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Expression of angiopoietins and vascular endothelial growth factors on bone marrow mononuclear cells from acute myeloid leukemia patients and their clinical significance
No full text急性骨髓性白血病 (acute myeloid leukemia, AML) 是一種骨髓細胞功能異常且癌症變化的疾病。因此骨髓中缺乏正常的幹細胞，故無法製造足夠的正常血球，因此病患往往死於貧血、出血或感染的問題。很多的科學研究指出：血管新生(angiogenesis) 在固態腫瘤 (solid tumor)之形成、擴散及轉移中扮演舉足輕重的角色。近來的研究報告也顯示：在血液的惡性腫瘤疾病裡，血管新生對於其致病機轉也有其重要地位。一般認為是血癌細胞及其附近之間質細胞 (stromal cell) 分泌許多促使血管新生的物質(angiogenic substances)，其會分布在microenvironment中，而這些物質會活化血管內皮細胞 (endothelial cells)以產生血管新生，進而加速腫瘤之生長。
因此為了了解急性骨髓性白血病其血管新生現象與其腫瘤間之關聯、臨床症狀及表徵與存活分析，故藉者收集急性白血病病患骨髓 (bone marrow ,BM) 之檢体，利用即時定量聚合酶連鎖反應合成法(Real-time quantitative polymerase chain reaction, RQ-PCR)、免疫細胞化學染色法(immunocytochemical stain, ICS) 及酵素免疫吸附檢測方法 (Enzyme-linked Immunosorbent Assay, ELISA) 的方法，希望能找到AML與血管新生 (angiogenesis) 間之關係。
我們從民國八十四年至九十五年間台大醫院血液科診斷為急性骨髓性白血病且接受標準化學治療之病患共計126位，利用即時定量聚合酶連鎖反應合成(RQ-PCR)的方法分析骨髓芽細胞中血管新生因子訊息核醣核酸 (mRNA) 的含量，包括研究 Angiopoietin 1 (Ang-1)、Angiopoietin 2 (Ang-2)、Tie2 receptor、Vascular endothelial growth factor A (VEGF-A) 與 VEGF-C 五種血管新生因子。此外針對 Ang-2的表現，我們額外作有關芽細胞之免疫細胞化學染色法及血漿蛋白之酵素免疫吸附檢測方法分析。至於臨床上分析的指標包含性別、年齡、FAB 分類、白血球數目、芽細胞總量、血色素、血小板數目、LDH數值、染色體變化、及治療上之效果 (含括血管新生因子數值高低與存活率(overall survival)、緩解率 (complete remission)、復發率(relapse rate)、無復發狀況之存活率(relapse-free survival)之相關性)。
我們的結果發現：急性骨髓性白血病之病人在Ang-1、Ang-2與VEGF-A之表現上都高於正常對照組 (Ang-1： P<0.0001; Ang-2： P=0.009; VEGF-A：P<0.0001)。而 VEGF-C 的表現則低於正常對照組上(P=0.011)，至於 Tie2 的表現則在兩組間無統計學差異(P=0.9142)。此外，利用免疫細胞化學染色法 (immunocytochemical staining) 分析九位病患白血病芽細胞之Ang-2 蛋白表現，發現Ang-2蛋白的表現與利用即時定量聚合酶連鎖反應合成法 (RQ-PCR)所測量之訊息核醣核酸 (mRNA)表現呈現一致。換言之，Ang-2 蛋白表現在較高之訊息核醣核酸之病患中，芽細胞的染色呈現高訊號及強度；相反地在較低之訊息核醣核酸之病患中，芽細胞的染色呈現低訊號及強度。同時在所有三十一位擁有骨髓血清之病患中，我們發現利用酵素免疫吸附檢測方法 (ELISA)而得到之Ang-2蛋白量，它們的數值與訊息核醣核酸 (mRNA)之表現量具有高度一致性 (P=0.022 by Spearman’s rank)。
在臨床表徵方面：我們發現Ang-1 與 Ang-2 的表現與絕對周邊芽細胞之總數 (absolute peripheral blast counts) 呈現正相關 (分別為P= 0.0044 及 0.0041)，而VEGF-A 與 VEGF-C 的表現與絕對周邊芽細胞之總數 (absolute peripheral blast counts) 呈現負相關 (分別為P= 0.011 及 0.019)。其他包括性別、年齡、白血球數目、血色素、血小板數目、LDH數值、染色體變化及FAB classification 皆與五種新生血管因子之數值沒有特別的關係。
