Defining the impact of secondary mutations in CEBPA-mutated acute myeloid leukaemia.

急性髓系白血病(AML)是一种血癌，在英国每年影响3000人。目前的治疗方法是强化化疗，这可能导致长达数月的住院时间。在诊断时，寻求有关每个患者白血病的遗传学信息。这些信息主要用于权衡患者白血病复发的风险与骨髓移植的风险。然而，进一步了解这些基因变化如何破坏血细胞的正常功能，可以帮助我们开发新的、更有针对性的治疗方法。反过来，这可能会改善所有患者的预后--特别是那些不适合进行强化化疗或在治疗后复发的患者。AML是从正常的血液干细胞发展而来的，这种干细胞获得了连续的基因异常，最终导致白血病的发展。下一代测序(NGS)是一种允许我们查看白血病遗传学的微小细节的方法。NGS已经在AML中发现了250多个基因异常，这些异常在不止一个人身上观察到。要患上白血病，每个患者似乎都需要5-20个这样的突变。涉及的基因频率不同，在10%的散发性AML病例中只有5个基因发生突变(Kihara等人，2014年)。其中一个基因被称为CEBPA。虽然大多数情况下CEBPA的变化发生在个体血液干细胞出生后，但在少数个体中CEBPA突变是可以遗传的。这意味着CEBPA的遗传错误存在于所有细胞中，并导致白血病的易感性。遗传了CEBPA突变的AML患者是独一无二的，因为我们可以确定白血病发展过程中的第一个遗传事件是什么。这一点很重要，因为第一个遗传事件可能是所有后续变化的触发因素。研究表明，与CEBPA一起突变的一个基因是TET2基因。在这个项目中，我们将研究CEBPA和TET2突变的组合对正常血液发育和白血病发展的影响。斑马鱼是一种用途广泛的淡水鱼。这对于快速评估多种基因异常的影响是非常有用的。这是因为它很小，完全透明，每周产生数百只后代。血液在生命的前5天内发育，这使得我们的实验非常快速和容易。我们的实验室使用斑马鱼来研究特定的基因突变是如何导致骨髓衰竭和急性白血病的临床症状的。我们将使用斑马鱼来确定TET2和cebpa的联合作用。在三分之一CEBPA突变的AML患者中，导致白血病发展的额外突变尚不清楚。我们的项目还将调查哪些其他基因可能在CEBPA突变的AML的发展中起重要作用。我们将通过系统回顾已发表的散发性AML DNA测序数据和我们在巴茨癌症研究所的合作者在8名家族性CEBPA突变AML患者上所做的工作来做到这一点。然而，到目前为止，只有17例CEBPA病例完成了完整的DNA测序。我们有一个超过100个患者样本的大队列，我们将在这些样本上收集关于NGS的AML突变情况的进一步信息。这将与我们对CEBPA/TET2突变斑马鱼的研究同步进行。这些测序研究的目的既是为了全面识别CEBPA突变的AML中哪些基因发生了反复突变，并将其与患者的临床结果相关联，也是为了利用这些信息推进我们的斑马鱼CEBPA模型的功能评估。

Acute myeloid leukaemia (AML) is a blood cancer that affects 3,000 people each year in the UK. Current treatment is with intensive chemotherapy, which can result in prolonged hospital admissions for many months. At diagnosis, information regarding the genetics of each patient's leukaemia is sought. This information is mainly used to weigh up the risk of a patient's leukaemia relapsing against the risks of a bone marrow transplant. However, further understanding of how these genetic changes disrupt the normal functioning of blood cells can help us develop new more targeted treatments. In turn this may improve outcomes in all patients - particularly for those who are unfit for intensive chemotherapy or who relapse following treatment.AML develops from a normal blood stem cell that has acquired successive genetic abnormalities eventually leading to the development of leukaemia. Next generation sequencing (NGS) is an approach that allows us to look at the genetics of the leukaemia in minute detail. NGS has identified more than 250 genetic abnormalities in AML that are observed in more than one individual. To develop leukaemia, each patient seems to need between 5-20 of these mutations. The genes involved vary in their frequency, with only 5 genes found to be mutated in >10% of sporadic AML cases (Kihara et al 2014). One of these genes is called CEBPA. Although most commonly the changes in CEBPA occur after birth to an individual blood stem cell, in a small number of individuals CEBPA mutations can be inherited. This means the genetic error in CEBPA is in all cells and leads to a predisposition to the development of leukaemia. Patients who develop AML that have inherited CEBPA mutations are unique because we can be certain what was the first genetic event in the development of leukaemia. This is important because the first genetic event may be the trigger of all the subsequent changes.Studies have shown that a gene commonly mutated along with CEBPA is the TET2 gene. In this project we will be investigating the effects of the combination of CEBPA and TET2 mutations on normal blood development and the development of leukaemia. The zebrafish is a highly versatile fresh water fish. It is extremely useful for rapid assessment of the effect of multiple genetic abnormalities. This is because it is small, totally transparent and produces many hundreds of offspring each week. Blood develops within the first 5 days of life making it very quick and easy to perform our experiments. Our laboratory has used the zebrafish to investigate how specific genetic mutations lead to clinical syndromes of bone marrow failure and acute leukaemia. We will use the zebrafish to define the combined effects of tet2 and cebpa.In a third of CEBPA mutated AML patients the additional mutations that lead to the development of leukaemia are unknown. Our project will also investigate which other genes might be important in the development of CEBPA mutated AML. We will do this by a systematic review of already published data on DNA sequencing in sporadic AML and work performed by our collaborators at Barts Cancer Institute on 8 patients with familial CEBPA mutated AML. However, only 17 cases of CEBPA have been fully DNA sequenced to date. We have a large cohort of over 100 patient samples, on which we will collect further information on the mutational landscape of AML by NGS. This will be performed in parallel with our studies on the cebpa/tet2 mutated zebrafish. The intention of these sequencing studies will be both to comprehensively identify which genes are recurrently mutated in CEBPA mutated AML and correlate this to clinical outcomes of patients, and to use this information to move forward to functional assessment in our zebrafish cebpa model.