臺灣博碩士論文加值系統

神經母細胞瘤(Neuroblastoma, NB)是源自於交感神經系統前驅細胞的胚胎性惡性腫瘤。罹患第四期NB的病童即使遵循常規治療預後仍差，其五年存活率低於40%，亟待找出更有效的治療標的物。多年來在NB的研究都指出造成治療失敗的特徵是腫瘤具有MYCN增幅。近年來許多研究認為ALK (Anaplastic lymphoma kinase)可能是另一個與NB致病相關的重要基因：臨床上發現high-risk NB若高度表現ALK，其病患存活率低於不表現ALK者。造成ALK基因異常表現的原因除了ALK基因gain/amplification (12.2% / 1.5% in NB)，尚與ALK基因突變( 8% in NB)或其他未知原因有關。ALK是一酪胺酸激酶接受器 (receptor tyrosine kinase)，當ALK的tyrosine kinase domain發生突變(如：F1174、F1245及R1275)會造成ALK過度活化，誘導細胞趨於癌化。ALK基因的調控經常與MYCN做連結，研究指出ALK的活化能促進MYCN表現；而MYCN亦能促進ALK轉錄表現。以ALK作為標的物的小分子抑制劑Crizotinb，臨床上已使用於有ALK轉位突變的非小細胞肺癌(NSCLC)之治療。在細胞實驗及小鼠xenograft模型中Crizotnib皆能有效抑制NB細胞及腫瘤生長，使ALK抑制劑具潛力為high-risk NB提供有效的輔助治療。然而，具有ALK F1174突變的轉殖小鼠NB動物模型中，Crizotinb無法抑制腫瘤發展，顯示ALK抑制劑對某些ALK突變的NB抑制效果不好；若改採用ALK小分子抑制物TAE-684則具有抑制ALK F1174突變的NB腫瘤。本研究目的為檢查台灣地區神經母細胞瘤中ALK基因表現及突變情形。將目前收集到34位健康成人PBMC以及61例NB病患檢體，針對ALK基因Exon20~25區域(kinase domain)及與其相鄰之Intron部分分段進行PCR，並進行核酸定序(Sanger’s sequencing)。在61例NB樣本中發現7例(11.3%)有ALK突變：3例為F1174L突變、3例為F1245突變，1例A1274T突變，未見到R1275突變；在Exon20~25中發現6個單一核苷酸多型性(SNPs)(3個SNPs位於外顯子之同義SNPs；3個SNPs位在Intron20及Intron25)，此6個SNPs變異型基因分布頻率在NB腫瘤與正常PBMC無差異。將ALK基因突變與病人臨床資料進行分析，發現ALK基因突變與惡性NB(advanced stage、high risk、poorly differentiation、MYCN amplification)有關聯性。腫瘤帶有ALK突變病患存活率較差(EFS：HR 4.2，p=0.0021；OS：HR 5.6， p=0.0015)。並且在advanced stage、high-risk NB的次群分析中，帶有ALK突變的腫瘤病患顯著有存活期較短。以ALK基因表現量與病人臨床資料進行分析，ALK高度表現在腫瘤細胞分化程度低的NB(UNB、PDNB)較顯著(p=0.0012)，其餘預後因子皆與ALK表現量高或低較無關聯。而ALK基因表現量與MYCN基因表現具有高度正相關(r=0.5831，p<0.0001)，但與ALK基因突變或基因多型性無顯著關聯。分析ALK基因表現量與NB病人存活率，結果則顯示腫瘤中ALK表現量高與病人存活率較差(EFS：HR 2.3，p=0.0600；OS：HR 4.8，p=0.0240)。透過本研究一系列實驗數據與臨床資料的統計分析，我們得知：(1) ALK基因突變常出現在惡性神經母細胞瘤，病患的預後較差；在惡性(advanced stages, high risk)神經母細胞瘤中， ALK突變為造成疾病較快進展的重要因子。透過檢測病患腫瘤是否具ALK基因突變及為何種突變，能在診斷早期預測病人的預後及選擇何種ALK抑制劑作為病患的輔助治療。(2) 不論ALK基因是否突變，神經母細胞瘤腫瘤皆可能有ALK高度表現；而ALK的高表現量在ALKWT及ALKMut+病患中皆可發現。(3) 在NB腫瘤中ALK表現與MYCN表現呈顯著正相關。 (4) 腫瘤中ALK表現量高為不良預後因子。(5)國人的NB病例帶有對Crizotinib具抗藥性的ALK F1174L突變比例較國外為高，未來若要使用ALK抑制劑治療國人神經母細胞瘤，ALK基因kinase domain突變之篩檢將十分重要。

Neuroblastoma (NB) is an embryonal malignancy derived from precursor cells of the sympathetic nervous system. Children suffer from the stage 4 disease display a poor 5-years survival (less than 40%) even following multi-modality treatments. For years, studies of neurblastoma show that MYCN gene amplification consistently associated with treatment failure. Recently, anaplastic lymphoma kinase (ALK) has been evolved as an important factor in carcinogenesis of neuroblastoma. Previous studies showed that NB with high ALK immune reactivity was associated with clinical outcome. Aberration in ALK, including ALK gene gain/ amplification (12.2%/ 1.5% in NB), ALK gene mutations (8% in NB) and others, makes ALK the second most commonly mutated gene in neuroblastoma. ALK is a receptor tyrosine kinase, mutations on the tyrosine kinase domain of ALK (eg. F1174, F1245, and R1275), would cause ALK overactivation and predispose to carcinogenesis. ALK and MYCN showed regulatoty loop: ALK was able to stimulate MYCN promoter via activation of ERK signaling, and MYCN was found to bind onto ALK promoter region to regulate ALK transcription. Targeting