在病患之預後方面：利用單一變數分析法發現：不好之染色體 (unfavorable karyotype)、較高之Ang-2 的族群、較高之Ang-1 的族群及較高之Tie2 的族群擁有較差的無復發狀況之存活率 (relapse-free survival)。至於性別、年齡、白血球數目、血色素、血小板數目、LDH數值及VEGF-A & VEGF-C 的高低與無復發狀況之存活率之長短無關。至於在 存活率 (overall survival) 方面，年紀超過40歲、不好之染色體 (unfavorable karyotype)、較高之Ang-1 的族群及較高之Tie2 的族群擁有較差的存活率。
至於使用Cox proportional hazards 之多變項分析可以發現，在無復發狀況之存活率方面：唯一的獨立變項因子，即是擁有不好之染色體 (unfavorable karyotype)，其 hazard ratio 為3.1 (95% CI 1.78-5.40, P<0.0001)。其他血管新生因子的表現皆非獨立因子。在存活率方面：除了擁有不好之染色體 (unfavorable karyotype)，其 hazard ratio 為2.19 (95% CI 1.78-5.40, P<0.0001)外，較高之Ang-2 的族群亦是較差之獨立因子，其 hazard ratio 為2.05 (95% CI 1.20-3.52, P=0.009)。
利用Kaplan-Meier curves存活分析，可以顯示Ang-2 的表現高低與存活時間有明顯相關。換言之；有較高Ang-2 表現的病患平均存活時間為 38±16.8 個月，較低Ang-2 表現的病患平均存活時間為 15.7±3.27 個月 (P=0.005)。而在同時擁有血清檢體的病患中，若是其蛋白質分泌表現大於 3 ng/ml之病患比那些蛋白質分泌表現小於 3 ng/ml之病患有較差之存活時間，統計上有明顯之差距。(平均存活時間, 5±1.47 versus 13±5.24 months, P=0.018)。此外，在那些擁有一般染色體變化之病患中，Ang-2 的表現對於病患之存活率有更大之影響 (P=0.004)。換言之，在這群族群中，有較高Ang-2 表現的病患平均存活時間為 55±20.57 個月，而較低Ang-2 表現的病患平均存活時間只有 16±2.92 個月。
由於血管新生因子會彼此交互影響，我們同時做次族群之分析 (subgroup analysis)。我們發現Ang-2 的表現高低在較低之Ang-1與Tie2的族群中其存活時間之差距特別明顯相關(P=0.003 及 P=0.003)，而在較高之Ang-1與Tie2的族群中其存活時間之差距並無明顯相關。另外方面，Ang-2 的表現高低在較高之VEGF-A與VEGF-C的族群中其存活時間之差距特別明顯相關(P=0.038 及 P=0.05)，而在較低之VEGF-A與VEGF-C的族群中其存活時間之差距則無明顯相關。
我們的實驗是第一篇研究急性骨髓性白血病病患之骨髓中血管新生因子的表現。病患中血管新生因子的表現與正常骨髓有明顯之差異。此外，不論是訊息核醣核酸或是蛋白質的測量，Ang-2 的表現更是與病患預後有顯著相關，這樣的相連性在擁有一般染色體變化之病患中更為清楚。此外，我們的研究也發現血管新生因子間的合作，亦會影響病患的臨床表現與預後。因此，Ang-2 的表現可能可以視為急性骨髓性白血病病患的一個生物指標，可望當作病患疾病評估、追蹤與治療的參考。BACKGROUD. Concerted expression of angiopoietins, their receptor Tie2 and vascular endothelial growth factor (VEGF) family plays an essential role in normal and pathologic angiogenesis, but its clinical implication in AML remained unclear. .
METHODS. We investigated the RNA expression of genes encoding angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), the receptor Tie2, VEGF-A and VEGF-C by real-time quantitative polymerase chain reaction (RQ-PCR) in a cohort of 126 patients with newly diagnosed de novo AML and 22 normal marrow donors. The results were correlated with clinical features and outcome of the patients.
