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

结直肠癌是西方世界第二高的癌症死亡率原因：最重要的生存决定因素是确诊时的疾病阶段，早期结直肠癌患者的存活率超过90%。随机试验，包括粪便血液检测和结肠镜检查(使用外科内窥镜检查结肠)，表明早期发现和适当的治疗结果可将死亡率降低约16%。不幸的是，这种干预措施的影响受到一些因素的限制，包括大多数患者直到疾病晚期才被诊断出来，目前检测所需的家庭粪便采样对患者的接受度较低(摄取率：英国57%；欧洲34%)，以及现有粪便检测的敏感性和特异性较低。同样，尽管另一种结肠成像方法(乙状结肠镜检查)似乎可以使死亡率降低(22%-31%)，但患者的接受度仍然很低(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测试，与粪便筛查相比，它将提高公众的接受度，同时提供更高的灵敏度和特异度。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袭的免疫系统也会对肿瘤内癌细胞过度生产和释放的分子做出反应。这种反应包括产生对这些肿瘤相关分子的抗体。因为抗体识别的分子来自患者，所以抗体被称为自身抗体。我们和其他组织有很好的证据表明，检测患者血液中的这些自身抗体可以为早期发现肿瘤提供一条改进方法的途径。我们的方法只需要血液样本，避免了粪便采样或侵入性结肠成像技术。因此，它极有可能极大地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提供更好的成本效益。我们的方法是生产一种能够同时测试多个肿瘤分子的自身抗体的测试方法。这是以微阵列的形式(在玻璃片表面上选定的肿瘤相关分子的规则间隔的点)的形式。来自患者血液的血清被洗涤在阵列上，如果存在抗体，它们就会结合到一个或多个与肿瘤相关的分子斑点。结合的抗体本身用荧光报告器检测，并测量信号强度。与基于已知结直肠癌患者和正常对照检查的临界值进行比较，可以识别出肿瘤分子自身抗体阳性的样本，这表明需要进一步的研究。微阵列和斑点的小尺寸允许复制阵列上每个肿瘤相关分子的斑点，但与每次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。在我们的初步工作中，使用32个肿瘤相关分子的有限小组和262名已知癌症/正常个体的小样本集，展示了这种基于微阵列的结直肠癌自身抗体筛查的潜力。该项目将从32个肿瘤相关分子中确定适合用作结直肠癌筛查工具的最佳小组，并使用比我们初步研究更大的、临床定义明确的样本集来验证所得到的小组。我们的预测是，患者依从性为90%，检测特异度为85%，敏感性为65%，这将导致检测到58.5%的CRC总数--是目前检测到的CRC总数的两倍多。

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.