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Introduction: Numerous studies have sought potential breath biomarkers for lung cancer and many have promising results but, to date, no validated biomarkers with clear connections to cancer metabolism have been discovered. We aim to show the feasibility of an Exogenous Volatile Organic Compounds (EVOC®) Probe targeting tumor-associated extracellular β-glucuronidase, a glycosidase enzyme that usually resides intracellularly within lysosomes in healthy tissues. Material and Methods: To further assess the suitability of β-glucuronidase as a target for a non-invasive breath test, the enzyme activity was evaluated by enzymatic assay in lung cancer PDX specimens and healthy tissues. In addition, β-glucuronidase was assessed by immunostaining a large panel of primary lung cancers, lymph node metastasis and normal lung tissue, including from smokers and those with chronic obstructive pulmonary disease (COPD). We developed a hydrophilic non-cell permeable substrate probe, D5-ethyl-βD-glucuronide (D5-EtGlu), that upon hydrolysis by the target enzyme releases D5-ethanol, a unique volatile reporter molecule. This provides a readout of tumour-associated enzyme activity using breath analysis. Administering D5-EtGlu to mice resulted in tumour-specific release of D5-Ethanol enabling discrimination between healthy and tumour-bearing animals. D5-EtGlu has been administered intravenously to healthy and lung cancer patients followed by breath analysis. A phase 1a clinical trial administered D5-EtGlu to 21 healthy individuals in a single ascending dose study to establish safety and background D5-ethanol levels in healthy individuals. The phase 1b trial is a proof of mechanism study in humans, in which D5-ethanol breath levels in lung cancer patients will be compared to cancer-free individuals receiving the same D5-EtGlu dose. Results and Discussions: Using ex-vivo specimens, higher β-glucuronidase activity was detected in washouts of PDX specimens compared with healthy tissue samples from mice. Higher β-glucuronidase intracellular and extracellular expression was observed in lung cancers compared with lung normal tissue, with low to moderate expression intracellularly in bronchial tissue and in emphysema and COPD. These results reinforce the presence of β-glucuronidase in the tumour microenvironment and its suitability as a target for a breath-probe. A phase 1a resulted in no adverse events and low/no D5-ethanol signal verifying the inaccessibility of D5-EtGlu to intracellular β-glucuronidase. In the phase 1b study, using the highest probe dose, we have detected a D5-ethanol signal in the majority of the cancer patients. Conclusion: β-glucuronidase can be targeted by the non-cell permeable substrate probe D5-ethyl-βD-glucuronide (D5-EtGlu) with potential to be used as a non-invasive breath test for lung cancer diagnosis including a role in screening. Citation Format: Christiaan Labuschagne, Rob Smith, Neelam Kumar, Max Allsworth, Billy Boyle, Sam Janes, Philip Crosbie, Robert Rintoul. Breath-based detection of lung cancer using exogenous volatile organic compound targeting β-glucuronidase in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3319.

2569 Background: Lung cancer is a leading cause of mortality with 5-year survival less than 20%, largely a result of many cases being diagnosed late. Early detection can increase cancer survival up to 13-fold underscoring the need for effective screening. Targeted Low dose computed tomography (LDCT) has been shown to be effective but its impact to date has been limited due to slow adoption and variable uptake in high-risk populations. Breath analysis represents a non-invasive screening approach either alone or alongside LDCT. Numerous studies have investigated potential endogenous breath biomarkers of lung cancer. Many have produced promising results but to date, no validated biomarkers with clear connections to cancer metabolism have been revealed. We have explored an alternative, probe-based approach based around Exogenous Volatile Organic Compound Probes (EVOC Probes). The probes target tumour associated extracellular b-glucuronidase, a glycosidase enzyme that normally resides within lysosomes. Methods: We use a hydrophilic non cell permeable substrate probe D5-ethyl-βD-glucuronide (D5-EtGlu) that upon hydrolysis by the target enzyme releases D5-ethanol, a unique volatile reporter molecule detectable on breath. This provides a readout of tumour associated enzyme activity using breath analysis. Results: Administering D5-EtGlu to mice resulted in tumour specific release of D5-ethanol, enabling discrimination between healthy and tumour bearing animals. Increased expression of b-glucuronidase in lung cancer tissue and the tumour microenvironment was confirmed with immunohistochemistry (IHC) in clinical samples. A phase 1a clinical trial administered D5-EtGlu to healthy individuals in a single ascending dose study to establish safety and background D5-ethanol levels in healthy individuals. This resulted in no adverse events and low/no D5-ethanol signal verifying the inaccessibility of D5-EtGlu to intracellular b-glucuronidase. The next stage, currently ongoing, is a proof of mechanism in humans. D5-EtGlu is administered intravenously to confirmed lung cancer patients followed by breath analysis. D5-ethanol breath levels will be compared to cancer free individuals receiving the same dose of D5-EtGlu. Conclusions: Non-invasive breath testing has great potential to contribute to diagnosis for lung cancer including a potential role in screening. Our current work is evaluating the use of an administered probe to stimulate tumour-specific enzyme activity and produce a marker detectable on breath. Continued success could result in a sensitive and highly specific method for lung cancer early detection.