RESULTS. Expression of Ang-1, Ang-2 and VEGF-A was significantly higher and that of VEGF-C was lower in AML patients than in normal controls. Only unfavorable karyotype and higher expression of Ang-2, but not other angiogenic factors, were independent prognostic factors for overall survival by multivariate analysis, with a hazard ratio of 2.19 (95%CI, 1.27-3.77, P=0.005) and 2.05 (95%CI, 1.20-3.52, P=0.009), respectively. The prognostic significance of Ang-2 expression was more obvious in the subgroup of patients with intermediate-risk cytogenetics (P=0.004). Subgroup analysis showed that Ang-2 expression had prognostic impact on patients with low (but not high) Ang-1 or Tie2 levels, and on patients with high (but not low) VEGF-A or VEGF-C levels.
CONCLUSIONS. These results provide evidence that high pre-treated levels of Ang-2 in the bone marrow indicate an unfavorable prognosis in AML.一、中文摘要 (Chinese Abstract)----------------------P3-5
二、緒論 (Introduction)-------------------------------P6-11
三、研究方法與材料 (Methods and Materials)----------P12-16
四、結果 (Results)------------------------------------P17-19
五、討論 (Discussion)---------------------------------P20-21
六、展望 (Perspective)-----------------------------------P22
七、英文簡述 (English Summary)-----------------------P23-30
八、參考文獻 (References)---------------------------P31-33
九、圖表 (Tables and Figures)-----------------------P34-4
Genetic alterations and their clinical implications in acute myeloid leukemia
No full text急性骨髓性白血病是一種惡性的骨髓造血功能異常，在病理機轉、臨床表現及治療效果上都是一個相當分歧的疾病。分子研究上的進步可進一步擴展我們對於急性骨髓性白血病致癌機轉的瞭解。傳統上染色體的核型異常已被證實在診斷上及預後上為重要的生物標記。儘管如此，約有 60% 至 75% 的病患屬於中度風險染色體核型變化，這個族群的基因變異甚大且預後也不盡相同，如何進一步剖析這群病患是最重要的臨床課題。近年來由於分子生物技術的進步，次世代定序的蓬勃發展，揭露出更多基因變異在急性骨髓性白血病的角色。
本研究的主題在於釐清不同基因變異在急性骨髓性白血病的致癌角色、臨床表徵與治療預後，譜劃出急性骨髓性白血病的基因譜，繼而彙集這些基因變異與染色體核型變化的結果，建構整合預後評估風險系統，當作病患風險評估、預後判斷及治療選擇的標的。首先我們擬針對目前尚不明瞭的 3 個重要基因，包括 RUNX1 基因突變、WT1 基因突變及 DNMT3A 基因突變，研究其在急性骨髓性白血病的致癌角色及其特有的臨床生物表徵。同時分析 CEBPA 單雙基因突變對於急性骨髓性白血病病患預後的影響。次而我們將整合完成現有 17 個不同基因變異在急性骨髓性白血病的角色，搭配染色體核型變化的結果，針對急性骨髓性白血病病患，尤其是中度風險染色體核型變化族群做風險評估的標的及未來治療選擇的依循。最後根據不同基因在診斷時及復發時的變化，研究其穩定性及評估是否可能成為未來微量殘餘疾病分析的生物標記。
