants. Limburg Clinical Research Program (LCRP) & VITO