to ALK, by small molecular inhibitor, Crizotinib has been applied in the treatment of NSCLC harboring ALK translocation. In cell experiments and mice xenograft model, crzotinib can also inhibit the growth of NB cell lines and NB tumors and make ALK inhibitors a potential effective adjuvant therapy for high-risk NB. However, in the MYCN-transgenic mice harboring ALK F1174 mutation, Crizotinib couldn’t inhibit the development of NB tumor, point out that Crizotinib is ineffective in inhibit ALK with certain mutation within kinase domain. ALK inhibitors have limitation in treatment. The goal of this research is to inspect the situation on ALK gene expression and mutations in neuroblastoma in Taiwan. 61 NB tumor samples sand PBMCs from 34 healthy adults were analyzed, PCR amplification of ALK gene fragments spanning exon20 to exon25 and theirs neighboring intronic regions, followed by Sanger’s sequencing, In 61 tumor DNA samples, 7 (11.3%) were found to have heterozygotic ALK mutation: 3 F1174L mutation, 3 with F1245 mutation, 1 with A1274T mutation, but there is no R1275 mutation found. Moreover, we find 6 SNPs in exon20 to 25 of ALK gene, 3 SNPs are synonymous variants which located in exon20, 21 and23; the other 3 SNPs are intron variants, located in intron20 and 25. The allelic distribution of the ALK SNPs is not significantly different between NB tumors and normal PBMCs. We find that ALK mutation is associated with adverse clinical features (advanced stage, high risk, poorly differentiation, and MYCN amplification), and inferior survival (EFS: HR 4.2, p=0.0021; OS:HR 5.6, p=0.0015). Subset analysis of advanced stage, high-risk NB showed that patient harboring ALK mutation displayed a shorter 5-year survival. Relative mRNA expression of ALK and MYCN were determinate by q-RT-PCR. ALK expression was found positively correlated with MYCN gene expression (r=0.5831, p<0.0001). High ALK expression was associated with undifferentiated/ poorly differentiated NB (p=0.0012), but not with ALK mutation, ALK genetic polymorphism, nor other clinical features. We found ALK high expression correlated with worse patients’ survival (EFS: HR 2.3, p=0.0600; OS: HR 4.8, p=0.0240).
In Summary, we obtain information in our research: (1) ALK gene mutation occurs in NB with adverse clinical features. Further, ALK mutation is an important factor that cause faster disease progression in advanced stage and/or high-risk NB. Detection of ALK mutational spectrum in early diagnosis, could predict patients’ prognosis and choice suitable ALK inhibitor as adjuvant therapy in neuroblastoma. (2) ALK high expression could exist in ALK WT or mutant+ tumors. ALK high expression was not necessary found in ALKMut+ tumor. (3) ALK expression is highly correlated with MYCN expression in NB tumor, (4) High ALK expression in NB tumor predicts poor clinical outcomes. (5) ALK F1174L (known Crizotinib-resistant) is a prevalent type of mutation in NB in Taiwan. Screening ALK mutation to evaluate the use of ALK inhibitor as therapentics is very important for NB patients in Taiwan.

