臺灣博碩士論文加值系統

中文摘要
子宮頸癌仍是世界上婦女死亡的主要原因之一，抹片雖然降低了子宮頸鱗狀上皮癌的發生率，但臨床上仍然存在有腺癌在年輕女性發生率緩慢上升、因擔心抹片對於不典型腺體細胞的潛在病灶及缺乏判讀的一致性導致過度使用侵犯性治療(如子宮頸圓錐狀切除)而造成病人過度焦慮及浪費醫療資源並增加後續懷孕早產之情形以及大部分子宮頸侵襲癌發生在未接受抹片的婦女身上這幾個重要問題亟待解決。在這次研究中我們發現PAX1,PTPRR,SOX1,ZNF582不僅在子宮頸鱗狀上皮癌而且同時在腺癌都有高度甲基化之情形，這對未來子宮頸癌(腺癌)篩檢有其重要之潛力。另外，我們藉由台灣婦癌聯合研究組織(TGOG)多中心臨床試驗發現，甲基化生物指標SOX1及POU4F3 兩者在偵測出子宮頸上皮重度贅疣以上病灶(CIN3+）及子宮內膜複雜性增生皆可以達到100%的敏感度，在專一性上也分別有可 接受的表現(67% and 52%)，在未來對於抹片AGC減少病人接受侵犯性治療會有一定之幫助。最後，我們藉由病人自我採集之陰道檢體與醫師採集之子宮頸檢體上細胞之定量甲基化分析發現兩者之一致性是合理到好的，將來是否可能透過病人自我採集陰道檢體之方式來降低未受檢率進而降低子宮頸癌發生率可藉由前瞻性隨機臨床試驗來進行驗證。

Abstract
Cervical cancer remains one of the main causes of death among women worldwide. Although cytology-based screening has successfully reduced the incidence of cervical squamous cell carcinoma. However, there are still some questions need to be solved, included the incidence of cervical adenocarcinoma (AC) has risen, especially in young women, atypical glandular cell (AGC) are over treated due to worried about occult disease and AGC interpretation is reproduced poorly between observers, which result in patient anxiety, increased medical expense, and increasing the risk of preterm delivery in subsequent pregnancies, the majority of cases of cervical cancer are still associated with absent or deficient screening, non-attendance at gynecological clinics is a major limitation of cervical cancer screening and self-collection of samples may improve this situation. In our study, cervical ACs carry aberrantly high methylation rates of PAX1, PTPRR, SOX1 and ZNF582—commonly methylated in SCCs—which might help for AC screening. Methylated (m) SOX1m and POU4F3m could be new methylation biomarkers for detection of CIN3+ and endometrial complex hyperplasia in AGC in multicenter study of Taiwanese Gynecologic Oncology Group (TGOG). Methylation biomarker analysis of PAX1, SOX1 and ZNF582 for detection of CIN3+ lesions shows reasonable to good concordance between the self-collected and physician-collected samples. Therefore, self-collection of samples could be adopted to decrease non-attendance and improve cervical screening.

Contents

Abstract (Chinese) …………………………………………........................................................................……. .………I
Abstract……………………………………………………………………. ......................................................................………. . …II
Chapter 1 Introduction …………………………….......................................................................…….. … ….1
1.1 Cervical Cancer Screening………………......................................................................…….. …..……1
1.2 Human Papillomavirus and Cervical Cancer........................................................... ………… ………………2
1.3 Epigenetic and Cervical Cancer ………………………………........................................................… ……..…5

