臺灣博碩士論文加值系統

近年來，利用血中DNA當作研究癌症的偵測工具被視為可行的方向。這是因為健康的正常人血液中DNA會呈現小片段，且相較於癌症的病人血中DNA會具有多樣化片段的現象。我們採用75個已做過化學治療的頭頸部癌病人與34個健康人的血漿，使用及時定量PCR的方法以及β-actin基因作DNA integrity index(Dii)的研究。我們發現頭頸部癌病人的平均 Dii 為1.03，健康個體的平均Dii為0.38，兩者之間有顯著的差異 (p < 0.0001)。表示頭頸部癌病人的血漿DNA片段較健康個體的血漿DNA片段有多樣性，此多樣性來自於癌症細胞的necrosis。另外在接受者操作特性曲線下的面積為0.80，也證明了Dii可以明確的分辨頭頸部癌病人與健康個體。我們也探討了此Dii可能可以應用在臨床上面，因為在陽性結果相似比的最佳敏感度分析中，當Dii值大於0.76時，真正罹病者檢查結果為陽性之比例除以真正沒病者但檢查結果為陽性之比例為20 : 1。此種分析DNA片段多樣性的偵測方法具有相當的潛力，可能可以在未來當作偵測頭頸部癌症的工具。

Tumor-released DNA in blood represents a hopeful molecular biomarker for cancer detection. It has been postulated that necrosis of tumor cells causes DNA of varying sizes to be released, which is in contrast with apoptosis in normal tissue that releases uniform and shorter DNA fragments. Plasma of seventy - five head and neck cancer patients with paired pre- and post-chemotherapy and 43 healthy individuals were analyzed by using real time PCR to determine the DNA integrity index (Dii). We found that the mean DNA integrity index was significantly higher in the plasma from head and neck cancer patients, 1.04 (95% CI: 1.22, 0.83), when compared to plasma from the healthy individuals, 0.38 (95% CI: 0.47, 0.16), p < 0.0001 using t-test analysis. The area under the ROC curve for β-actin was 0.80. The optimal sensitivity (defined as the value for which sensitivity equals specificity) was found with DNA integrity index greater than 0.49: sensitivity, 68%; specificity, 76.7%. The optimal sensitivity of positive likelihood ratio was found with DNA integrity index higher than 0.76: sensitivity, 46.7%; specificity, 97.7%. The DNA integrity index might be a potential tool for detecting head and neck cancer.

Chapter 1 Introduction 1
1. Head and neck cancer 1
2. Symptoms of head and neck cancer 1
3. Epidemical situation of head and neck cancer in Taiwan 1
4. Risky factors for head and neck cancer 2
5. Squamous cell carcinoma of head and neck cancer 2
6. Treatment of head and neck cancer 2
7. Diagnosis of head and neck cancer 3
8. DNA integrity index assay 4
9. Real time PCR of β-actin 4
10. DNA released from apoptosis or cancer cell necrosis 5
11. β-actin 7
12. Hypothesis 8
Chapter 2 Materials and methods 9
1. Samples collection 9
2. Extraction of DNA from plasma samples 9
3. Extraction of DNA from blood samples 11
4. DNA strand integrity analysis 11
5. Statistical analysis 12
Chapter 3 Results 14
1. Patient characteristics 14
2. Real time PCR 15
3. Calculation method of real time PCR 16
4. DNA integrity index as a diagnostic tool in head and neck cancer 16
Chapter 4 Discussions 21
1. Diagnostic assays on blood analysis in recent years 21
2. The defect of DNA DipStick TM Kit method 21
3. Real time PCR method with hTERT gene 22
4. The defect of absolute DNA concentration 22
5. DNA integrity index of head and neck cancer patients 24
6. DNA integrity index of head and neck cancer patients after chemotherapy 25
7. DNA integrity index of Karnofsky scale 26
8. DNA integrity index of serum albumin level 27
9. DNA integrity index of SCC-antigen 27
10. DNA integrity index of regional lymph node staging analysis 28
11. DNA integrity index of primary tumor stage 28
12. DNA integrity index of primary disease free survival curve, neck disease free survival curve, metastasis disease free survival curve, and overall survival curve 28
13. ROC curve for β-actin 29
Chapter 5 References 30
Chapter 6 Figures 35
1. Fluorescent data for 100bp β-actin gene of plasma DNA 35
2. Fluorescent data for 400bp β-actin gene of plasma DNA 36
3. Fluorescent data for 100bp β-actin gene of blood cell DNA 37
4. Fluorescent data for 400bp β-actin gene of blood cell DNA 38
5. Electrophoresis of 100bps (a) and 400bps (b) products of β-actin gene 39
6. Plasma DNA integrity index of healthy individuals compared with head and neck cancer patients 40
7. ROC curve for analysis of DNA integrity index in detecting head and neck cancer patients in plasma 41
8. Response of head and neck cancer patients after chemotherapy 42
9. Mean plasma Dii of 71 male and 4 female 44
10. Dii of Karnofsky scale 45
11. Recurrence of head and neck cancer patients 46
12. Dii of serum albumin level 47
13. Dii of serum tumor marker (SCC-antigen) 48
14. Dii of regional lymph node stage 49
15. Dii of primary tumor stage 50
16. Survival curve of pre- chemotherapy Dii 51
17. Survival curve of pre- chemotherapy Dii subtracted from post- chemotherapy Dii 55
Chapter 7 Tables 59
1. Sample calculation with comparative Ct method 59
2. Primers of β-actin gene 60
3. Overall statistical comparison of head and neck cancer patients groups and controls 61
4. Characteristic of head and neck cancer patients 62

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