Augmentation of Volatile Biomarkers of Oesophageal Adenocarcinoma from the Tumour Lipidome

在英国，每年有近1万名患者死于下食道癌症，即食道腺癌（OAC）。OAC发现较晚，因为症状非特异性，经常被误认为是常见的问题，如胃灼热。这意味着每10名被诊断为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、准确地检测出OAC并可供所有患者使用的检测方法。OAC患者的呼吸中富含预示癌症的挥发性化学物质（VCs）。在呼气测试中，OAC的检测率为80 / 100。虽然令人鼓舞，但可以通过使用外部刺激物（如饮料）来提高检出率，这种刺激物只会放大肿瘤特异性vc的产生，从而增加它们在呼吸中的水平，并提高呼吸测试中的检出率。这使得可以直接比较来自同一患者的呼吸样本（兴奋剂前和兴奋剂后），而不是与其他人进行比较，从而提高了测试的可靠性。这就是所谓的“增强”，是我们提高呼吸测试检测OAC的能力的新方法，特别是在癌症很小的早期OAC。为了扩大OAC风险投资，我们必须针对其产生背后的机制，并加强这一途径。脂质对健康的细胞结构至关重要，然而OAC中的脂质被重新编程以获得不同的成分，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类产生vc的主要机制。这表明OAC脂质和/或OAC细菌脂质选择性不稳定，并分解产生呼吸中检测到的vc；然而，这一点尚未得到证实。如果这是真的，这一过程可能会被一种促进这种分解的简单兴奋剂所增强。为了验证这一理论，我开发了一种强大的方法来从OAC患者的细胞中分解脂质，并分析产生的vc。结果表明：(1)脂质自发分解生成vc；ii)与健康脂质相比，OAC脂质增强了这一过程，表明它们在癌症中的不稳定性；iii) OAC脂质产生的vc与OAC患者呼吸中检测到的vc相同，iv)使用兴奋剂溶液增加OAC脂质可显著增加诊断性vc的产生。这些结果表明，呼吸试验中检测到的vc起源于OAC脂质，这一过程可以增强。该项目的总体目标是生产一种改进的第二代呼吸测试，具有更好的能力，可以通过增强检测OAC。这将提高患者的长期生存从癌症使用完全非侵入性测试。这一建议将通过三个相互关联的阶段来实现。第一阶段将全面鉴定产生vc的OAC肿瘤中的脂质种类，使用专门为此目的设计的定制质谱技术。鉴定的脂质将用于OAC患者的2期“稳定同位素实验”。这些实验从根到枝追踪脂质向VC转化的分解。这些实验将在增强条件下进行，以评估增强对风险投资的影响，并确定最佳的增强方法。第三阶段将把科学知识转化为造福患者。患者将被给予一种优化的兴奋剂饮料，并在服用前后对他们的呼吸进行采样。病人的反馈会对呼吸测试的设计产生很大的影响。有了这种第二代呼吸测试，症状模糊的患者可以进行快速、无创的测试，以安全、准确的方式对样本进行分析，随后根据其患OAC的风险进行分层，从而早期发现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.