Augmentation of Volatile Biomarkers of Oesophageal Adenocarcinoma from the Tumour Lipidome

在英国，每年有近10，000名患者死于食道癌，称为食道腺癌（OAC）。OAC被发现晚，因为症状是非特异性的，经常被误认为是常见的问题，如胃灼热。这意味着每10名被诊断患有OAC的患者中只有不到2人的寿命超过5年。这突出表明迫切需要一种能够早期、安全地准确检测OAC并可供所有患者使用的检测方法。 OAC患者的呼吸富含指示癌症的挥发性化学物质（VC）。当在呼吸测试中测量时，它在100次中检测到OAC 80次。虽然令人鼓舞，但可以通过使用外部刺激物（例如饮料）来提高检测率，该外部刺激物仅放大肿瘤特异性VC的产生，以增加它们在呼吸中的水平并提高呼吸测试中的检测。这使得能够直接比较来自同一患者的呼吸样本（刺激前和刺激后），而不是与其他人进行比较，这提高了测试的可靠性。这被称为“增强”，是我们提高呼吸测试检测OAC的能力的新方法，特别是当癌症很小时的早期OAC。为了增加OAC VC，我们必须瞄准其生产背后的机制并增强这一途径。 脂质对健康的细胞结构是必不可少的，然而OAC中的脂质被重新编程以获得不同的组成，这使得它们容易通过称为“过氧化”的过程被分解。这包括与癌症相关的细菌脂质的分解。脂质过氧化被认为是人体产生VC的主要机制。这表明OAC脂质和/或OAC细菌脂质是选择性不稳定的，并且分解以产生在呼吸中检测到的VC;然而，这尚未得到证实。如果是真的，这个过程可能会被一种简单的刺激物所增强，这种刺激物会促进这种分解。 为了验证这一理论，我开发了一种强大的方法来打破OAC患者细胞中的脂质，并分析产生的VC。结果显示：i）脂质自发分解以产生VC; ii）与健康脂质相比，OAC脂质增强了该过程，表明其在癌症中的不稳定性; iii）由OAC脂质产生的VC与在OAC患者的呼吸中检测到的VC相同，以及iv）使用刺激溶液增加OAC脂质显著增加了产生的诊断VC的量。这些发现表明，在呼气测试中检测到的VC源自OAC脂质，并且该过程可以增强。 该项目的总体目标是产生具有上级能力的改进的第二代呼吸测试，以检测增强的OAC。这将提高长期患者生存从癌症使用一个完全非侵入性的测试.这一建议将通过三个相互关联的阶段来实现。第一阶段将全面确定OAC肿瘤中的脂质种类，使用为此目的定制设计的定制质谱技术产生VC。鉴定出的脂质将在第2阶段用于OAC患者的“稳定同位素实验”。这些实验跟踪从根到分支的脂质分解到VC的转化。这些实验将在增强条件下进行，以评估增强对VC的影响，并确定最佳的增强方法。第三阶段将把科学知识转化为有益于患者的知识。患者将获得优化的兴奋剂饮料，并在饮用前后对其呼吸进行采样。将获得严重影响呼吸测试设计的患者反馈。 通过这种第二代呼吸测试，具有模糊症状的患者可以进行快速，非侵入性的测试，以安全和准确的方式分析样本，随后根据其患有OAC的风险进行分层，从而更早地检测OAC并改善临床结果。

Nearly 10,000 patients die each year in the United Kingdom from cancer of the lower gullet, known as oesophageal adenocarcinoma (OAC). OAC is detected late as symptoms are non- specific and often mistaken for common problems such as heartburn. This translates to fewer than 2 in every 10 patients diagnosed with OAC living longer than 5 years. This highlights the urgent need for a test that can accurately detect OAC early, safely and can be used by all patients. The breath of OAC patients is enriched with volatile chemicals (VCs) that indicate cancer. When measured in a breath test, it detects OAC 80 out of 100 times. Whilst encouraging, the detection rate can be improved by using an external stimulant, such as a drink, that amplifies the production of tumour specific VCs only, to increase their levels in breath and improve detection in the breath test. This enables direct comparison of breath samples from the same patient (pre and post stimulant) rather than comparison with others, which improves the reliability of the test. This is known as 'augmentation' and is our novel approach to improving the ability of the breath test to detect OAC, especially early-stage OAC when the cancer is small. To augment OAC VCs, we must target the mechanisms behind their production and enhance this pathway. Lipids are essential to healthy cell structure however the lipids in OAC are reprogrammed to acquire a different composition which leaves them vulnerable to breakdown via process known as 'peroxidation'. This includes breakdown of bacterial lipids associated with the cancer. Lipid peroxidation is considered a main mechanism for producing VCs in humans. This suggests OAC lipids and/ or OAC bacterial lipids are selectively unstable and breakdown to generate VCs that are detected in breath; however, this has not been proven. If true, this process may be augmented with a simple stimulant that promotes this breakdown. To test this theory, I developed a robust way to break lipids from the cells of OAC patients and analyse the VCs generated. Results showed i) lipids break down spontaneously to generate VCs; ii) this process was enhanced with OAC lipids in comparison to healthy lipids, indicating their instability in cancer; iii) the VCs produced from OAC lipids are the same ones detected in the breath of OAC patients and iv) augmentation of OAC lipids using a stimulant solution profoundly increased the quantity of diagnostic VCs produced. These findings suggest that VCs detected in the breath test originate from OAC lipids and this process can be augmented. The overall aim of this project is to produce an improved second-generation breath test with superior ability to detect OAC with augmentation. This will improve long term patient survival from cancer using an entirely non- invasive test. This proposal will be achieved through three interconnected phases. Phase 1 will comprehensively identify the lipid species in OAC tumours that produce VCs using bespoke mass spectrometry techniques custom designed for this purpose. The identified lipids will be used in Phase 2 for 'stable isotope experiments' in OAC patients. These experiments track the breakdown of lipids to VC conversion from root to branch. These experiments will be performed in augmented conditions to evaluate the effect of augmentation on VCs and identify the optimal augmentation approach. Phase 3 will translate the scientific knowledge to benefit patients. Patients will be given an optimised stimulant drink and their breath will be sampled before and after consumption. Patient feedback will be obtained that will heavily influence the design of the breath test. With this second-generation breath test, patients with vague symptoms can undergo a quick, non- invasive test, have samples analysed in a safe and accurate manner and be subsequently stratified based on their risk of having OAC, leading to earlier OAC detection and improved clinical outcomes.