臺灣博碩士論文加值系統

乳癌是最常威脅女性健康的癌症之一。一般來說，乳癌能區分成兩大類，包括荷爾蒙受體(雌激素受體與/或黃體激素受體)陽性與陰性兩類。乳癌屬於荷爾蒙受體陽性者通常預後較好，但也有一部份乳癌為荷爾蒙受體陽性的病患對荷爾蒙療法的反應不佳，並且預後也較差。利用分析乳癌基因表現模組(Gene expression profiling)的方式來鑑定乳癌特徵已被證實能夠預測乳癌的預後。許多基因已被發現能用來預測罹患乳癌的西方婦女的遠端復發機率；因此，我們將分析這些基因在乳癌為荷爾蒙受體陽性的台灣婦女中的表現量，並分析這些基因與臨床因子的關係性。我們利用即時偵測同步定量之聚合酶連鎖反應器(real-time polymerase chain reaction)來定量這些基因的表現。在一開始，我們先構築標準曲線所需的標準質體，共包含了十六種乳癌相關基因(survivin, MYBL2、BAG1、PGR、CTSL2、SCUBE2、STK15、CCNB1、CD68、MKI67、MMP11、GRB7、GSTM1、BCL2、ERBB2 與ESR1)與五種參照基因(ACTB、TFRC、RPLP0、GAPDH 與GUSB)的質體。接著我們建立了在LightCycler 與ABI7900 系統平台上的分析系統。在測試過這些標準質體的線性後，我們共分析了三十九對的臨床檢體。我們發現這二十一個基因在癌症組織中的表現量都比在正常組織之中的表現量高。我們也利用統計軟體來分析實驗的數據，我們發現有許多基因在癌症組織中的表現量與乳癌的期數、淋巴結轉移的顆數以及組織學分級有關。除此之外，我們還發現BCL2 基因與SCUBE2 基因的表現量和發病年齡相關(P=0.010 與P=0.036)。PGR 基因與黃體激素受體的表現(P=0.008)，以及HER2 基因與HER2 受體的表現(P=0.015)的關係性也被確立了。這些發現顯示這些乳癌相關基因的確與乳癌病程的進展有關，並且有可能與預後有關。我們另外計算了復發積分(recurrence score)，並由積分的高低將三十九位病患區分成三組，高復發風險佔二十一位(54%)、中復發風險與低復發風險各佔九位(23%)。我們也發現發病年齡較早的病患會有較高的復發風險(P=0.048)。在這個研究中，我們成功建立了定量分析多基因表現模組的系統，並利用這個系統發現許多的臨床特徵與這二十一基因的表現或復發積分有關。

Worldwide, breast cancer is the most common type of cancer in the females.Traditionally, breast cancer is classified into hormone receptor (estrogen receptor and/or progesterone receptor) -positive and -negative groups. The patients with hormone receptor –positive breast tumors have relatively good prognosis, due to responsiveness to hormone therapy. Gene expression profiling can identify breast cancer signatures and wasreported having prognostic significance. Several genes were recognized to have strong correlation with distant recurrence of breast cancer in Caucasian women. At present the literature contains little regarding gene expression profiling in Taiwan, an area with low incidence but poor prognosis. In this study, we analyzed multiple breast cancer related genes and investigated the gene expression pattern of hormone receptor positive breast cancer women and the relation with clinical characters and outcome. Real-time polymerase chain reaction (real-time PCR) was used to quantify the levels of expression of genes. At first we constructed the standard plasmids for generation of external standard curves,including 16 targeted genes (surviving, MYBL2, BAG1, PGR, CTSL2, SCUBE2, STK15,CCNB1, CD68, MKI67, MMP11, GRB7, GSTM1, BCL2, ERBB2 and ESR1) and 5 reference genes (ACTB, TFRC, RPLP0, GAPDH and GUSB). Then we established the quantification assays on LightCycler and ABI7900 system, respectively. After verification of methodology, we quantitatively measured multiple gene expression levels of 39 paired breast tissues. The resulted showed that all 21 genes were overexpressed in tumor tissue.By using Kruskal-Wallis test to analyze these data, we found many genes were expressed higher in elevated tumor stage, metastatic axillary lymph node and histologic grade in tumor tissues. In addition, we found higher expression of BCL2 and SCUBE2 in patients with early on-set age (P=0.010 and P=0.036), PGR in PR positive tumors (P=0.008) and HER2 in HER2 overexpression tumors (P=0.015). Those finding indicated that these breast cancer related genes might play important roles in tumor cell progress and thus might potentially affect prognosis in Taiwanese breast cancer females. By calculating the recurrence score (RS) from each tumor, we classified 54% tumors (n=21) into high risk group, 23% (n=9) into intermediate risk group and 23% (n=9) into low risk group. We also found that patients with early on-set age had higher RS (P=0.048) and there was significant correlation between RS and on-set age in linear regression model (P=0.002). In this study,we successfully set up a quantitatively assay for multigenes expression profiling and found several clinical characters were correlated with the expression levels of these 21 genes or the recurrence score.

