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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 蘇剛毅 | |
dc.contributor.author | Chi-Ching Wu | en |
dc.contributor.author | 吳奇靜 | zh_TW |
dc.date.accessioned | 2021-06-15T12:30:57Z | - |
dc.date.available | 2018-08-26 | |
dc.date.copyright | 2016-08-26 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-08-04 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/50149 | - |
dc.description.abstract | 在非小細胞肺癌當中,約有3-7%的病人腫瘤存有間變性淋巴瘤激酶(Anaplastic lymphoma kinase, ALK) 融合蛋白。在肺癌中,除了棘皮動物微小管相關蛋白樣4 (Echinoderm microtubule-associated protein-like 4, EML4),EML4-ALK,還有TRK融合基因(TRK-fused gene, TFG; Tropomyosin receptor kinase, TRK, 原肌球蛋白受體激酶),TFG-ALK、驅動蛋白家族成員(KIF5B, kinesin family member 5B),KIF5B-ALK、驅動蛋白輕鏈 1 (KLC1, Kinesin light chain 1)KLC1-ALK,皆有被發現並報導。另一方面,先天性巨結腸原癌基因(RET, RET proto-oncogene)、ROS1原癌基因(ROS1, ROS proto-oncogene 1)等的融合蛋白也佔據肺癌的一定比例。病人罹有ALK基因突變的人,已有美國食品與藥品管理局(Food and Drug Administration, FDA)核准的標靶藥物可以用來治療。此外,尚有許多針對此三種融合基因突變的標靶藥物,正進行臨床試驗中,目前效果良好並值得期待。因此,ALK、RET及ROS1的融合突變顯得相當重要。然而,現今利用染色體異常來診斷融合基因的方法,例如螢光原位雜合技術(Fluorescence in situ hybridization, FISH)、免疫染色法(Immunohistochemistry, IHC)、逆轉錄聚合酶鏈式反應(Reverse transcription – polymerase chain reaction, RT-PCR)等,受限於技術層面、低靈敏度、檢體品質,和無法區分子型態等方面,故而開發新的檢驗方式,來偵測此三種融合基因突變有其必要性。在本次的研究中,我們致力於開發一個多重聚合酶鏈式反應(Multiplex polymerase chain reaction, Multiplex PCR)及高敏感性的檢驗平台,以克服前述診斷方法的不足。我們的目標在於使用福馬林固定和石蠟包埋(Formalin-fixed, paraffin-embedded, FFPE)的檢體,和核酸質譜儀技術平台(Matrix-Assisted Laser Desorption/Ionization Time-of-Flight, MALDI-TOF MS),來檢測ALK、RET,及ROS1原癌基因的融合突變。在此次研究中,我們使用這項新開發的檢測平台,及已被臨床端診斷,罹有ALK融合基因的病人檢體(兩位病人分離出的細胞檢體,及四位病人的福馬林固定和石蠟包埋檢體)做融合基因的子型態分型,並且成功檢測出其子型態(2/2病人細胞檢體,3/4病人的福馬林固定和石蠟包埋檢體)。此外,我們近來收到一位肺癌病人的胸水檢體,而此病人先前被診斷為不具有表皮生長因子接受器(Epidermal growth factor receptor, EGFR)、KRAS、ALK融合基因等突變。經過我們開發的新平台做檢測,此病人罹有CCDC6-RET,RET融合基因突變;而此檢測結過亦經核酸定序(Sanger Sequencing)作確認。
總觀而言,這個新的檢驗平台具高度敏感性、檢體需求量低,且低品質檢體如福馬林固定和石蠟包埋組織即可上機檢測。此外,這項檢驗平台具有一相當優越的特點:可以利用設計不同的探針(probe),在「一個反應中」,檢測多種融合基因突變、及細分其變體形式(variant form)。如該反應可檢測出檢體具有EML4-ALK融合,並且進一步分型出他為變體形式1 (variant 1)。更甚者,如果後續研究發現新的融合基因突變,或新型態的變體形式,我們可以在不干擾先前的任何設計的情況下,藉由加入新設計的探針,來檢測新的突變基因。透過同樣的簡易設計原理、及該功能強大的檢驗平台,我們得以在一個反應中,利用檢體品質相對不佳的福馬林固定和石蠟包埋組織,來一同檢測融合基因突變,或是其他非融合基因的突變。 | zh_TW |
dc.description.abstract | Approximately 3-7% of lung tumors harbor anaplastic lymphoma kinase (ALK) fusions in the subgroup of non-small cell lung cancer (NSCLC). In addition to echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion, TRK-fused gene (TFG)-ALK, kinesin family member 5B (KIF5B)-ALK and kinesin light chain 1 (KLC1)-ALK had been reported in lung cancer. On the other hand, RET proto-oncogene (RET) and ROS proto-oncogene 1 (ROS1) fusion proteins also have prevalence in lung cancer. Food and Drug Administration (FDA)-approved ALK target drugs are available to treat those patients who harbor ALK fusions. Moreover, there are several target drugs for these