慢性髓细胞白血

发布时间：2018-07-05 05:21

本文选题：慢性髓细胞白血病 + 星形细胞瘤　；参考：《天津医科大学》2016年博士论文

【摘要】：目前,随着研究的深入,我们对肿瘤的生物学和基因组学的认识也有了改变。要将肿瘤的基因组学研究转化到肿瘤的临床治疗,需要考虑到肿瘤细胞的复杂性以及其动态的演化特征。肿瘤演化过程中不同的肿瘤细胞克隆竞争空间和资源,最终具有生长优势的克隆形成新的肿瘤亚群。肿瘤以不同的时间和速度演化进展,在任何一个患者中其克隆结构、基因型和表型都随着时间改变。肿瘤克隆演化的研究除了使我们对肿瘤的发生发展有更全面的了解,同时也有着重要的应用价值,比如发现对治疗更有预测价值的标志以提高个体化治疗效果、以及阻止肿瘤对药物的治疗抵抗。而肿瘤克隆演化的特征,如克隆结构或驱动突变等,或可以用于肿瘤预后的判定。慢性髓细胞白血病(chronic myeloid leukemia,CML)是一种骨髓细胞增殖异常的血液疾病。该疾病存在特征性的9号和22号染色体的易位t(9;22)(q34;q11),即费城染色体(Philadelphia chromosome,Ph),在分子水平上形成BCR/ABL1融合基因。该融合基因编码的蛋白具有酪氨酸激酶活性,在无生长因子的情况下能够促使细胞增殖。Ph染色体是造成CML肿瘤表型的主要染色体畸变,几乎所有的CML均存在BCR/ABL1融合基因。针对BCR/ABL1的靶向治疗能够使的CML患者缓解,但是Ph染色体的形成机制以及靶向治疗后Ph染色体克隆演化还不是很清楚。胶质瘤(glioma)是一类起源于胶质细胞的肿瘤,星形细胞瘤(astrocytoma)是最常见的神经上皮性肿瘤。低级别的星形细胞瘤一般生长缓慢,但是经过一段时间会进展为高级别的恶性肿瘤,引起肿瘤的复发从而降低整体生存率。星形细胞瘤手术切除后极易复发,且恶性程度增高,但复发和恶性程度增高的机制尚不明确。大多数星形细胞瘤患者死于肿瘤的复发和恶变,研究其细胞遗传水平的克隆演化对认识星形细胞瘤的生物学行为、进行针对性的治疗有重要意义。目的:肿瘤的克隆演化是肿瘤的一个重要特征。为了研究克隆演化对于疾病的进展以及治疗的影响,本研究选取了两个肿瘤类型:慢性髓细胞白血病和星形细胞瘤。前者存在特征性的染色体异常即费城染色体;后者的疾病特征为手术切除后的复发恶变。通过全基因组的测序,一是研究CML中BCR/ABL1融合基因的形成机制以及针对Ph靶向治疗后的克隆演化;二是通过研究星形细胞瘤的克隆演化,探寻影响该肿瘤复发恶变的分子遗传因素。方法:本研究选取了一例CML患者,该患者确诊CML后清除其自身骨髓行全相合异体造血干细胞移植(hematopoietic stem cell transplantation,HSCT)(供体为患者姐姐),疾病进入Ph(-)慢性期;后患者发生急淋变,荧光原位杂交(fluorescence in situ hybridization,FISH)实验显示BCR/ABL1(+);对患者进行针对Ph染色体的靶向治疗后疾病得以缓解,Ph染色体消失。本研究收集了该患者姊妹间全相合异体造血干细胞移植后慢性期(Ph-)、急变期(Ph+)以及针对Ph靶向治疗后再缓解期的骨髓组织活检样本,并获得患者的皮肤活检组织作为正常对照。本研究选取一例星形细胞瘤患者,该患者初诊时肿瘤组织病理检测显示右侧额叶星形细胞瘤(WHO II级),后患者肿瘤复发,组织病理检测显示右额叶胶质母细胞瘤合并局灶间变型星形细胞瘤(WHO IV级)。收集患者初诊肿瘤组织样本(星形细胞瘤WHO II级)以及复发后肿瘤组织样本(WHO IV级),并收集患者的外周血样品作为正常对照。对CML患者的骨髓组织皮肤组织的DNA和星形细胞瘤患者的肿瘤组织和外周血DNA进行全基因重测序(测序深度≥50×)。首先将测序数据利用BWA软件对比到参考基因组(UCSC hg19)上,应用samtools、control-FREEC、mu Tect、Strelka、crest软件分析样品存在的遗传学改变,包括点突变、拷贝数变异(copy number variation,CNV)、插入/缺失(insertion-deletion,INDEL)和染色体易位;并与对照比较,基于肿瘤样本的体细胞单位点突变和拷贝数的位点集合,应用Ex PANd S软件分析肿瘤样品存在的亚克隆结构,包括亚克隆的数目、亚克隆大小以及每个亚克隆中包含的突变。应用Sci Clone软件对成对样本进行亚克隆分析。结果:本研究选取的CML患者,在行姊妹间全相合异体骨髓移植后疾病进入慢性期(Ph-),后发生急变(Ph+),针对Ph靶向药物治疗后疾病再缓解。分析测序数据发现骨髓样品中存在一系列的SNPs、INDEL、CNVs和SVs,急变期(Ph+)比慢性期(Ph-)出现更多的遗传学异常,经分析,急变期费城染色体的出现来源于患者本身(骨髓受体)。在靶向治疗后一些急变期特有的遗传异常随着Ph染色体的消失而消失;患者Ph(+)的急变期和皮肤组织样本中独有DNA修复基因BRIP1框移缺失突变;供体骨髓和患者的骨髓样本共同存在BCR基因上的一些插入/缺失,仅患者Ph(+)的急变期存在BCR形成融合基因的断裂点。Ex PANd S软件在Ph(+)急变期和Ph(-)缓解期均有8个亚克隆结构,克隆进化的发生图均为“树状”结构。本研究中选取的星形细胞瘤患者,初诊时为星形细胞瘤WHO II级,复发时为胶质母细胞瘤合并局灶间变型星形细胞瘤WHO IV级。对其肿瘤组织DNA进行测序、数据分析,发现初诊和复发阶段肿瘤均存在基因NOTCH和ATRX的错义突变;初诊时存在基因TP53的杂合终止突变,复发时存在基因TP53的纯合终止突变以及基因IDH1的杂合错义突变;同时复发肿瘤中有较多的染色体易位。Ex PANd S软件分析显示,在初诊和复发阶段肿瘤分别有4和3个亚克隆结构,初诊阶段的克隆进化的发生图为“树状”结构,而复发阶段克隆进化的发生图为“并行”结构,提示该阶段肿瘤克隆存在较高的异质性。结论:在该CML患者复发、靶向治疗的疾病进展过程中,BCR/ABL1融合基因为特征性的克隆改变,随着其产生而出现一些列继发性的突变或者染色体畸变,针对其靶向治疗后以一些急变期特有的遗传学异常随着Ph染色体的消失而消失;基因BCR存在微缺失/突变的前提下,BRIP1等DNA修复基因的异常最终导致BCR/ABL1融合基因的形成。对于星形细胞瘤,特异性的突变可用于肿瘤分子分型的依据,但IDH1与星形细胞瘤预后的关系值得进一步探讨;肿瘤组织亚克隆结构的分析可以为肿瘤的临床预后判断和靶向治疗提供分子遗传学依据;放疗、化疗可能对肿瘤克隆演化产生选择压力,从而使TP53突变的克隆产生选择优势。星形细胞瘤复发阶段克隆的异质性增强,研究结果提示初次手术后的放疗要慎重。
[Abstract]:At present, with the deepening of research, our understanding of the biology and genomics of tumors has changed. To convert the tumor genomics research into the clinical treatment of the tumor, the complexity of the tumor cells and its dynamic evolution should be taken into consideration. The competitive space and resources of different tumor cells cloned during the process of tumor evolution The clones, which eventually have growth advantages, form a new subgroup of tumor. The tumor evolves at a different time and speed. In any patient, the clone structure, genotypes and phenotypes change over time. The study of the tumor cloning and evolution has a more comprehensive understanding of the development of the tumor, but also important for the development of the tumor. Applied values, such as the discovery of more predictive value for treatment to improve the effectiveness of individualized treatment, and to prevent cancer resistance to drugs. The characteristics of the tumor clone evolution, such