致謝…………………………………………………………………………………I
摘要…………………………………………………………………………………II
Abstract……………………………………………………………………………IV
縮寫表………………………………………………………………………………VI
目錄…………………………………………………………………………………X
表目錄………………………………………………………………………………XIV
圖目錄………………………………………………………………………………XV
附錄目錄……………………………………………………………………………XVI
第一章 緒論………………………………………………………………………1
1.1 神經母細胞瘤………………………………………………………………1
1.1.1 神經母細胞瘤簡介……………………………………………………1
1.1.2 神經母細胞瘤的致病機轉……………………………………………1
1.1.3 神經母細胞瘤的臨床表現……………………………………………2
1.1.4 神經母細胞瘤的診斷與分期…………………………………………2
1.1.5 神經母細胞瘤的病理特徵……………………………………………3
1.1.6 神經母細胞瘤的治療…………………………………………………4
1.1.7 神經母細胞瘤的分子標記及預後指標………………………………5
1.2 Anaplastic lymphoma kinase (ALK)基因……………………………………7
1.2.1 ALK基因的簡介………………………………………………………7
1.2.2 ALK基因的腫瘤學歷史………………………………………………8
1.2.3 ALK基因與神經母細胞瘤……………………………………………9
1.2.4 ALK基因的調控………………………………………………………10
1.2.5 ALK抑制劑在神經母細胞瘤中的潛力………………………………10
1.3 研究假說……………………………………………………………………11
第二章 研究目的與實驗設計……………………………………………………12
2.1 研究目的……………………………………………………………………12
2.2 實驗設計……………………………………………………………………12
第三章 材料與方法………………………………………………………………13
[實驗材料]…………………………………………………………………………13
3.1 臨床檢體……………………………………………………………………13
3.2 試劑、試藥、試劑套組與工作清單………………………………………13
3.2.1 試劑試藥清單…………………………………………………………13
3.2.2 試劑套組………………………………………………………………14
3.2.3 試劑配方………………………………………………………………14
(a) DNA製備………………………………………………………………14
(b) Polymerase Chain Reaction (PCR)…………………………………….15
3.2.4 聚合酶連鎖反應之引子序列…………………………………………15
(a) ALK基因………………………………………………………………15
(b) Control gene……………………………………………………………16
3.2.5 反轉錄聚合酶連鎖反應/即時定量反轉錄聚合酶連鎖反應之引子…16
(a) ALK基因………………………………………………………………16
(b) MYCN基因……………………………………………………………16
(c) Control gene……………………………………………………………16
3.2.6實驗儀器………………………………………………………………17
3.2.7軟體與網路工具………………………………………………………17
[實驗方法]…………………………………………………………………………18
3.3檢體DNA、RNA製備………………………………………………………18
3.3.1傳統常規DNA萃取……………………………………………………18
3.3.2傳統常規RNA萃取……………………………………………………18
3.3.3 以DNA、RNA及蛋白質萃取套組萃取神經母細胞腫瘤樣本………18
3.3.4 cDNA製備……………………………………………………………19
(a) SuperScript® III First-Strand Synthesis System………………………19
(b) Maxima First Strand cDNA Synthesis Kit with dsDNase………………19
3.4聚合酶連鎖反應引子設計…………………………………………………19
3.5聚合酶連鎖反應 (Polymerase Chain Reaction, PCR)………………………19
3.6反轉錄聚合酶連鎖反應 (Reverse Transcription-PCR, RT-PCR)…………20
3.7聚合酶連鎖反應產物純化與定序…………………………………………20
3.8即時定量聚合酶連鎖反應…………………………………………………20
3.9數據統計分析………………………………………………………………20
第四章 結果………………………………………………………………………22
[臨床資料及檢體收案情形]………………………………………………………22
4.1 神經母細胞瘤預後因子與病患存活率之分析……………………………22
4.2 ALK基因突變及基因多型性分析…………………………………………23
4.2.1 神經母細胞瘤病人腫瘤中ALK基因突變分析………………………23
4.2.2 ALK突變基因型的表現………………………………………………23
4.2.3神經母細胞瘤病人ALK基因突變與預後分子相關性之分析………24
4.2.4 ALK基因多型性分析…………………………………………………25
4.3 ALK基因表現量分析………………………………………………………26
4.3.1 神經母細胞瘤中ALK基因表現量之分析……………………………26
4.3.2 ALK基因表現量與神經母細胞瘤預後因子之關聯性………………27
4.3.3 ALK基因表現量與ALK基因突變之關聯性…………………………28
4.3.4 ALK基因表現量與ALK基因多型性之關聯性………………………29
4.3.5 ALK基因表現量與ALK組織化學染色之關聯性……………………29
4.3.6 ALK基因表現量與MYCN基因增幅及其MYCN表現量之相關性分析……29
4.4 ALK基因變異及基因表現量與神經母細胞瘤病患存活率之分析………30
4.4.1 ALK基因突變與神經母細胞瘤病患不良存活率具顯著相關性……30
4.4.2 ALK基因多型性與神經母細胞瘤病患存活率之相關性……………32
4.4.3 ALK基因表現與神經母細胞瘤病患存活率之相關性………………32
第五章 討論………………………………………………………………………33
第六章 參考文獻…………………………………………………………………38
圖與表………………………………………………………………………………52
附錄…………………………………………………………………………………80

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