three fusion genes on clinical trial currently. Therefore, the diagnosis of ALK, RET or ROS1 fusion genes shows quite important. However, nowadays methods of detecting ALK fusions by chromosomal abnormalities, such as fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and reverse transcription - polymerase chain reaction (RT-PCR), are limited to technique, low sensitivity, sample quality as well as subtype classification. Here, we are dedicated to setting up a multiplex and highly sensitive panel to overcome these difficulties. We aim to use formalin-fixed, paraffin-embedded (FFPE) samples to detect major types of ALK, RET and ROS1 fusions by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF mass spectrometry). In this study, we used 2 patients’ cell cDNA and 4 patients’ lung FFPE samples cDNA, which had been diagnosed as ALK fusion before, to be detected by this panel, and then identified their variant types successfully (2/2 of cell cDNA, 3/4 of FFPE sample cDNA). Furthermore, we recently received one lung cancer patient’s pleura effusion sample, which was identified as non-EGFR, KRAS, and ALK mutation before. After tested by our panel, we identified that this patient harbors CCDC6-RET fusion mutation, and this sample had been confirmed by Sanger Sequencing as well.
To be concluded, this new panel has high sensitivity and allows little and poor quality samples for detecting. Moreover, it is very powerful that this new panel can detect not only fusion gene types, like EML4-ALK, but also the fusion variants, such as EML4-ALK variant 1, in only one reaction by using different probes. What’s more, we can detect new discovered types by adding new probes in the reaction without disturbing previous designs. Through the same design principle, we can detect other mutations and fusion genes together with ALK, RET or ROS1 fusions in one reaction with FFPE samples by this powerful and highly sensitive panel. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T12:30:57Z (GMT). No. of bitstreams: 1 ntu-105-R03424005-1.pdf: 2541148 bytes, checksum: 4c3676b9962034eed5fad6ab1d6fb8a8 (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | 目錄
中文摘要 I 英文摘要 III 1. Introduction 1 1.1 Cancer 2 1.2 Lung Cancer 2 1.2.1 Small-Cell Lung Cancer (SCLC) 3 1.2.2 Non-Small-Cell Lung Cancer (NSCLC) 3 1.3 Targeted Drugs and Genetic Alteration in Lung Cancer 4 1.4 Fusion Genes in Lung Cancer 5 1.5 Target Therapies and Detection of Fusion Genes in Lung Cancer 7 1.6 MALDI-TOF Mass Spectrometry 10 2. Specific aim 12 3. Material and Methods 14 3.1 Material 15 3.1.1 Experimental Apparatus 15 3.1.2 Materials 15 3.2 Methods 17 3.2.1 Sample Preparation 18 3.2.2 Primer Designation 21 3.2.3 Probe Designation 22 3.2.4 MALDI-TOF Mass Spectrometry 24 3.2.5 Cell Culture 29 3.2.6 Limit of Detection 30 4. Results 32 4.1 Panel Designs 33 4.2 Probe Adjustment 35 4.3 Tested by Human Cancer Cell Line