成人急性骨髓性白血病的臨床特徵和基因變異

Clinical Characteristics and Genetic Mutation in Adult Patients with Acute Myeloid Leukemia

陳建源(Chien-Yuan Chen)

指導教授 ： 田蕙芬

共同指導教授 ： 林亮音(Liang-In Lin)

國立臺灣大學/醫學院/臨床醫學研究所/博士(2013年)

https://doi.org/10.6342/NTU.2013.02512

摘要

緒論
急性骨髓性白血病 (Acute myeloid leukemia，AML），是成年人最常見的急性白血病，也是一種骨髓性造血芽細胞異常增殖的血液惡性腫瘤。急性骨髓性白血病的病患，常常因為身體無法製造出足夠數量之正常分化的嗜中性白血球，容易造成患者感染。顱內出血則是急性骨髓性白血病患，次常見的併發症。我們嘗試建立台灣血液腫瘤患者，這些臨床議題的流行病學基礎資訊，希望對日後血液腫瘤病患的支持療法將有所助益。
癌症是複雜且棘手的一群疾病，急性骨髓性白血病的發生過程，常常是多階段逐漸地累積許多的基因變異而造成。基因變異在急性骨髓性白血病的發生機轉中，扮演重要的角色。我們目前對癌症的所知有限，瞭解那些致癌基因會導致癌症發生，將有助於未來急性骨髓性白血病的診斷及追蹤。最近幾年，透過染色體變異分析和基因分子檢測，可以將急性骨髓性白血病，區分成不同預後的類別。利用聚合酶鍊反應和定序，尋找台灣急性骨髓性白血病患之基因變異和臨床特徵的相關性。另一方面，複雜染色體變化之急性骨髓性白血病患者的預後不好，然而相關的致癌基因所知卻不多，為了找出複雜染色體變化之急性骨髓性白血病致癌基因，我們利用轉錄體次世代定序的技術，找到一些新的轉位基因。
方法或程序
1.調查成人急性骨髓性白血病之伺機性感染、顱內出血的流行病學和臨床特徵的相關性，建立台灣急性骨髓性白血病患者的基礎資訊。
2.偵測成人急性骨髓性白血病的基因變異，利用傳統的聚合酶鍊反應和Sanger定序 方式，來偵測急性骨髓性白血病患者的基因變異；利用次世代定序的方式，嘗試找出複雜染色體變化之急性骨髓性白血病的致癌基因，和臨床之相關性。
結果與討論
成人急性骨髓性白血病，男性發生率每十萬人口為2.93人，女性每十萬人口為2.62人。在西元1996到2008年間，這十三年來台灣本土之急性骨髓性白血病發生率，在男性成長50%，女性成長67%，有逐漸增加的趨勢。
感染是急性骨髓性白血病患最常見的併發症。我們針對成人急性骨髓性白血病患的細菌感染，在西元1996~2001及西元2002~2006年期間，進行兩次大規模回溯性研究調查。這是本土第一次，大規模血液腫瘤成人病患血行性感染的流行病學研究。革蘭氏陰性細菌約佔全部致病菌60%，革蘭氏陽性細菌佔全部致病菌三成，其它一成是念珠菌和厭氧菌。革蘭氏陰性細菌中最主要的是大腸桿菌、克雷白氏菌。革蘭氏陽性細菌中以凝固酶陰性葡萄球菌、草綠色鏈球菌為主要的致病菌。廣泛抗藥性的鮑氏不動桿菌和萬古黴素抗藥性的腸球菌，在這段期間也逐漸出現。另一方面，我們也對台灣本土成人急性骨髓性白血病患的黴菌、結核菌和非結核分枝桿菌感染作相關的研究調查。熱帶念珠菌是血液惡性腫瘤患者念珠菌菌血症最常見的致病菌。黃麴菌則是侵入性黴菌鼻竇炎最常見的致病黴菌。急性骨髓性白血病病患較其他惡性血液病患者，容易發生侵入性黴菌鼻竇炎和結核病。非結核分枝桿菌感染在各種惡性血液腫瘤患者的發生率則沒有差異。