針對第一部份 (RUNX1 基因突變的角色)：我們的研究成果是第一篇針對大規模病患做全面 exon 3至 exon 8 的完整分析。RUNX1 基因突變在非 M3 亞型急性骨髓性白血病的發生率為 13.2%。RUNX1 基因突變好發於男性、年長者、擁有 FAB M0/M1 亞型或 +8 染色體核型變化的病患中。大多 runt homology domain (RHD) 的突變屬於 missense 基因突變 (64.5%)，相對地 transcription activation domain (TAD) 的突變則多為 frameshift 基因突變 (76.5%)。Missense基因突變預測會影響到去氧核醣核酸的結合能力或與 CBFβ 形成異合體 (heterodimerization) 的能力。而 frameshift 基因突變則會造成 TAD 部分或全部的遺失，失去 transactivation potential。在 62 位具有 RUNX1 基因突變的病患中， 31 位 (50%) 會伴隨其他的分子基因變異；其中最常見的是 Class I 基因變異 (83.9%)，包括 FLT3/ITD、 FLT3/TKD 與 NRAS基因突變等。在存活分析方面，我們確認了 RUNX1 基因突變是一個獨立的不良預後標的，病患會有較差的完全緩解率、無病況之存活時間與整體存活時間。
針對第二部份 (CEBPA 基因突變的角色)：我們的研究指出三分之二病患的 CEBPA 基因突變為雙基因突變，同時影響 TAD1 及 basic leucine zipper domain (bZIP) 的區域。我們的結果顯示擁有 CEBPA 雙基因突變的病患比那些 CEBPA 單基因突變或是沒有 CEBPA 基因突變之病患，他們的完全緩解率較高且整體存活時間及無病況之存活時間較長。進一步在多變項分析的結果顯示，具有 CEBPA 雙基因突變，但並非 CEBPA 單基因突變，是一個獨立預後好的存活因子。由於單基因突變比較容易存在於芽細胞有 CD56 表現的病患，同時常與一些不好的基因變異存在，諸如 FLT3/ITD、FLT3/TKD、MLL/PTD及 RUNX1 基因突變。或許可以部分解釋為何 CEBPA 單基因突變的病患預後較差的原因。
針對第三部份 (WT1 基因突變的角色)：這是第一篇收納最多染色體核型異常變化之急性骨髓性白血病病患研究，我們的研究顯示 WT1 基因突變在所有病患的發生率為 6.8%，在年輕且染色體核型正常變化的病患中其發生率為 8.3%；而突變率在染色體核型正常變化與染色體核型異常變化的兩族群中無差別。 WT1基因突變好發於年輕者或擁有 FAB M6 亞型的病患，而較少發生在 M0 亞型的病患中。除了發現 t(7;11)(p15;p15) 常見於 WT1 基因突變的病患外，大多數 (72%) 擁有 WT1 基因突變的病患會合併其他分子基因的變異，常見於 Class II 基因變異 (69.6%) 與 Class I 基因變異 (56.5%)，其中 FLT3/ITD 與 CEBPA 基因突變是最常與 WT1 基因突變同時出現的。在存活分析方面，不論在全部或是染色體核型正常變化的病患， WT1 基因突變皆是一個獨立的不良預後標的。病患會有較高的復發率及較短的無復發病況之存活時間與整體存活時間。藉著整合 4 個生物預後標記，包括年紀、WT1 基因突變、NPM1/FLT3-ITD 與 CEBPA 基因突變，我們可將病患做更有效的危險預後分組。
針對第四部份 (DNMT3A 基因突變的角色)：我們的研究指出 DNMT3A 基因突變會出現在 30 個不同的位置，最常見在 MTase domain。此處所有的 nonsense、frameshift 及 in-frame 基因突變預測會造成 truncated peptide，會破壞這個酵素的活性。其中最常見的 R882 突變會造成酵素功能的失調。DNMT3A 基因突變的發生率在所有的病患、非 M3 亞型的病患、帶有中度風險染色體核型變化的病患及染色體核型正常變化的病患，各為 14%、15.2%、19.5% 及 22.9%。 DNMT3A基因突變好發於年長者、擁有 FAB M4/M5 亞型或染色體核型正常變化的病患中。有趣地，在急性骨髓性白血病的病人身上，DNMT3A 基因突變很少單獨存在，高達 97.1% 會合併其他分子基因變異。除了 DNMT3A 基因突變會與 NPM1 基因突變或 FLT3/ITD 基因變異密切相關外，發現擁有 DNMT3A 基因突變的病患比那些沒有 DNMT3A 基因突變的病患，同時具有較高的 IDH2 或 PTPN11 基因突變；相反地， CEBPA 基因突變很少出現在擁有 DNMT3A 基因突變的病患身上。在存活分析方面，不論在全部或是染色體核型正常變化的病患， DNMT3A 基因突變皆是一個獨立的不良預後的標的，病患有較高的復發率及較短的無復發病況之存活時間與整體存活時間。藉著整合 8 個生物預後標的，包括年紀、白血球數目、 NPM1/FLT3-ITD、 CEBPA 基因突變、DNMT3A 基因突變、WT1 基因突變、RUNX1 基因突變與 IDH2 基因突變，我們可進一步將病患做更好的危險預後評估分組。
針對第五部份 (整合預後風險評估系統)：針對 318 位非 M3 亞型的急性骨髓性白血病且接受標準化學治療的病患，我們進行 17 個基因的檢測，發現 RUNX1 基因突變、WT1 基因突變及 DNMT3A 基因突變皆是獨立預後不良之存活因子。最後採用 8 個與預後有關的基因變異包括 FLT3/ITD、CEBPA、NPM1、RUNX1、WT1、IDH2、ASXL1 及 DNMT3A 基因突變，可將非常分歧的中度風險染色體核型變化之病患分成預後截然不同的三組。同時可將傳統上用染色體核型變化分類中占 72% 的預後中等族群，減少到利用整合分析而來之 24.5%。進一步發現：當病患屬於中度風險染色體核型變化卻同時擁有預後良好的分子基因變化 (在沒有 FLT3/ITD 基因變異下，具有 NPM1 基因突變、IDH2 基因突變或是 CEBPA 雙基因突變)者之治療成績與那些擁有預後良好之染色體核型變化者一樣好。相反地，當病患屬於中度風險染色體核型變化卻同時擁有預後不佳的分子基因變化 (具有 RUNX1 基因突變、WT1 基因突變、ASXL1 基因突變或 DNMT3A 基因突變) 者之治療成績與那些擁有預後不佳之染色體核型變化者一樣差。