Abstract (in Chinese)-------------------------------------I
Abstract (in English)------------------------------------II
Acknowledgements----------------------------------------III
Index----------------------------------------------------IV
Tables/Figures Index-------------------------------------VI
Reagents and Instruments-------------------------------VIII
Chapter 1: Introduction-----------------------------------1
A. Breast Anatomy-----------------------------------------2
B. Epidemiology of the Breast Cancer----------------------2
C. Medical Treatment of Breast Cancer---------------------3
D. Classification of Breast Tumors According to Gene Expression------------------------------------------------4
E. Recurrence Score Assay (Oncotype DXTM assay)-----------5
F. Introduction of 16 Cancer-Related Genes and 5 Reference Genes-----------------------------------------------------6
1. ER Group-----------------------------------------------7
2. HER2 Group---------------------------------------------8
3. Proliferation Group------------------------------------9
4. Invasion Group----------------------------------------10
5. Other Group-------------------------------------------11
6. Reference Group---------------------------------------12
G. Specific Aim of this study----------------------------13
Chapter 2: Materials and Methods-------------------------14
A. Sample Collection and Establishment of Data Bank------16
1. Sample Collection-------------------------------------16
2. Data Processing---------------------------------------16
B. Establish Real time-PCR Quantification Assays---------16
1. Construct Standard Plasmids for Real-time PCR Standard
Curves---------------------------------------------------16
I. Cell Culture------------------------------------------17
(1) Cell Subculture--------------------------------------17
(2) Preparation of Frozen Cells--------------------------18
(3) Culture of Frozen-Defrozen Cells---------------------19
II. Total RNA Extraction from Cells----------------------20
III. Reverse Transcription (RT)--------------------------20
IV. Polymerase Chain Reaction (PCR)----------------------21
V. DNA Electrophoresis-----------------------------------21
VI. Gel Extraction---------------------------------------22
VII. Ligation--------------------------------------------23
VIII. Transformation-------------------------------------23
IX. Small-scale Preparation of Plasmids------------------23
2. Test Amplification Efficiency of Plasmids for Quantification Standard Curves---- ----------------------24
I. Real-Time Polymerase Chain Reaction In LightCycler system---------------------------------------------------24
II. Real-Time Polymerase Chain Reaction In ABI 7900------25
C. Quantify Gene Expression Level of Clinical Samples and Breast Cancer Cell Lines---------------------------------26
1. Total RNA Extraction from Tissues---------------------26
2. RNA Electrophoresis-----------------------------------27
3. Reverse Transcription for Real-Time Polymerase Chain
Reaction Analysis----------------------------------------28
D. Statistical Analysis----------------------------------28
1. Wilcoxon Signed Ranks Test----------------------------28
2. Kruskal-Wallis test-----------------------------------29
Chapter 3: Results---------------------------------------30
A. Test Amplification Efficiency of Plasmids for Quantification Standard Curves---------------------------31
B. Adjust the Difference between Different Runs of Reverse Transcription--------------------------------------------31
C. Statistically Analyze the Correlation between Paired Clinical Samples-----------------------------------------32
D. Statistically Analyze the Relation between Gene Expression Levels and Clinical Characters----------------32
E. Calculate the Recurrence Score of Tumor Samples-------36
F. Statistically Analyze the Relation between Recurrence Score and Clinical Characters----------------------------37
Chapter 4: Discussion------------------------------------39
A. The Gene Expression Profiling of 21 Genes in Clinical Samples--------------------------------------------------40
B. The Comparison of Recurrence Score in Taiwanese and Western Breast Cancer Women------------------------------41
C. Conclusion--------------------------------------------42
References-----------------------------------------------43
Tables/Figures-------------------------------------------50
Author---------------------------------------------------81

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