as the clone structure or the driving mutation, may be used to determine the prognosis of the tumor. Chronic myeloid leukemia (CML) is one of the ones. A hematological disease of abnormal proliferation of bone marrow cells. The disease is characterized by the translocation of chromosome 9 and 22 (9; 22) (q34; Q11), the Philadelphia chromosome (Philadelphia chromosome, Ph), to form a BCR/ABL1 fusion gene at the molecular level. The protein encoded by the fusion gene has tyrosine kinase activity in the absence of growth factors. The.Ph chromosome is the main chromosome aberration that causes the phenotype of CML tumor, and almost all CML has BCR/ABL1 fusion gene. The CML patients can be alleviated by targeting therapy for BCR/ABL1, but the mechanism of Ph chromosome formation and the cloning and evolution of Ph dyed body after targeting therapy are not very clear. MA) is a class of tumors originating from glial cells. Astrocytoma (astrocytoma) is the most common neuroepithelial tumor. Low grade astrocytoma usually grows slowly, but it progresses to high grade malignant tumors for a period of time, causing tumor recurrence and reducing the overall survival rate. Astrocytoma is excised after surgical excision. The mechanism of recurrence and malignancy is not clear. Most astrocytomas die from the recurrence and malignancy of the tumor. It is of great significance to study the cloning and evolution of the genetic level of the astrocytoma for the understanding of the biological behavior of astrocytoma. It is an important feature of the tumor. In order to study the progress of the disease and the effect of the treatment, two types of tumor are selected: chronic myelocytic leukemia and astrocytoma. The former has characteristic chromosomal abnormalities in Philadelphia chromosome; the latter is characterized by recurrent malignant changes after surgical excision. The whole genome sequencing, one is to study the formation mechanism of BCR/ABL1 fusion gene in CML and the clone and evolution after targeted therapy for Ph; two is to explore the molecular genetic factors that affect the tumor recurrence by studying the cloning and evolution of astrocytoma. Method: This study selected a case of CML patients, and the patient cleared its self after CML. Hematopoietic stem cell transplantation, HSCT (the donor's sister), the disease entered the Ph (-) chronic phase, and the postoperative patients had acute lymphoblastic changes, and the fluorescence in situ hybridization (fluorescence in situ hybridization, FISH) experiment showed BCR/ABL1 (+). The disease was relieved and the Ph chromosome disappeared. The study collected the chronic phase (Ph-), acute phase (Ph+) and bone marrow biopsy samples of the patients after the Ph target treatment, and the skin biopsy tissue of the patients as a normal control. In the patients with cytomatoma, the tumor tissue pathological examination showed the right frontal astrocytoma (grade WHO II) at the first visit. The tumor recurred in the patients. The histopathological examination showed that the right frontal glioblastoma was combined with the WHO IV grade. The tumor tissue samples (astrocytoma WHO II) were collected and the recurrence was collected. The posterior tumor tissue samples (WHO IV grade) and the peripheral blood samples of the patients were collected as normal controls. The whole gene resequencing of the tumor tissue and peripheral blood DNA of the DNA and astrocytoma patients in the bone marrow tissue of CML patients (the sequence depth was more than 50 *). First, the sequencing data were compared to the reference genome by BWA software to the reference genome (UCSC Hg1). 