Chapter 2 Specific Aim …………………………………….....................................................................………… … ….9
Chapter 3 Materials and Methods…………………....................................................................………… … ……10
3.1 Patient for Adenocarcinoma of Cervix……............................................................…………………… ..…10
3.2 Patient for Triage of AGC………………………………...............................................................………………. .…11
3.3 Patient for self-collected vaginal samples…………………........................................................…… ……11
3.4 Real-time Quantitative Methylation-specific Polymerase Chain Reaction (QMSP) Amplification for adenocarcinoma of cervix………….………………….……………………..………………..................................................……….....................…………………12
3.5 Amplification of DNA with real-time quantitative methylation-specific polymerase chain reaction (QMSP) for AGC patients and self-collected vaginal samples…………………………………............................................……………………. …… ……14
3.6 Bisulfite Pyrosequencing for adenocarcinoma of cervix…...................................................… ………15
3.7 Ethics statement ……………………..………………………………………......................................................... ..... .…16
3.8 Statistics …….…………………………………..………..…………………………................................................................ …..17
3.8.1 Statistical Analysis for adenocarcinoma of cervix…………………............................................... ....…..17
3.8.2 Statistical Analysis for triage of AGC…………………….............................................................. ..18
3.8.3 Statistical Analysis for self-collected vaginal samples….............................................………...… ..18
Chapter 4 Results ……………………………………………................................................................…………...…… ……20
4.1 Result for adenocarcinoma of cervix ………….………........................................................…………...……… ……20
4.1.1 Demographic characteristics of patients and flow chart of the study sampling procedure………… ………….…………………...……. ..20
4.1.2 Real-time quantitative methylation-specific polymerase chain reaction (QMSP) analysis of LMX1A, NKX6-1, PAX1, PTPRR, SOX1 and ZNF582 in normal cervical scrapings and cervical adenocarcinomas ………………………………………………………………………………………………..........................................................................… ...……21
4.1.3 Validation of methylation status in adenocarcinoma tissues and normal cervical scrapings by bisulfite pyrosequencing ………………………………………………………………….….……………………………....................................................... .....…21
4.1.4 Methylation status of PAX1, PTPRR, SOX1, and ZNF582 in normal and adenocarcinoma cervical cells…….………...…………………..…….................................................................................................…................…23
4.2 Result for AGC………………………………..…………………................................................................…………………..… ……23
4.2.1 The histopathology and HPV results of AGC patients………….......................................................……23
4.2.2 Methylation index of SOX1, PAX1, ZNF582, PTPRR, AJAP1, HS3ST2, POU4F3, and ADRA1D genes………………………………………....……………24
4.2.3 The clinical accuracy of SOX1, PAX1, ZNF582, PTPRR, AJAP1, HS3ST2, POU4F3, and ADRA1D genes in the triage of AGC patients………………….….…………..……………………………………………...............................................................…………........…25
4.3 Results for self-collected vaginal samples……….....................................................………………....……………26
4.3.1 Population, study design flowchart and cytology/histology of study samples………………………………………………………………………….….………………26
4.3.2 Validation of clinical performance and concordance analysis of methylation biomarkers in self-collected and physician-collected samples…………………………………………………………………..………………….......................................................…26
4.3.3 Optimization of the clinical accuracy of methylation biomarkers using the cutoff values of the self-collected group………………………….....................................................................................................…27
Chapter 5 Discussion …………………….......................................................................……………………………………29
5.1 DNA methylation and adenocarcinoma of cervix……………………............................................................…29
5.2 Triage of AGC with DNA methylation biomarker………………............................................................………32
5.3 Feasibility of DNA methylation biomarker by self-collected vaginal samples………...…………………………………………………………….………………………37

Chapter 6 Conclusions ……………..………………………………………….......................................................................…43
References…………………………………………………………………………...............................................................................45
Tables & Figures ……………………………………....................................................................………………………….55
Table 1. High methylation levels of six genes in cervical adenocarcinoma tissue samples……………………………………………………..............….........................................................................……55
Table 2. High methylation levels of four genes in adenocarcinomas of the cervix (from scrapings)……..................................…………......................................................................……………….56
Table 3. Pathology and HPV results of AGC patients………......................................................…………..……….57
Table 4. Clinical performance of methylation biomarker and HPV to detect CIN3+ /CIS and complex hyperplasia of endometrium in AGC………………………………………………………………...………………….............................................................……….58
Table 5. Summary of clinical performance of methylation biomarker, HPV and CA-IX in triage of AGC………………………….......………………..………..........................................................................................................59
Table 6.Cytology / histology and mean age of self-collected vaginal samples……….…………………………………………………………..…………………..……….60
Table 7. Comparison the detection of CIN3+ between the physician-collected and self-collected samples using methylation of PAX1, SOX1 and ZNF582 genes…………………………………....…………………….…..….......................................................…….61
Supplemental Table1. Demographic characteristics in three sets of adenocarcinoma of cervix….……………………………………………………………………62

Figure 1. Flow chart of the study sampling procedure (adenocarcinoma of cervix)………………………………………………………………………………….……...….63
Figure 2. QMSP analysis of 6 genes in normal cervical scrapings and cervical adenocarcinomas in set A (adenocarcinoma of cervix)……...........................................................................................................……64
Figure 3. Validation of methylation status in adenocarcinoma tissues and normal cervical scrapings by bisulfite pyrosequencing…………..................................................................................................….65
Figure 4. Methylation status of PAX1, PTPRR, SOX1, and ZNF582 in normal and adenocarcinoma cervical cells……………………………………….……...............................................................................................66
Figure 5. Study flowchart of AGC…….……..................................................................…………………………………….67
Figure 6. Methylation index (meth-index) on a log scale of methylated (m) genes by histopathology of AGC…………………………….….68
Figure 7. Proposed algorithm for management of AGC in TGOG study………………….……..................………..…………………………………………………….69
Figure 8. The study design flowchart of self-collect vaginal samples….................................................70
Figure 9 Heat map of study patients in self-collected and physician-collected samples………………………………………...................... ……………..................................................................….71
Figure 10. Concordance in the clinical performance of methylation biomarkers between the self-collected and physician-collected groups………………………………………………………………………………................................................................……….……..72


Appendix I
Letter of Approval from Institutional Review Board of Tri-Service General Hospital National Defense Medical Center
Appendix II
Published Article

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