針對第六部份 (基因變異的配對檢體系列檢驗分析)：我們利用系列性的病患檢體 (診斷、完全緩解及復發時) 做基因的檢測，來探討常見基因突變在急性骨髓性白血病發生及病程進展時所扮演的角色，尤其是 RUNX1、WT1、CEBPA 及 DNMT3A 等 4 個基因。我們發現在所有 117 位病患中，61 位病患出現基因改變 (包括基因遺失或基因獲得) 的現象。Class I 基因遠比 Class II 基因更容易出現基因改變的現象 (50.8% vs. 15.6%, P=0.001)。此外，復發時有 15 位 (12.8%) 病患獲得新的基因變異，其中多數是獲得新的 Class I 基因變異，其中 3 位獲得新的 WT1 基因突變。然而卻沒有任何 1 位獲得新的 Class II 基因變異。這部分所收納的病患皆是目前研究數目最多且最完整的系列檢驗。我們的研究發現相對於 FLT3/ITD 基因變異的不穩定性， DNMT3A 基因突變、NPM1 基因突變、CEBPA 基因突變及 IDH1/2 基因突變皆維持在癌化過程中的穩定地位。此外 RUNX1 基因突變也被證實在此疾病進展時扮演著重要角色，但在復發時的角色較不明顯。相反地，WT1 基因突變與 TET2 基因突變會在復發時消失或是出現。這些結果可協助判斷何種基因變異可做為微量殘餘疾病分析時的理想生物標記。
本研究的成果首先提供不同基因變異在急性骨髓性白血病病患的發生率、臨床表徵、與染色體核型變化及合併不同基因變異的關聯性，次而論及這些基因變異對於病患的預後影響。綜合這些資料我們可以譜出急性骨髓性白血病病患詳細的基因變異圖表，進一步可瞭解在診斷及復發時基因變異的狀況，建構整合預後評估風險系統，當做個人化醫療的基石及未來轉譯醫學的重要參考。Acute myeloid leukemia (AML) is a heterogeneous group of neoplastic disorders with great variability in the pathogenesis, clinical course and response to therapy. Advances in molecular research have greatly improved our understanding of the leukemogenesis in AML. The aim of the present doctoral thesis is to first investigate the detailed molecular genetic alterations in patients afflicted with AML. We plan to elucidate the role of RUNX1, WT1 and DNMT3A mutations, three molecular alterations not well studied before, and single or double CEBPA mutation (CEBPAsingle-mut or CEBPAdouble-mut) in the leukemogenesis of AML. Second, it is of utmost significance to understand the clinic-biologic features, associated cytogenetic and molecular abnormalities and clinical relevance of these molecular aberrations in AML patients. Further, we can depict the comprehensive picture of total 17 frequent molecular genetic alterations in AML patients from the project and other researches from our team. In addition to the importance of these genetic alterations in the development of AML, we also perform sequential studies to explore the role of these events in the disease progression. As we aware, the integrated prognostic system is more power than a single biomarker to risk-stratify the heterogeneous AML patients into different groups with distinct outcome. Therefore, the integration of cytogenetic and molecular changes we design will improve the prognostic stratification of AML patients, especially those with intermediate-risk cytogenetics, and may lead to better decision on therapeutic strategy.