without ALK, RET, and ROS1 Fusion Mutation 35 4.4 Patient cDNA with ALK Fusion Mutation 37 4.5 Formalin-fixed, paraffin-embedded (FFPE) Sample 38 4.6 Well-Known EML4-ALK Mutation Cell Line: H2228 Cell 40 4.7 Cut-Off Value – 20 Healthy People Leukocyte cDNA 41 4.8 Limit of Detection – H2228 Cell mixed with Hop-62 cell 42 4.9 One Lung Cancer Patient without Harboring EGFR, KRAS, ALK mutation 43 5. Discussion and Conclusion 44 5.1 Discussion 45 5.2 Future Application 48 5.3 Conclusion 49 6. Tables 50 Table 1.1.1 Well 1 Primers (for Revised and Old Panel) 51 Table 1.1.2 Well 2 Primers (for Revised Panel) 52 Table 1.2.1 Well 2 Primers (for Old Panel, Part I) 53 Table 1.2.2 Well 2 Primers (for Old Panel, Part II) 54 Table 2.1.1 Well 1 Probes Sequence (for Revised Panel) 55 Table 2.1.2 Well 2 Probes Sequence (for Revised Panel) 56 Table 2.2 Probes Sequence (for Old Panel) 57 Table 3.1 Condition of Well 1 Probe Mix 58 Table 3.2 Condition of Well 2 Probe Mix 59 Table 4.1.1 Well 1 Data Analysis Table (for Revised Panel) 60 Table 4.1.2 Well 2 Data Analysis Table (for Revised Panel) 61 Table 4.2 Data Analysis Table (for Old Panel) 62 Table 5.1 CV value of PC and NC cell lines – Well 1 63 Table 5.2 CV value of PC and NC cell lines – Well 2 64 Table 6. The Possible False Positive Variants 65 Table 7.1 Cut-Off Value: 20 Healthy People Leukocyte cDNA – Well 1 66 Table 7.2 Cut-Off Value: 20 Healthy People Leukocyte cDNA – Well 2 67 7. Figures 68 Figure 1. Flow Chart 69 Figure 2. Designation of ALK, RET, ROS1 Fusion Gene Detection 70 Figure 3. Detection Probes and Their Targets 76 Figure 4. Probe mix after probe adjustment 79 Figure 5. Panel tested by H322M cell cDNA 81 Figure 6. Mass Spectrograms of ALK fusion mutation patient 85 Figure 7. Mass Spectrograms of FFPE Sample from ALK Fusion Mutation Patients 88 Figure 8. Mass Spectrograms of H2228 Cell 91 Figure 9. Mass Spectrograms of Limit of Detection 94 Figure 10. Lung Cancer Patient Harboring CCDC6-RET Fusion Mutation 95 8. Reference 96 | |
dc.language.iso | en | |
dc.title | "利用核酸質譜儀建立肺腺癌融合基因ALK, RET, ROS1偵測平台" | zh_TW |
dc.title | Detection of ALK, RET, and ROS1 Fusion Mutation by MALDI-TOF Mass Spectrometry in Lung Adenocarcinoma | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 林亮音,楊雅倩,俞松良 | |
dc.subject.keyword | 肺癌,ALK 基因,RET 基因,ROS1 基因,融合基因,福馬林固定和石蠟包埋,組織核酸質譜儀,高敏感性檢測平台, | zh_TW |
dc.subject.keyword | lung cancer,ALK gene,RET gene,ROS1 gene,fusion gene,MALDI-TOF MS,FFPE sample,highly sensitive panel/platform, | en |
dc.relation.page | 108 | |
dc.identifier.doi | 10.6342/NTU201601936 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2016-08-04 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 醫學檢驗暨生物技術學研究所 | zh_TW |
顯示於系所單位: | 醫學檢驗暨生物技術學系 |
文件中的檔案:
檔案 | 大小 | 格式 | |
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ntu-105-1.pdf 目前未授權公開取用 | 2.48 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。