Unravelling signatures of clonal response, resistance and evolution of high-risk essential thrombocythaemia at single-cell resolution

近年来，基因检测的重大进展已经改变了我们从微量遗传物质中分析整个人类DNA代码（人类基因组）及其镜像RNA序列（转录组）的能力，这是基因表达的替代品。这推动了我们在全基因组/转录组水平上分析单细胞的能力，现在可以以前所未有的规模和分辨率进行分析。这些技术现在已准备好用于精准医学的临床应用，以改善诊断、风险分层、疾病监测和药物发现。然而，将这种强大的新技术转化为直接的临床应用需要整合临床和科学专业知识，以及这种方法如何使特定疾病领域的患者受益的证据。理想情况下，这应该在前瞻性临床试验的背景下进行评估。这是我们在本研究中旨在解决的首要问题/挑战，即提供单细胞基因组分析在特定疾病（高风险原发性血小板增多症（ET））中临床实用性的原则证明。ET是一种慢性骨髓增生性肿瘤（MPN），其特征是血小板计数高，血栓形成，出血和进展为侵袭性血癌（如骨髓纤维化（MF）和急性髓性白血病（AML））的风险增加。MPN起源于骨髓中最早的血细胞形式，即干细胞，其获得遗传变化，导致基因表达的变化，从而促进不受抑制的细胞生长。ET患者的病程可变，约20%的高风险患者对标准一线治疗（含羟基脲（HC））产生耐药性。HC耐药ET患者疾病进展为MF或AML的风险增加，总生存率显著降低（10年时为26%）。目前的治疗方法通过减少血细胞来预防出血或血栓形成，但没有一种显示出预防疾病进展的能力。鲁索利替尼是JAK抑制剂，是首个获批用于MF和真性红细胞增多症（PV）MPN的靶向治疗药物。在MAJIC研究中，已在高风险HC耐药或不耐受ET中进行了评价，观察到不同的反应，并且在一定程度上，该治疗发生了疾病进展。该项目旨在应用单细胞技术来了解MAJIC研究中HC耐药/不耐受ET患者的细胞和分子差异，并确定ruxolitinib反应（或缺乏反应）和疾病进展的机制。首先，我将这些患者的临床信息与超过30个与MPN相关的基因的突变检测进行比较，以了解这些基因是否影响鲁索替尼的反应和疾病进展。在这些结果的指导下，我将对患者亚组的突变骨髓干细胞和祖细胞进行广泛的单细胞基因组分析，以了解在单细胞水平上突变和非突变干细胞之间的基因表达差异是否确定了影响反应和疾病进展的机制。越来越多的证据表明，血癌患者的骨髓环境异常，并可能影响疾病的进程。因此，我也将使用单细胞分析来研究HC耐药/不耐受ET患者中的非突变干细胞，以确定这些细胞数量的增加或其基因表达的改变是否与治疗反应、疾病进展或治疗副作用有关。该临床研究培训奖学金将提供单细胞基因组学在临床医学中应用的最先进培训。我们预计，通过更好的方法来诊断，监测（识别早期复发的生物标志物）和治疗高危ET，将对患者的预后产生直接，切实的影响。

In recent years, substantial advances in genetic testing have transformed our ability to carry out analysis of the entire human DNA code (human genome) and its mirroring RNA sequence (transcriptome), a surrogate for gene expression, from minute amounts of genetic material. This has propelled our ability to analyse single-cells at a genome/transcriptome wide level, which is now possible with unprecedented scale and resolution. These techniques are now poised for clinical use in precision medicine to improve diagnosis, risk stratification, disease monitoring and drug discovery. However, translating this powerful new technology through to direct clinical application requires integration of clinical and scientific expertise as well as evidence of how such an approach might benefit patients in a specific disease area. Ideally, this should be assessed within the context of a prospective clinical trial. This is the overarching problem/challenge that we aim to address in this research i.e. to provide proof of principle of the clinical utility of single-cell genomic analysis in a specific disease with an unmet need: high-risk essential thrombocythaemia (ET). ET is a form of chronic myeloproliferative neoplasm (MPN) characterised by a high platelet count, increased risk of thrombosis, bleeding and progression to aggressive blood cancers such as myelofibrosis (MF) and acute myeloid leukaemia (AML). MPNs arise from the earliest form of a blood cell, a stem cell, in the bone marrow which acquires a genetic change, leading to changes in gene expression which promotes uninhibited cell growth. Patients with ET have a variable disease course, with approximately 20% of high-risk patients developing resistance to standard first line therapy with hydroxycarbamide (HC). HC-resistant ET patients have an increased risk of disease progression to MF or AML and significantly reduced overall survival (26% at 10 years). Current therapies prevent bleeding or thrombosis through reduction of blood cells but none have shown ability to prevent disease progression. Ruxolitinib, a JAK inhibitor, is the first targeted treatment approved in MPNs in MF and polycythaemia vera (PV). It has been evaluated in high-risk HC-resistant or intolerant ET in the MAJIC study with variable responses observed and in a proportion, disease progression occurred on this treatment. This project aims to apply single-cell technology to understand the cellular and molecular differences in patients with HC-resistant/intolerant ET on the MAJIC study and in doing so, determine mechanisms for ruxolitinib response (or lack of response) and disease progression. Firstly, I will compare clinical information of these patients with mutation testing of over 30 genes associated with MPNs to understand if these influence ruxolitinib response and disease progression. Guided by these results, I will then carry out extensive single-cell genomic analysis of mutated bone marrow stem and progenitor cells of subgroups of patients to understand whether gene expression differences between mutated and non-mutated stem cells at a single-cell level identifies mechanisms influencing responses and disease progression. There is increasing evidence to support that the bone marrow environment is abnormal in blood cancers and may influence the disease course. Therefore, I will also study non-mutated stem cells in HC-resistant/intolerant ET patients using single-cell analysis to determine if increased number of these cells or their altered gene expression may be associated with response to treatment, disease progression or side effects of the treatment.This clinical research training fellowship will provide a state-of-the-art training in the application of single-cell genomics in clinical medicine. We anticipate a direct, tangible impact on patient outcomes through better approaches to diagnose, monitor (identify biomarkers of early relapse) and treat high-risk ET.