臺灣博碩士論文加值系統

癌症轉移是導致癌症患者死亡的主因，而患者血液中的微量循環腫瘤細胞是源自於原發腫瘤位置的特殊細胞種，此細胞早已被證實與癌轉移及癌症復發有相當密切的關連。臨床上，循環腫瘤細胞的數目或者特殊表面抗原的表現，可以用來判斷癌症預後、復發及癌症治療成效，以便提供醫師預測癌症病況，即時調整癌症治療方式。近年來，癌症的藥物治療，隨著特定有意義基因的發現，例如肺癌的EGFR突變，大腸癌的KRAS突變等，治療與藥物的選擇，變得較有效率且治療反應改善。然而，目前臨床上仍必須依照反覆切片的癌症組織才能進行診斷，對患者來說，是一個有風險、具相當侵入性的醫療行為，因此透過循環腫瘤細胞偵測與分離技術可以抽血就不斷追蹤癌症細胞多寡與基因變化，有改變臨床常規處置的劃時代意義，但此技術目前仍不成熟、穩定，甚至能否與腫瘤組織對應都是個未解的問題。因此，為了解決上述基本的問題，在博士班的訓練中，研究了幾個方向，依著主題為循環腫瘤細胞鑑別、分群、分離、培養與基因檢測，期待循環腫瘤細胞協助達到精細掌握癌症變化的目標。
本博士論文在訓練期間，參與開發了高純度分離的光介電泳力分選平台、傳統負篩選技術之改良、加入流式細胞儀協助判讀與篩選(專利申請中)與癌症團塊組織與循環腫瘤細胞三維培養的技術，基於此技術，又進行了四個分別為食道癌、大腸癌與頭頸癌P16、人類乳突病毒偵測、mRNA、DNA偵測與頭頸癌循環腫瘤細胞培養的四個研究成果，整體而言，此博士論文涵蓋了數個基礎循環腫瘤細胞分離技術的開發與臨床試驗驗證，甚至進展到培養與基因檢測的部分，我們期待本研究所建立的循環腫瘤細胞辨識、分離與培養技術能更加成熟，減少反覆切片風險，在精準醫學的目標下，最終運用到廣大的癌症患者幫助治療的抉擇。

Distant metastasis of cancer remains the major cause of cancer death. One of the evidence is that some rare cells shed from primary tumor exist in the circulation of cancer patients, which has been proven to be related to cancer relapse and distant metastasis. The number of circulating tumour cells (CTCs) or the expression status of the specific marker(s) on them also correlated with the disease prognosis and treatment effects, which might change the decision of treatments. In recent years, as specific disruptive genes were discovered, such as EGFR in non-small cell lung cancer, KRAS in colorectal cancer, the response rate to treatment, disease control and survival have been much improved. However, the molecular information obtained from cancer tissue depends on repeated biopsies, which is very risky and invasive to cancer patients. By means of the advances of CTCs sampling technique, we hypothesized that repeated follow-ups for cancer status with even specific gene profiles are becoming possible. However, the protocol has not been well-established and mature, even the correlation between primary cancer tissue and CTCs remains unknown. To tackle the clinical problems and validate the hypothesis, we focused on several directions (CTC isolation methodology, identification, culture and genetic detection) in my doctoral training period to help manage complex changes of cancer evolution.
After these training years, we have successfully involved high-purity isolation platform (optically di-electrophoresis, ODEP), revised negative selection protocol, flow cytometry(patent under application) and technology of 3-dimensional culture of cancer tissues and CTCs. Based on advances in these technologies, we go further to conduct 4 clinical trials to elucidate the roles of CTCs in oesophageal cancer, colorectal cancer, P16 expression status on CTCs, mRNA, DNA mutation detection from CTCs and preliminary reports of CTCs culture in head and neck cancer patients. In summary, the doctoral thesis consists of several fundamental technology developments with their clinical validation trials. Hopefully, we expect that these technologies become widely known and practically useful to help cancer patients decide optimal treatments.