In the subproject one (RUNX1 mutation in AML), we found that RUNX1 mutations could be detected in a substantial proportion (13.2%) of patients with de novo non-M3 AML. RUNX1 mutations were closely associated with male gender, older age, immature FAB subtypes (M0 and M1) and trisomy 8. Most runt homology domain (RHD) mutations were missense mutations (64.5%), while most transcription activation domain (TAD) mutations were of frameshift mutations (76.5%). Among the 62 patients with RUNX1 mutations, 31 (50%) showed additional molecular abnormalities at diagnosis. They were mutually exclusive with CEBPA and NPM1 mutations, but were closely associated with MLL/PTD. Furthermore, the RUNX1 mutation predicted lower complete remission (CR) rate and was an independent poor-risk factor for overall survival (OS) and disease-free survival (DFS).
In the subproject two (CEBPA mutation in AML), about two third of patients with CEBPA mutations had CEBPAdouble-mut both at TAD1 and bZIP domains. CEBPAdouble-mut patients had a higher CR rate and a significant longer DFS and OS than those with CEBPAwild or CEBPAsingle-mut. CEBPAsingle-mut was closely associated with CD56 expression but inversely correlated with HLA-DR, CD7 and CD15 expression. Compared to the patients with CEBPAdouble-mut, those with CEBPAsingle-mut had a higher incidence of concurrent FLT3/ITD, FLT3/TKD, MLL/PTD or RUNX1 mutation and had a poorer prognosis. This study provides evidences independently from previous ones, stressing the differences in biologic characteristics between CEBPAsingle-mut and CEBPAdouble-mut AML and their possible prognostic implication.
In the subproject three (WT1 mutation in AML), 29 different kinds of WT1 mutations were detected in 32 (6.8%) patients. We showed that WT1 mutations occurred with similar frequencies in patients with normal cytogenetics (CN-AML, 7.0%) and those with abnormal cytogenetics (6.3%). The mutation was closely associated with younger age, FAB M6 subtype and t(7;11)(p15;15), but inversely related to M0 subtype. Among the 32 patients with WT1 mutations, 23 (72%) showed additional molecular abnormalities at diagnosis; sixteen (69.6%) of them had at least one concurrent Class II mutation and 13 (56.5%), Class I mutation. Furthermore, the WT1 mutation predicted higher relapse rate and was an independent poor-risk factor for OS and relapse-free survival (RFS) among total cohort and CN-AML patients. Incorporation of the these gene mutations, including NPM1/FLT3-ITD, CEBPAdouble-mut, WT1 mutation and age at diagnosis that are closely associated with prognosis, into survival analyses can better stratify patients into different risk groups.
In the subproject four (DNMT3A mutation in AML), DNMT3A mutations at 30 different positions, most commonly in the MTase domain, were demonstrated. All the nonsense, frameshift, and in-frame mutations generated truncated peptide with complete or partial deletion of the MTase domain and were suggested to abolish the catalytic activity of this enzyme. The missense R882 mutations, the most common DNMT3A mutations, resulted in impaired enzyme activity. DNMT3A mutations could be detected in a substantial proportion of patients with de novo AML (14% of total patients; 15.2% of non-M3 AML; 19.5% of intermediate-risk cytogenetics and 22.9% of CN-AML). DNMT3A mutations were closely associated with older age, FAB M4/M5 subtypes and intermediate-risk cytogenetics and CN-AML. Among the 70 patients with DNMT3A mutations, 68 (97.1%) showed additional molecular abnormalities at diagnosis. They were mutually exclusive with CEBPA mutation, but were closely associated with FLT3/ITD, NPM1, PTPN11 and IDH2 mutations. Furthermore, the DNMT3A mutation was an independent poor-risk factor for OS and RFS among total cohort and CN-AML patients. Incorporation of DNMT3A mutation with eight other prognostic factors, including age, WBC counts, cytogenetics, NPM1/FLT3-ITD, CEBPA, RUNX1, WT1, and IDH2 mutations, into survival analyses can better stratify AML patients into different risk groups.