9) the genetic changes in samples of samtools, control-FREEC, mu Tect, Strelka, crest software were applied, including point mutation, copy number variation (copy number variation, CNV), insertion / deletion (insertion-deletion, INDEL), and chromosome translocation, and compared with the control, the unit point mutation and the copy number based on the tumor samples. Point set, using the Ex PANd S software to analyze the subclonal structure of the tumor samples, including the number of subclones, the size of the subclone and the mutations contained in each subclone. The Sci Clone software was used to subclone the paired samples. Results: the CML patients selected in this study were infected with the disease after the sister allograft bone marrow transplantation. A series of SNPs, INDEL, CNVs and SVs in the bone marrow samples were found in the bone marrow samples, and the sudden change period (Ph+) appeared more genetic abnormalities than the chronic phase (Ph-), and the occurrence of the Philadelphia chromosome appeared in the patient itself (bone marrow). The analysis showed that the emergence of the Philadelphia chromosome was derived from the patient itself (bone marrow). The specific genetic abnormalities in some rapid changes after the target therapy disappear with the disappearance of the Ph chromosome; the patient's Ph (+) and the skin tissue samples are unique to the DNA repair gene BRIP1 frame deletion mutation; the donor bone marrow and the patient's bone marrow samples have some insertion / deletion on the BCR basis, only the patient Ph (+) rapid change period. The.Ex PANd S software with BCR fusion gene has 8 subcloned structures in the Ph (+) and Ph (-) remission period. The genetic map of the clone evolution is a "tree" structure. The astrocytoma in this study was first diagnosed as astrocytoma WHO II grade, and the recurrence was glioblastoma with the local variant star. WHO IV level of tumor cell tumor. Sequencing of its tumor tissue DNA, data analysis showed that there were missense mutations of gene NOTCH and ATRX in both primary and recurrent tumors. At first diagnosis, there was a heterozygous termination mutation of gene TP53, the homozygous termination mutation of gene TP53 and misheterozygous mutation of the genetic IDH1 in the relapse, and the recurrence of the tumor at the same time. The analysis of.Ex PANd S software with more chromosomal translocation showed that there were 4 and 3 subcloned structures in the primary and recurrent stages. The clone evolution of the first diagnosis was "tree like" structure, and the occurrence of clones in the relapse stage was "parallel", suggesting that there was a high heterogeneity in the tumor clones at this stage. In the course of the disease progression of the CML patients, the BCR/ABL1 fusion gene is a characteristic clone change during the course of the target therapy. With its production, some secondary mutations or chromosomal aberrations occur. After targeted therapy, the specific genetic anomaly of some sudden changes disappears with the disappearance of the Ph chromosome, and the gene BCR exists. On the premise of microdeletion / mutation, the abnormal DNA repair gene such as BRIP1 eventually leads to the formation of BCR/ABL1 fusion gene. For astrocytoma, specific mutations can be used as the basis for tumor molecular typing, but the relationship between the prognosis of IDH1 and astrocytoma deserves further discussion; the analysis of the subclonal structure of the tumor tissue may be the tumor's presence. The molecular genetic basis is provided for the prognosis and target therapy of the bed. Radiotherapy and chemotherapy may produce selective pressure on the clonal evolution of the tumor, thus making the clone of the TP53 mutation dominant. The heterogeneity of the clones in the recurrence stage of astrocytoma is enhanced. The results suggest that the radiotherapy after the first operation should be careful.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R733.72

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