顱內出血是在急性骨髓性白血病患者併發症的第二大原因。從1995至2007年間，回朔研究841位急性骨髓性白血病患者。大部份顱內出血患者為頑固難治或復發的急性骨髓性白血病病患。多變項分析顯示有四個危險因子是急性骨髓性白血病患者顱內出血的獨立預後因素，包括凝血酶原時間的國際標準化比值延長1.5倍以上(P <0.001）、腦幹出血（P = 0.001）、蛛網膜下腔出血（P = 0.017）和硬膜外出血（P = 0.014）。 急性骨髓性白血病病患顱內出血的發生位置包括大腦皮層（60例，83％）、基底神經核(13例，18％）、小腦(10例，14％）和腦幹（5例，7％），總共有33位（46％）患者發生多處顱內出血。顱內出血的位置和類型和預後有關，神經影像學能夠協助預測急性骨髓性白血病患者之顱內出血的預後。
急性骨髓性白血病是否能夠治癒與白血病的染色體以及基因變異息息相關。我們在台灣調查了NPM1、CEBPA、RUNX1等等基因突變的流行病學、臨床特徵和預後。我們也針對NPM1細胞表面抗原的表現，進行相關性分析。層次聚類分析將NPM1突變的患者分為兩組免疫群集，第一組CD34和 CD7的表現為陰性，但有不等量的HLA-DR的表達量差異;第二組HLA DR、CD34和 CD7的表現為陽性。在平均追蹤53個月後，第二組患者比對照組第一組患者，有顯著較短的無復發存活時間（中位數為3個月和23個月，P = 0.006）和總體存活時間（中位數為11個月與40個月，P = 0.02）。多變項分析顯示，免疫群集是NPM1突變之急性骨髓性白血病患者，一個獨立的預後因子（無復發存活時間P = 0.002; 總體存活時間P = 0.024）。
我們應用次世代定序的技術，透過生物資訊的資料篩選，發現未曾被報告的融合基因。初步在8個轉錄體，有5個融合基因被發現，有2個是已知急性骨髓性白血病的融合基因，NSD1-NUP98和CBFB-MYH11。另外有3個是未曾被報告的融合基因，TPM4-KLF2，TALDO1-DSN1, KIAA0430-CES3。TALDO1基因位於染色體11 p15的位置，DSN1基因位於染色體20q11的位置。TALDO1-DSN1基因轉位和該樣本的染色體報告互相吻合，並且已經由聚合酵素鍊反應來證實。細胞分裂的階段，染色體排列在細胞中間，動粒(kinetochore)的內板附著在染色體中間的著絲點，紡錘體的紡錘絲則附著在動粒的外板，牽引姊妹染色體向細胞兩側的中心體移動、將染色體拉向兩個子細胞，完成細胞分裂。DSN1基因是構成動粒的MIND複合蛋白的結構之一，動粒結構的異常，可能會造成細胞分裂時，姊妹染色體的不平均分配，造成非整倍體(aneuploidy)。本研究為複雜染色體變化的急性骨髓白血病，染色體的第七對的長臂缺失，因此，我們推測複雜染色體變化之急性骨髓白血病的非整倍體，可能和細胞分裂相關的(DSN1)基因變異有關。
結論 我們在過去這段時間，調查本土的急性骨髓性白血病病患細菌、黴菌和結核菌等感染的情況和臨床預後的變因；以及急性骨髓性白血病病患顱內出血時，臨床預後和神經影像學的關聯；還有，急性骨髓性白血病基因突變的流行病學，可以用以預測病患的化療效果。初步建立了台灣本土急性骨髓性白血病臨床特徵和基因突變的流行病學。我們也應用次世代定序的技術，發現新的融合基因。希望未來能對病患臨床治療和處置有所幫助，以改善急性骨髓性白血病病患的預後。

