本文選題：慢性髓細(xì)胞白血病 + 星形細(xì)胞瘤�。� 參考：《天津醫(yī)科大學(xué)》2016年博士論文

【摘要】：目前,隨著研究的深入,我們對(duì)腫瘤的生物學(xué)和基因組學(xué)的認(rèn)識(shí)也有了改變。要將腫瘤的基因組學(xué)研究轉(zhuǎn)化到腫瘤的臨床治療,需要考慮到腫瘤細(xì)胞的復(fù)雜性以及其動(dòng)態(tài)的演化特征。腫瘤演化過(guò)程中不同的腫瘤細(xì)胞克隆競(jìng)爭(zhēng)空間和資源,最終具有生長(zhǎng)優(yōu)勢(shì)的克隆形成新的腫瘤亞群。腫瘤以不同的時(shí)間和速度演化進(jìn)展,在任何一個(gè)患者中其克隆結(jié)構(gòu)、基因型和表型都隨著時(shí)間改變。腫瘤克隆演化的研究除了使我們對(duì)腫瘤的發(fā)生發(fā)展有更全面的了解,同時(shí)也有著重要的應(yīng)用價(jià)值,比如發(fā)現(xiàn)對(duì)治療更有預(yù)測(cè)價(jià)值的標(biāo)志以提高個(gè)體化治療效果、以及阻止腫瘤對(duì)藥物的治療抵抗。而腫瘤克隆演化的特征,如克隆結(jié)構(gòu)或驅(qū)動(dòng)突變等,或可以用于腫瘤預(yù)后的判定。慢性髓細(xì)胞白血病(chronic myeloid leukemia,CML)是一種骨髓細(xì)胞增殖異常的血液疾病。該疾病存在特征性的9號(hào)和22號(hào)染色體的易位t(9;22)(q34;q11),即費(fèi)城染色體(Philadelphia chromosome,Ph),在分子水平上形成BCR/ABL1融合基因。該融合基因編碼的蛋白具有酪氨酸激酶活性,在無(wú)生長(zhǎng)因子的情況下能夠促使細(xì)胞增殖。Ph染色體是造成CML腫瘤表型的主要染色體畸變,幾乎所有的CML均存在BCR/ABL1融合基因。針對(duì)BCR/ABL1的靶向治療能夠使的CML患者緩解,但是Ph染色體的形成機(jī)制以及靶向治療后Ph染色體克隆演化還不是很清楚。膠質(zhì)瘤(glioma)是一類(lèi)起源于膠質(zhì)細(xì)胞的腫瘤,星形細(xì)胞瘤(astrocytoma)是最常見(jiàn)的神經(jīng)上皮性腫瘤。低級(jí)別的星形細(xì)胞瘤一般生長(zhǎng)緩慢,但是經(jīng)過(guò)一段時(shí)間會(huì)進(jìn)展為高級(jí)別的惡性腫瘤,引起腫瘤的復(fù)發(fā)從而降低整體生存率。星形細(xì)胞瘤手術(shù)切除后極易復(fù)發(fā),且惡性程度增高,但復(fù)發(fā)和惡性程度增高的機(jī)制尚不明確。大多數(shù)星形細(xì)胞瘤患者死于腫瘤的復(fù)發(fā)和惡變,研究其細(xì)胞遺傳水平的克隆演化對(duì)認(rèn)識(shí)星形細(xì)胞瘤的生物學(xué)行為、進(jìn)行針對(duì)性的治療有重要意義。目的:腫瘤的克隆演化是腫瘤的一個(gè)重要特征。為了研究克隆演化對(duì)于疾病的進(jìn)展以及治療的影響,本研究選取了兩個(gè)腫瘤類(lèi)型:慢性髓細(xì)胞白血病和星形細(xì)胞瘤。前者存在特征性的染色體異常即費(fèi)城染色體;后者的疾病特征為手術(shù)切除后的復(fù)發(fā)惡變。通過(guò)全基因組的測(cè)序,一是研究CML中BCR/ABL1融合基因的形成機(jī)制以及針對(duì)Ph靶向治療后的克隆演化;二是通過(guò)研究星形細(xì)胞瘤的克隆演化,探尋影響該腫瘤復(fù)發(fā)惡變的分子遺傳因素。方法:本研究選取了一例CML患者,該患者確診CML后清除其自身骨髓行全相合異體造血干細(xì)胞移植(hematopoietic stem cell transplantation,HSCT)(供體為患者姐姐),疾病進(jìn)入Ph(-)慢性期;后患者發(fā)生急淋變,熒光原位雜交(fluorescence in situ hybridization,FISH)實(shí)驗(yàn)顯示BCR/ABL1(+);對(duì)患者進(jìn)行針對(duì)Ph染色體的靶向治療后疾病得以緩解,Ph染色體消失。本研究收集了該患者姊妹間全相合異體造血干細(xì)胞移植后慢性期(Ph-)、急變期(Ph+)以及針對(duì)Ph靶向治療后再緩解期的骨髓組織活檢樣本,并獲得患者的皮膚活檢組織作為正常對(duì)照。本研究選取一例星形細(xì)胞瘤患者,該患者初診時(shí)腫瘤組織病理檢測(cè)顯示右側(cè)額葉星形細(xì)胞瘤(WHO II級(jí)),后患者腫瘤復(fù)發(fā),組織病理檢測(cè)顯示右額葉膠質(zhì)母細(xì)胞瘤合并局灶間變型星形細(xì)胞瘤(WHO IV級(jí))。收集患者初診腫瘤組織樣本(星形細(xì)胞瘤WHO II級(jí))以及復(fù)發(fā)后腫瘤組織樣本(WHO IV級(jí)),并收集患者的外周血樣品作為正常對(duì)照。