指導教授推薦書
口試委員會審定書
誌謝 …………………………………………………………………. iii
中文摘要 ……………………………………………………………. iv
英文摘要 ……………………………………………………………. vi
縮寫表 ………………………………………………………………. viii
目錄 …………………………………………………………………. xii
表目錄………………………………………………………………. . xvii
圖目錄……………………………………………………………….. xviii

第一章 前言(Background) ................................................................. 1
1.1 The Most Serious Problem Worldwide: Cancer Death ............... 1
1.2 The Era of Circulating Tumor Cells Helping Clinical Diagnosis and Treatment................................................................................... 2
第二章 文獻探討(Literature Review)................................................. 4
2.1 Introduction of Circulating tumour cells .................................... 4
2.1.1 Brief History of CTC researches .......................................... 4
2.1.2 Natures of CTCs ................................................................... 5
2.1.3 CTCs in cancer progression ................................................. 9
2.1.4 The impact of CTCs on multidrug resistance ....................... 11
2.2 The strategies for CTC isolation and enumeration ..................... 13
2.2.1 Label-free isolation strategy ................................................. 13
2.2.2 Positive versus Negative Selection-based CTC isolation ..... 15
2.2.2.1 Positive selection methods ............................................. 15
2.2.2.2 Negative selection methods ........................................... 17
2.2.2.3 Other methodologies ...................................................... 19
2.2.3 Comparison of different strategies ......................................... 20
2.3 The main elements in negative selection plus microfluidic CTCs isolation ..................................................................................... 22
2.3.1 Immunomagnetic beads-based methods................................ 22
2.3.2 Preparation of the magnetic beads......................................... 23
2.3.3 Positive immunomagnetic separation technique................... 23
2.3.4 Negative immunomagnetic separation technique................. 24
2.3.5 Microfluidic-based methods for the high purity CTC isolation…………………………………………………….. 24
2.3.6 Perspective for future of CTC technology: Combinations of several methods ..................................................................... 25
2.4 The applications of CTC testing in clinical cancer researches.................................................................................. 27
2.4.1 Breast cancer ........................................................................ 27
2.4.2 Lung cancer .......................................................................... 29
2.4.3 Gastrointestinal tract cancer ................................................. 31
2.4.4 Head and neck cancer ........................................................... 33
2.4.5 Liver cancer .......................................................................... 34
2.4.6 Genitourinary tract cancer…………………………………. 35
2.4.7 Skin cancer and melanoma ................................................... 35
2.4.8 Other cancers ........................................................................ 36
2.5 Unanswered Questions in the field of CTCs, technically and clinically ................................................................................... 37
2.6 Concluding remarks for the introduction. ................................... 38
第三章 材料與方................................................................................. 39
3. 1 Method of Circulating Tumour Cell Counting .......................... 39
3.2 Sorting for Specific Group Analysis ........................................... 40
3.3 Using 3-D cell culture for further CTC isolation and purification………………………………………………..…. 41
3.4 Demonstration of using the two-step cell isolation process for CTC isolation: clinical sample tests………....................................... 43
3.5 Identification of P16INK4a status on CTCs from Oropharyngeal Cancer Patients. ........................................................................ 44
第四章 循環腫瘤細胞在食道癌的臨床意義 ................................... 48
4.1 Study Design for Circulating Tumour Cells in Esophageal Cancer Patients………………………………………………………... 48
4.2 Esophageal Cancer Patient Enrolment…………………………. 48
4.3 Chemotherapy Regimens of Chemoradiotherapy in Oesophageal Cancer…………………………......……........…...................... 49
4.4 Clinical Assessment of Chemoradiotherapy Effectiveness and Consolidation Therapy for Patients Who Did Not Undergo Oesophagectomy……………………………………………… 50
4.5 Statistical Analysis of CTC in Esophageal Cancer Patients…... 50
4.6 Results of Findings…………………………….......................... 51
4.6.1 Patient Enrolment…………………………......................... 51
4.6.2 The Impacts of Initial Staging and Surgery After Chemoradiotherapy ……………………............................... 52
4.6.3 Clinical Relevance of Circulating Tumour Cells Before Chemoradiotherapy……………............................................ 52
4.6.4 Summary and Conclusion of this study……………………. 53
第五章 循環腫瘤細胞在大腸癌的臨床意義..................................... 55
5.1 Colon Cancer Patient selection and treatment of systemic chemotherapy………………………….................................... 55
5.2 Statistical analysis………………................................................ 56
5.3 Results of Circulating Tumour Cells in Colorectal Cancer Patients……………………………………………………….. 57
5.4 Summary and Conclusion of the Present Study........................... 59
第六章 循環腫瘤細胞培養之進展 ................................................... 60
6.1 Quantification of erythrocytes, CD45pos leukocytes, and CD45neg PBMCs in blood samples of healthy donors and head-and-neck cancer patients after negative selection-based CTC isolation... 60
6.2 Use of 3-D cell culture for further CTC isolation and purification after negative selection-based CTC isolation process: feasibility evaluation based on cell line model........................................... 62
6.3 Demonstration of the two-step cell isolation process for CTC isolation: clinical tests............................................................... 66
6.4 Summary and Conclusion of the Current Study.......................... 68
第七章 人類乳突病毒或 P16 在循環腫瘤細胞的偵測進展 ……. 70
7.1 The establishment of P16 staining methods …………………… 70
7.1.1 Apply conventional methods (IHC) of p16 and HPV detection on CTCs ................................................................................. 70
7.1.2 Apply immunofluorescent staining of p16 expression on CTCs ...................................................................................... 71
7.1.3 Apply conventional methods (genotyping) of HPV detection on CTCs ...................................................................................... 71
7.2 CTC detection in clinical cancer patients with SqCC.................. 73
7.2.1 The Protocol Efficiency, Recovery Rate, and Purity of CTC isolation ................................................................................. 73
7.2.2 Patient Enrolment.................................................................. 73
7.2.3 The Impacts of Initial Staging and Surgery After Chemoradiotherapy................................................................ 73
7.2.4 Clinical Relevance of CTCs Before Chemoradiotherapy...... 74
7.2.5 Summary and Conclusion .................................................... 75
第八章 結論 (Conclusion) ................................................................. 76
參考文獻(References) ......................................................................... 77
表格 (Tables) ....................................................................................... 117
圖與註解(Figures and Legends) .......................................................... 136