In the subproject five (integrated prognostic system in AML patients), early assessment of cytogenetics and mutational profiling of eight relevant genes, including CEBPA, NPM1, FLT3, RUNX1, WT1, IDH2, ASXL1 and DNMT3A, may provide a framework for risk stratification in 318 non-M3 AML patients. Using the proposed classification by integrating cytogenetic and mutational profiles, we reduced the proportion of patients in intermediate-risk group, as defined by cytogenetics alone, from 72% to 24.5%, defined by both cytogenetic and molecular changes. Three fourth of intermediate-risk patients defined by cytogenetics alone can be reliably reclassified into favorable- (35.2%) or unfavorable-risk (40.2%) group according to the molecular genotype. The patients with intermediate-risk cytogenetics but favorable molecular genotype (mutation of NPM1, IDH2 or CEBPAdouble-mut in the absence of FLT3/ITD) had similar treatment response to those with favorable-risk cytogenetics. Similarly, the prognosis of the patients with intermediate-risk cytogenetics but unfavorable molecular genotype (mutation of RUNX1, WT1, ASXL1, or DNMT3A) was as poor as those with unfavorable-risk cytogenetics. The integrated cytogenetic and molecular classification further refines the prognostic prediction models and may guide the therapeutic decision.
In the subproject six (comparative analyses of genetic alterations in paired samples), we found that mutational shifts, including mutational loses and gains, occurred in 61 (52.1%) of 117 patients at relapse. Class I mutations, which activate signal transduction, were lost more frequently (50.8%) than Class II mutations (15.6%), which cause transcriptional deregulation. Genetic evolution with acquisition of novel mutations at relapse were identified in 15 individuals (12.8%), all involving Class I, WT1 or ASXL1 mutations, but not Class II or NPM1 mutations. Sequential study showed that mutations of CEBPA, RUNX1, IDH1/2 or DNMT3A remained quitely stable during the clinical course. In contrast, the instability of WT1 and TET2 mutations was noticed during disease progression. Our findings distinctly help judge which genetic alteration can be used as a biomarker for minimal residual disease (MRD) monitoring.
In conclusions, the present doctoral thesis combined genetic association studies, molecular results and clinical data to demonstrate how the genetic alterations, especially RUNX1, WT1 and DNMT3A mutations are involved in the leukemogenesis of AML. Incorporation of cytogenetic changes and molecular alterations as an integrated prognostic system can better risk-stratify the heterogeneous AML patients, especially intermediate-risk cytogenetics into different prognostic groups with distinct outcome. Finally, the gene mutations which are stable during treatment courses can also be used as biomarkers to monitor MRD.目錄
口試委員會審定書……………………………………………………...……………….i
誌謝………………………………………………………………...………………….ii
目錄…………………………………………………………..………..………………..iii
表目錄……………………………………………….……………………………...…...v
圖目錄…………………………………………………………………………………viii
中英文及縮寫對照表…………………………...………………………………………x
中文摘要………………………………………………………………….…………...xiii
Abstract…………………………………………………….…………...…………...xviii
第一章 緒論……….……………………………………………………………..1
第一節 急性骨髓性白血病的重要性....................…….…………………………1
第二節 急性骨髓性白血病的致癌機轉….………………………...……………3
第三節 染色體核型變化在急性骨髓性白血病的致病角色………………….....5第四節 基因變異的研究與急性骨髓性白血病的致癌關係……..……………...8第五節 急性骨髓性白血病的危險因子及臨床分類的重要性………..……….16第六節 不同基因變異在急性骨髓性白血病診斷時及復發時的表現……….19
第七節 研究目的與假說……………..…...………………………..……...….....19
第二章 研究方法及材料……………………………………………………….........22
第一部份 收納研究的急性骨髓性白血病病患族群…..…………….….…..22
第二部份 血癌細胞的染色體核型分析…..…………….….…………………..23
第三部份 血癌細胞的細胞表面抗原分析…...………..……………………..23
第四部份 血癌細胞基因的變異分析……………………………...……………23
第五部份 血癌細胞的 TA cloning 分析.............................................…..……..27
第六部份 血癌細胞的Gene Scan分析............................................…..……..28
第七部份 即時定量聚合酶連鎖反應合成法……………….............…..……..28
第八部份 統計分析模式………………………………………..………..……...29
第三章 研究結果……….……………………………………………………...…….31
第一部份 RUNX1 基因突變在急性骨髓性白血病的致癌角色及臨床意義….31
第二部份 CEBPA 基因突變在急性骨髓性白血病的致癌角色及臨床意…....35
第三部份 WT1 基因突變在急性骨髓性白血病的致癌角色及臨床意義……..37
第四部份 DNMT3A 基因突變在急性骨髓性白血病的致癌角色及臨床意義..41
第五部分 不同基因變異在急性骨髓性白血病的發生率與染色體核型變化的關連性－整合生物標記在急性骨髓性白血病的臨床預後評估…...46
第六部份 不同基因變異在急性骨髓性白血病配對檢體之系列綜合分析…...51
第四章 討論……….…………………………………………………………………56
第五章 結論及展望…….……………………………………………………………71
第六章 論文英文簡述….……………………………………………………………78
第七章 參考文獻..….………………………………………………………………107
第八章 圖表……….…………………………………………..……………………125
第九章 附錄……….…………………………………………..……………………20