對(duì)CML患者的骨髓組織皮膚組織的DNA和星形細(xì)胞瘤患者的腫瘤組織和外周血DNA進(jìn)行全基因重測(cè)序(測(cè)序深度≥50×)。首先將測(cè)序數(shù)據(jù)利用BWA軟件對(duì)比到參考基因組(UCSC hg19)上,應(yīng)用samtools、control-FREEC、mu Tect、Strelka、crest軟件分析樣品存在的遺傳學(xué)改變,包括點(diǎn)突變、拷貝數(shù)變異(copy number variation,CNV)、插入/缺失(insertion-deletion,INDEL)和染色體易位;并與對(duì)照比較,基于腫瘤樣本的體細(xì)胞單位點(diǎn)突變和拷貝數(shù)的位點(diǎn)集合,應(yīng)用Ex PANd S軟件分析腫瘤樣品存在的亞克隆結(jié)構(gòu),包括亞克隆的數(shù)目、亞克隆大小以及每個(gè)亞克隆中包含的突變。應(yīng)用Sci Clone軟件對(duì)成對(duì)樣本進(jìn)行亞克隆分析。結(jié)果:本研究選取的CML患者,在行姊妹間全相合異體骨髓移植后疾病進(jìn)入慢性期(Ph-),后發(fā)生急變(Ph+),針對(duì)Ph靶向藥物治療后疾病再緩解。分析測(cè)序數(shù)據(jù)發(fā)現(xiàn)骨髓樣品中存在一系列的SNPs、INDEL、CNVs和SVs,急變期(Ph+)比慢性期(Ph-)出現(xiàn)更多的遺傳學(xué)異常,經(jīng)分析,急變期費(fèi)城染色體的出現(xiàn)來(lái)源于患者本身(骨髓受體)。在靶向治療后一些急變期特有的遺傳異常隨著Ph染色體的消失而消失;患者Ph(+)的急變期和皮膚組織樣本中獨(dú)有DNA修復(fù)基因BRIP1框移缺失突變;供體骨髓和患者的骨髓樣本共同存在BCR基因上的一些插入/缺失,僅患者Ph(+)的急變期存在BCR形成融合基因的斷裂點(diǎn)。Ex PANd S軟件在Ph(+)急變期和Ph(-)緩解期均有8個(gè)亞克隆結(jié)構(gòu),克隆進(jìn)化的發(fā)生圖均為“樹(shù)狀”結(jié)構(gòu)。本研究中選取的星形細(xì)胞瘤患者,初診時(shí)為星形細(xì)胞瘤WHO II級(jí),復(fù)發(fā)時(shí)為膠質(zhì)母細(xì)胞瘤合并局灶間變型星形細(xì)胞瘤WHO IV級(jí)。對(duì)其腫瘤組織DNA進(jìn)行測(cè)序、數(shù)據(jù)分析,發(fā)現(xiàn)初診和復(fù)發(fā)階段腫瘤均存在基因NOTCH和ATRX的錯(cuò)義突變;初診時(shí)存在基因TP53的雜合終止突變,復(fù)發(fā)時(shí)存在基因TP53的純合終止突變以及基因IDH1的雜合錯(cuò)義突變;同時(shí)復(fù)發(fā)腫瘤中有較多的染色體易位。Ex PANd S軟件分析顯示,在初診和復(fù)發(fā)階段腫瘤分別有4和3個(gè)亞克隆結(jié)構(gòu),初診階段的克隆進(jìn)化的發(fā)生圖為“樹(shù)狀”結(jié)構(gòu),而復(fù)發(fā)階段克隆進(jìn)化的發(fā)生圖為“并行”結(jié)構(gòu),提示該階段腫瘤克隆存在較高的異質(zhì)性。結(jié)論:在該CML患者復(fù)發(fā)、靶向治療的疾病進(jìn)展過(guò)程中,BCR/ABL1融合基因?yàn)樘卣餍缘目寺「淖?隨著其產(chǎn)生而出現(xiàn)一些列繼發(fā)性的突變或者染色體畸變,針對(duì)其靶向治療后以一些急變期特有的遺傳學(xué)異常隨著Ph染色體的消失而消失;基因BCR存在微缺失/突變的前提下,BRIP1等DNA修復(fù)基因的異常最終導(dǎo)致BCR/ABL1融合基因的形成。對(duì)于星形細(xì)胞瘤,特異性的突變可用于腫瘤分子分型的依據(jù),但I(xiàn)DH1與星形細(xì)胞瘤預(yù)后的關(guān)系值得進(jìn)一步探討;腫瘤組織亞克隆結(jié)構(gòu)的分析可以為腫瘤的臨床預(yù)后判斷和靶向治療提供分子遺傳學(xué)依據(jù);放療、化療可能對(duì)腫瘤克隆演化產(chǎn)生選擇壓力,從而使TP53突變的克隆產(chǎn)生選擇優(yōu)勢(shì)。星形細(xì)胞瘤復(fù)發(fā)階段克隆的異質(zhì)性增強(qiáng),研究結(jié)果提示初次手術(shù)后的放療要慎重。
[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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R733.72

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