關鍵字

急性骨髓性白血病 ； 嗜中性白血球低下的發燒 ； 顱內出血 ； 基因變異 ； 次世代定序 ； 融合基因

並列摘要

Acute myeloid leukemia(AML）is the most common leukemia in adults. AML is a hematological malignancy characterized with abnormal proliferation of hematopoietic progenitors. Infection is the most common complication in the AML patients, and intracranial hemorrhage (ICH) is the second common complications. We investigated the epidemiological data of these clinical issues in Taiwan, and this result potentially can benefit the AML patients in the future.
Cancer is a heterogeneous clonal disease. Leukemia development is usually a multistep process and arises from accumulated several genetic mutations. Recently, the prognosis in AML patients could be stratified by cytogenetics and molecular gene mutations. To know the genetic mutations can help the disease diagnosis and follow-up. We used the polymerase chain reaction and Sanger sequencing to find the epidemiology of genetic mutations and clinical characteristics in AML patients. We also used the technique of next generation sequencing to find the novel fusion genes in the complex karyotype AML.
Method
1. Survey the epidemiology and clinical manifestations of opportunistic infection、 intracranial hemorrhage in the adult patients with AML.
2. Detect the genetic mutations in adult patients with AML by the PCR and Sanger sequencing and correlate with clinical characteristics; Use the technique of next generation sequencing to detect the genetic lesions in the complex karyotype AML.
Result and Discussion
The incidence of AML is increasing in Taiwan. The incidence is 2.93 per 100000 populations in male and 2.62 per 100000 populations in female.
Two large scale retrospective studies of blood stream infection in the patients with hematological malignancy were investigated in 1996~2001 and 2002~2006. Gram negative bacteria were about 60% of pathogens, Gram positive bacteria were 30%, and the others were candida and anaerobes. E. coli and Klebsiella were the most common pathogens of Gram negative bacteria. Coagulase negative staphylococcus and streptococcus viridans were the most common Gram positive bacteria.
We also investigated the clinical characteristics of fungus, mycobacterium tuberculosis and non-tuberculosis mycobacterium in Taiwan. Candida tropicalis was the most common pathogen of candidemia. Aspergillus flavus was the most common mold in the invasive fungal sinusitis. The patients with AML tended to developed invasive fungal sinusitis and tuberculosis than patients with other hematological malignancy.
We retrospectively reviewed 841 AML patients between 1995 and 2007. Most AML patients with ICH were in refractory and relapsed status. Multivariate analysis revealed four independent prognostic factors: prolongation of prothrombin time (INR>1.5)、brainstem hemorrhage、subarachnoid hemorrhage and epidural hemorrhage。 The most common sites of ICH in AML patients included cerebral cortex (60 cases, 83％）、basal ganglion (13 cases,18％）、cerebellum(10 cases, 14％）and brainstem（5 cases,7％）. There were 33 cases (46%) with multifocal hemorrhage. The locations and types of ICH in neuroimaging could predict prognosis in AML patients.
Cytogenetics and molecular genetic mutation can predict the prognosis in AML.We found distinct clinical characteristics and prognosis in AML patients with genetic mutation of NPM1, CEBPA, RUNX1, or so on. Hierachical cluster analysis of the immunophenotype could stratify AML patients with NPM1 mutation into two groups with distinct prognosis. Patients whose immunophenotypes showed positive expression of HLADR, CD34, and CD7 had worse prognosis.
We detected new fusion transcripts by the technique of bioinformatics and next generation sequencing. Five fusion transcripts were found, including NSD1-NUP98 and CBFB-MYH11, that had been reported before, and three novel fusion transcripts TPM4-KLF2, TALDO1-DSN1,and KIAA0430-CES3.
DSN1 is the component of kinetochore. Kinetochore involves in the mitotic process of chromatids during cell division into two daughter cells.We predict the complex karyotype of AML patients could be related to the dysfunction of mitosis associated (DSN1) gene.
Conclusion
We studied the clinical manifestations and prognosis of opportunistic infection in AML patients. The neuroimaging correlated with clinical outcomes of ICH in the AML patients. We investigated the genetic mutations and clinical characteristics in the AML patients. We also detected the new fusion transcripts by the technique of next generation sequencing in the complex karyotype AML. We set up the basis of clinical characteristics and epidemiology of genetic mutations in the AML patients in Taiwan, and we hope these findings will be beneficial to the clinical management and improve the outcome in the adult patients with AML.

並列關鍵字

Acute myeloid leukemia ； febrile neutropenia ； intracranial hemorrhage ； genetic mutation ； next generation sequencing ； fusion gene

參考文獻

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