Study of genetic and immunological events in Lynch syndrome progression to colorectal cancer

结直肠癌(CRC)是世界上第三大常见癌症，约3%的病例是由一种名为林奇综合征(LS)的常染色体遗传性疾病引起的。这种非息肉病是通过错配修复(MMR)基因(MLH1、MSH2、MSH6和PMS2)之一的胚系单等位基因突变或高甲基化来鉴定的。MMR缺乏症(MMR-d)是通过剩下的健康等位基因的躯体功能丧失而达到的，并导致微卫星束中重复序列的获得或丢失，最终发展为微卫星不稳定(MSI)癌症。确诊的LS患者一生都在接受监测，每隔几年接受一次结肠镜检查。在LS患者和其他非易感患者之间，腺瘤的生长没有增加，但癌症发生率大大增加，LS患者终生CRC风险为20-80%，而普通人群为~5%。由于这一点，人们对改进LS的预测技术、早期发现和化学预防非常感兴趣。为了解决这些问题，必须充分了解导致MMR-d的早期事件以及向癌症的持续演变。我的项目旨在研究LS患者导致CRC发生的最早事件。一些LS患者在癌前没有息肉形成的证据，这表明这些患者可能不会出现传统的腺瘤-癌-结直肠途径。此外，在非肿瘤性LS结肠隐窝中也记录到了完整的MMR-d。综上所述，这意味着LS患者的癌症形成有一个不规则的非经典根部。LS-CRCs的免疫环境既不同于散发性MSI-CRCs，也不同于其他家族性遗传综合征中的CRC。有趣的是，这种独特的免疫格局在来自LS携带者的MMR异源和MMR-d活检中是相似的，这可能意味着在任何可检测到的肿瘤发生之前，生命早期的免疫激活发生了变化。在LS患者中，一旦获得MMR-d，突变负荷、新抗原呈递和FOXP3+调节性T细胞就会增加。目的：1.确定LS患者在正常-腺瘤-癌症通路中除MMR-d外还发生了哪些事件，以及这些事件的发生顺序。免疫浸润物是否需要改变为抑制的调节表型才能发生进展？2.研究这些事件发生的顺序有多重要，还是这些事件的积累是重要的？3.研究新抗原呈递在正常-腺瘤-癌症途径中的持续进展。新抗原量在时间线上是否有特定的时间跃升？具体与什么事件相吻合？假设：林奇综合征患者在任何可检测到的MMR-d相关损害发生之前，都有创造有利于癌症的独特环境的既往事件。临床相关性：通过了解LS发生的早期事件，甚至在任何异常组织出现之前，就可以为LS携带者确定更好的预防和监测策略。这些目的将使用人类LS正常、息肉和癌症样本进行研究。将对整个组织进行MMR-d和一组免疫标记物的染色。将对免疫细胞与MMR-d细胞相关的空间组织进行分析，并分析MMR-d隐窝中的克隆扩张区，以此作为预测亚群适应性和突变时间的指标。单个隐窝的激光捕获将允许整个基因组测序，以识别新的抗原呈现和突变负担，并与整个患者样本的时间线上的免疫状况和MMR-d相关。

Colorectal cancer (CRC) is the 3rd most common cancer in the world, with around 3% of cases occurring due to a hereditary autosomal dominant disease called Lynch syndrome (LS). This non-polyposis condition is identified by a germline monoallelic mutation or hypermethylation in one of the mismatch repair (MMR) genes (MLH1, MSH2, MSH6 and PMS2). MMR deficiency (MMR-d) is reached through somatic loss of function in the remaining healthy allele and leads to a gain or loss of repeats in microsatellite tracts, which ultimately can develop into microsatellite instable (MSI) cancer. Identified LS patients stay on surveillance throughout their lifetime, undergoing colonoscopies every few years. No increase in adenoma growth is seen between LS patients and other non-predisposed patients, but the rate of carcinoma incidence is vastly increased with a 20-80% lifetime CRC risk in LS compared to ~5% in the general population. Due to this there has been much interest in improving predictive techniques, early detection and chemoprevention in LS. To address these issues, the early events leading to MMR-d as well as continuing evolution to carcinoma must be fully understood.My project aims to study the earliest events in LS patients that lead to the development of CRC. Some LS patients display no evidence of polyp formation prior to cancer, indicating the traditional adenoma-carcinoma colorectal pathway may not occur in these patients. In addition, complete MMR-d has been recorded in non-neoplastic LS colonic crypts. Together this implies an irregular non-classical root to cancer formation in LS patients. The immune landscapes of LS CRCs are distinct from both sporadic MSI CRCs and CRCs occurring in other familial inherited syndromes. Interestingly this unique immune landscape is similar in both MMR heterogenous and MMR-d biopsies from LS carriers which may imply changes in immune activation early in life, before any detectable neoplasia. Once MMR-d is gained in LS patients there is an increase in mutational burden, neoantigen presentation and FOXP3+ regulatory T cells. Aims:1. To determine the events, and in what order, occur in addition to MMR-d in LS patients in the normal-adenoma(?)-cancer pathway. Does immune infiltrate need to be altered to a suppressive regulatory phenotype for progression to occur?2. Investigate how essential the order in which these events occur, or is it the accumulation of the events that is important?3. Study the ongoing progression of neoantigen presentation throughout normal-adenoma(?)-cancer pathway. Are there certain times in the timeline that neoantigen amount jumps and what event exactly does this coincide with?Hypothesis: Lynch Syndrome patients have previous events creating a distinct environment favourable to cancer before any detectable presence of MMR-d related lesions occur. Clinical relevance: By understanding the early events that occur in LS, even before any presentation of abnormal tissue, a better strategy of prevention and surveillance can be determined for LS carriers. These aims will be investigated using human LS normal, polyp and cancer samples. Staining for MMR-d and a panel of immune markers throughout the tissue will be carried out. Analysis for the spatial organisation of immune cells in relation to MMR-d cells will be performed as well as analysis of clonal expansion areas in MMR-d crypts as a predictor of subpopulation fitness and time of mutation. Laser capture of individual crypts will allow for whole genome sequencing to identify neoantigen presentation and mutational burden in correlation to immune landscape and MMR-d throughout the timeline of patient samples.