 
表目錄


Table 1. Overview of analytical methodologies for the detection and molecular characterization of CTCs………………………… 117
Table 2. The coordinates of the curve and the area under the curve…. 121
Table 3. Basic Characteristics of Enrolled Patients………………….. 122
Table 4. Univariate and Multivariate Analysis for Survivals……….... 123
Table 5. Correlations Among CTC Number Category and First Responses to CCRT and Patterns of Failure……………………………. 125
Table 6. Colorectal Cancer Patients’ demographic data……………… 126
Table 7. Univariate analyses for overall survival (OS) and progression- free survival (PFS)………………………………………….. 128
Table 8. Point scoring of overall survival for the prognostic models… 130
Table 9. OECM-1-GFP cell purity analysis using flow cytometry….. 131
Table 10. Clinical test results………………………………………… 132
Table 11. Genotyping detection limit after negative selection……….. 133
Table 12. Basic Characteristics of Enrolled Patients…………………. 134
Table 13. Univariate and Multivariate Analysis for Survivals……….. 135


 
圖目錄


Figure 1. Illustration of circulating tumour cell detection method…... 136
Figure 2. The Design of the study of P16 expressing CTCs…………. 137
Figure 3. Tumour Staging, Treatment Response and Surgery Impacts on Survivals of Oesophageal Cancer patients………………… 138
Figure 4. Circulating Tumour Cell Counts and CTCR score Significantly Impacts on Survival……………………………………….. 139
Figure 5. Correlations among the number of circulating tumour cells (CTC), locations and chemotherapies and the survival impacts of the CTC plus response score (CTCR)………………….. 140
Figure 6. Multivariate analysis for overall survival and progression-free survival in Patients with Metastatic Colorectal Cancer….... 141
Figure 7. Circulating Tumour Cell-based Prognostic Score Predicts Progression-free Survival in Patients with Metastatic Colorectal Cancer……………………………………………………... 142
Figure 8. Circulating Tumour Cell-based Prognostic Score Predicts overall survival in Patients with Metastatic Colorectal Cancer.................................................................................... 143
Figure 9. Tumour response rate to chemotherapy, stratified according to the prognostic model............................................................. 144
Figure 10. Quantification of main cell populations in blood samples after negative selection-based CTC isolation…………………… 145
Figure 11. Feasibility evaluation of using a 3-D cell culture for further CTC purification………………………………………….. 147
Figure 12. Using two-step cell isolation process for CTC isolation from blood samples of cancer patients…………………………. 148
Figure 13. Demonstrated IHC cases of CK+ and p16+ CTCs………… 149
Figure 14. Representative imaging demonstrations by immunefluore- scence staining of EpCAM and p16………………………. 150
Figure 15. Flow controls and demonstrated CTC case of EpCAM and p16………………………………………………………… 151
Figure 16. The correlations among tumour staging, response, surgery, unresectable status and survival………………………..…. 152
Figure 17. The number of circulating tumour cells can differentiate oesophageal cancer patients and its impact on survival….... 153

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