Abstract 3319: Breath-based detection of lung cancer using exogenous volatile organic compound targeting β-glucuronidase in the tumor microenvironment

Abstract

Abstract 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.