Identification of in-vivo generated human autoantibodies for the screening and early detection of primary colorectal cancer (CRC)

CRC是西方世界癌症死亡率的第二高原因：生存的最重要决定因素是诊断时的疾病阶段，早期结直肠癌患者的生存率超过90%。随机试验，包括粪便血液检测，然后进行结肠镜检查（使用外科内窥镜检查结肠），已经显示早期检测和适当的治疗导致死亡率降低约16%。不幸的是，这些干预措施的影响受到许多因素的限制，包括大多数患者直到疾病晚期才被诊断出来，目前测试所需的家庭粪便采样的患者接受度低（接受率：英国57%;欧洲34%）以及现有粪便测试的灵敏度和特异性低。类似地，尽管替代结肠成像方法（乙状结肠镜检查）似乎可以降低更高的死亡率（22%-31%），但患者的可接受性仍然很低（45%-58%），存在显著的社会不平等。此外，检查通常仅限于部分结肠（远端结肠）。 我们的解决方案是提供一种血液检测，与粪便筛查相比，它将提高公众的可接受性，同时也提高了灵敏度和特异性。这将显著改善临床结果（提高存活率）和成本效益。保护我们免受微生物侵害的免疫系统也会对肿瘤内癌细胞过度产生和释放的分子做出反应。这种反应包括产生针对这些肿瘤相关分子的抗体。由于抗体识别的分子来源于患者，因此抗体被称为自身抗体。我们和其他研究小组有很好的证据表明，检测患者血液中的这些自身抗体可以提供一种改进肿瘤早期检测方法的途径。我们的方法只需要血液样本，避免粪便采样或侵入性结肠成像技术。因此，它极有可能大大提高患者的可接受性，这本身将提高检测率，同时简化测试过程，并为NHS提供更高的成本效益。我们的方法是生产一种检测方法，使我们能够同时测试多种肿瘤分子的自身抗体。这是以微阵列的形式（在载玻片表面上有规律间隔的选定肿瘤相关分子的斑点）。来自患者血液的血清在阵列上洗涤，如果存在抗体，则它们与一个或多个肿瘤相关分子斑点结合。结合的抗体本身用荧光报告物检测，并测量信号强度。根据已知结直肠癌患者和正常对照的检查，与截止值进行比较，可以鉴定肿瘤分子自身抗体阳性的样本，这表明需要进一步研究。微阵列和斑点的小尺寸允许在阵列上复制每个肿瘤相关分子的斑点，但与每次测试仅检查一个肿瘤相关分子的测试相比，需要显著减少的化学体积。使用有限的32种肿瘤相关分子组和262个已知癌症/正常个体的小样本组。该项目将从32种肿瘤相关分子组中确定最佳组。相关的分子，其适合用作结直肠癌筛查工具，并使用比我们的初步研究大得多的、临床上明确定义的样本集来验证所得到的面板。我们的预测是，患者依从率> 90%，检测特异性为85%，灵敏度为65%，这将导致检测到CRC总数的58.5%-比目前检测到的CRC总数增加一倍以上。

CRC is the second highest cause of cancer mortality in the western world: the most important determinant of survival is stage of disease when diagnosed, with over 90% survival for patients with early stage colorectal cancer. Randomised trials, involving faecal blood testing followed by colonoscopy (use of a surgical endoscope to examine the colon), have shown early detection with appropriate treatment results in mortality reduction of ~16%. Unfortunately the impact of such interventions is limited by a number of factors, including that fact that the majority of patients are not diagnosed until late stage disease, the low patient acceptability of home faecal sampling required for current tests (Uptake: UK 57%; European 34%) and the low sensitivity and specificity of existing faecal tests. Similarly, although an alternative colonic imaging method (sigmoidoscopy) appears to give higher mortality reductions (22%-31%), patient acceptability remains low (45%-58%) with significant social inequalities. Furthermore the examination is usually limited to only part of the colon (distal colon). Our solution is to provide a blood test that will improve public acceptability while also providing improved sensitivity & specificity compared to faecal screening. This will significantly improve clinical outcomes (improved survival rates) and cost-effectiveness.The immune system, which protects us from microbes, also mounts a response to molecules overproduced and released by cancer cells within a tumour. This response includes the generation of antibodies to these tumour-associated molecules. Because the molecules recognised by the antibodies originate from the patient, the antibodies are termed autoantibodies. We, and other groups, have good evidence that detecting these autoantibodies in the blood of patients can provide a route to improved methods for early detection of tumours. Our approach will require only a blood sample, avoiding faecal sampling or invasive imaging colon techniques. As such it is highly likely to dramatically increase patient acceptability, which in itself will improve detection rates and at the same time simplify the testing process and provide improved cost-effectiveness for the NHS.Our approach is to produce an assay enabling us to test for autoantibodies to multiple tumour molecules simultaneously. This in the form of a microarray (regularly spaced spots of selected tumour-associated molecules on a glass slide surface). Serum from patient blood is washed over the array, and if antibodies are present, they bind to one or more of the tumour-associated molecule spots. Bound antibodies are themselves detected with a fluorescent reporter and the signal intensity measured. Comparison to cutoffs based upon examination of known colorectal cancer patients and normal controls allows the identification of samples positive for tumour-molecule autoantibodies, indicating that further investigation is required. The small size of the microarray and spots allows for replication of spots for each tumour-associated molecule on the array, yet requires significantly reduced chemical volumes over tests which examine only one tumour-associated molecule per test.The potential of this microarray-based autoantibody screening in colorectal cancer is demonstrated in our preliminary work, using a limited panel of 32 tumour-associated molecules and a small sample set of 262 known cancer/normal individuals.This project will identify the optimal panel from the 32 tumour-associated molecules which are suitable for use as a colorectal cancer screening tool and validate that resultant panel using much larger, clinically well-defined sample sets than our preliminary study. Our prediction is that with a patient compliance rate of >90%, test specificity of 85% and sensitivity of 65% this would result in detection of 58.5% of the total number of CRCs -more than a doubling of the total number of CRCs currently detected.