Gene Ontology Annotation of Full-Text Biomedical Articles Using Word Proximity Relationship
No full text
Enhancing performance of protein and gene name recognizers with filtering and integration strategies
No full textHou Tianya
No full text학위논문(석사)--아주대학교 일반대학원 :산업공학과,2009. 8I INTRODUCTION-------------------------------------- 1
II METHODS------------------------------------------- 5
II-1 Semi-Supervised Learning--------------------- 6
II-2 Technical Indicators Transform----------------- 8
II-3 Feature Extraction (PCA/NLPCA)--------------- 10
III EXPERIMENTS-------------------------------------- 15
III-1 Artificial Data---------------------------------- 16
III-2 Benchmark Data------------------------------- 20
III-3 Oil Price Data--------------------------------- 25
IV CONCLUSION--------------------------------------- 35
REFERENCES----------------------------------------- 36MasterOil price prediction is an important issue for the regulators of the government and the related industries. When employing the time series techniques for prediction, however, it becomes difficult and challenging since the behavior of the series of oil prices is dominated by quantitatively unexplained irregular external factors, e.g., supply- or demand-side shocks, political conflicts specific to events in the Middle East, and direct or indirect influences from other global economical indices, etc. Identifying and quantifying the relationship between oil price and those external factors may provide more relevant prediction than attempting to unclose the underlying structure of the series itself. Technically, this implies the prediction is to be based on the vectoral data on the degrees of the relationship rather than the series data. This paper proposes a novel method for time series prediction of using Semi-Supervised Learning that was originally designed only for the vector types of data. First, several time series of oil prices and other economical indices are transformed into the multiple dimensional vectors by the various types of technical indicators and the diverse combination of the indicator-specific hyper-parameters. Then, to avoid the curse of dimensionality and redundancy among the dimensions, the well-known feature extraction techniques, PCA and NLPCA, are employed. With the extracted features, a timepoint-specific similarity matrix of oil prices and other economical indices is built and finally, Semi-Supervised Learning generates one-timepoint-ahead prediction. The proposed method was validated on one artificial- and five real-world- problems. And then the series of crude oil prices of West Texas Intermediate (WTI) was used to verify the proposed method, and the experiments showed promising results: 0.86 of the average AUC and 88% of the average classification accuracy
Patient Safety in Taiwan: A Survey on Orthopedic Surgeons
No full textBackground/Purpose: Patient safety is an important issue in medical quality control. To our knowledge, no studies have been conducted to specifically address patient safety in surgery in Taiwan. The purposes of this study were to determine the incidence of surgical errors in Taiwan, to evaluate the effectiveness of a campaign to mark the planned operation site, and the factors that influence the frequency of this preoperative safety maneuver. Methods: In March 2004, each member of the Taiwan Orthopaedic Association was given a 12-question survey regarding wrong- site, wrong-patient, and wrong-procedure errors to provide baseline data. We then implemented a campaign to encourage orthopedic surgeons to mark the planned operation site (" Mark op site" campaign). A follow-up survey was done in October 2004, and the results of both surveys were compared. Results: On the second survey, the number of surgeons who marked the incision site had significantly increased (p < 0. 05), and the incidences of reported wrong-site (0.5%) and wrong-procedure errors (2.4%) were lower than on the first survey (4.8%, p < 0.05 and 5.6%, p < 0.05). On the second survey, preoperative marking of the incision site was significantly correlated with the location of the surgeon's practice. Conclusion: Orthopedic surgeons marked the incision sites more frequently after our campaign than before, suggesting that the campaign was effective in changing their behavior. In Taiwan, this campaign reduced the risk of wrong-site and wrong-procedure errors
[Handwritten list of names by an unknown author #1]
No full textHandwritten note by an unknown author, listing various names
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