The interplay between an evolving cancer and a dynamic immune microenvironment remains unclear. Here we analyse 258 regions from 88 early-stage, untreated non-small-cell lung cancers using RNA sequencing and histopathology-assessed tumour-infiltrating lymphocyte estimates. Immune infiltration varied both between and within tumours, with different mechanisms of neoantigen presentation dysfunction enriched in distinct immune microenvironments. Sparsely infiltrated tumours exhibited a waning of neoantigen editing during tumour evolution, indicative of historical immune editing, or copy-number loss of previously clonal neoantigens. Immune-infiltrated tumour regions exhibited ongoing immunoediting, with either loss of heterozygosity in human leukocyte antigens or depletion of expressed neoantigens. We identified promoter hypermethylation of genes that contain neoantigenic mutations as an epigenetic mechanism of immunoediting. Our results suggest that the immune microenvironment exerts a strong selection pressure in early-stage, untreated non-small-cell lung cancers that produces multiple routes to immune evasion, which are clinically relevant and forecast poor disease-free survival.

Objective To describe the experience and acceptability of whole-body magnetic resonance imaging (WB-MRI) staging compared with standard scans among patients with highly suspected or known colorectal or lung cancer. Design Qualitative study using one-to-one interviews with thematic analysis. Setting Patients recruited from 10 hospitals in London, East and South East England between March 2013 and July 2014. Participants 51 patients (31 male, age range 40–89 years), with varying levels of social deprivation, were recruited consecutively from two parallel clinical trials comparing the diagnostic accuracy and cost-effectiveness of WB-MRI with standard scans for staging colorectal and lung cancer (‘Streamline-C’ and ‘Streamline-L’). WB-MRI was offered as an additional scan as part of the trials. Results In general WB-MRI presented a greater challenge than standard scans, although all but four patients completed the WB-MRI. Key challenges were enclosed space, noise and scan duration; reduced patient tolerance was associated with claustrophobia, pulmonary symptoms and existing comorbidities. Coping strategies facilitated scan tolerance and were grouped into (1) those intended to help with physical and emotional challenges, and (2) those focused on motivation to complete the scan, for example focusing on health benefit. Our study suggests that good staff communication could reduce anxiety and boost coping strategies. Conclusions Although WB-MRI was perceived as more challenging than standard scans, it was sufficiently acceptable and tolerated by most patients to potentially replace them if appropriate. Trial registration number ISRCTN43958015 and ISRCTN50436483.

Malignant pleural mesothelioma causes the greatest societal burden of all the asbestos-related diseases. Progress in better understanding tumour biology will be facilitated by the availability of quality-assured annotated tissue. MesobanK has been created to establish a bioresource of pleural mesothelioma tissue linked to detailed anonymised clinical data. When complete, the bioresource will comprise a 750-patient tissue microarray and prospectively collected tissue, blood and pleural fluid from 300 patients with mesothelioma. Twenty-six new cell lines have also been developed. MesobanK meets all appropriate ethical and regulatory procedures and has recently opened to requests for tissue and data.

The two primary cell lineages that make up the endodermally-derived single-layer epithelium of alveoli are alveolar type I (ATI) cells and alveolar type II (ATII) cells. The thin, frail and flat ATI cells cover more than 95% of the alveolar surface. As they are extremely thin, they permit efficient gas exchange by virtue of their squamous morphology. The smaller and cuboidal ATII cells (approximately 9 mm in diameter, 250 mm2 surface area) are more abundant than ATI cells, representing up to 60–88% of all alveolar epithelial cells by number and usually reside at the corners of alveoli. As the cytoplasm of ATII cells contains phospholipid multilamellar bodies, they have a foamier appearance than ATI cells. They have a large central nucleus, a high density of mitochondria and are connected to neighboring cells by intercellular and tight junctions. When these polarized epithelial cells are observed at electron microscopy, they have thin projections protruding from the apical cell surface called microvilli. A wonderful description of ATII cells comes from Robert Mason, who states the role they play as the defender of the alveolus microenvironment is similar to that of a tower in a medieval crenellated wall. Indeed, ATII cells maintain the alveolar space relatively fluid-free and prevent it from collapsing by secreting a lipoprotein material called surfactant (promoting lung expansion on inspiration and preventing lung collapse on expiration), serve as progenitor cells to repopulate the alveolar epithelium after injury and play an important role in the innate immune system response. The most updated scientific contributions in the field come from bioinformatics and omics data, that have added insights to the “non-surfactant-related functions” of ATIIs, emphasizing their pivotal role in lung repair/regeneration after injury. Dysfunctional alveolar epithelium is implicated to such an extent in almost every lung disease that, of late, it has been considered a possible therapeutic target. Therefore, studying its role from a cellular perspective can provide a novel understanding of lung diseases, implying that regenerative medicine in the treatment of degenerative parenchymal lung diseases may well become a reality.