跳到主要內容

臺灣博碩士論文加值系統

(3.87.33.97) 您好!臺灣時間:2022/01/27 15:55
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:闕淳美
論文名稱:利用癌症病患血漿DNA微衛星變異偵測微轉移之評估
論文名稱(外文):The assessment of microsatellite alterations in the plasma DNA from cancer patients as biomarkers for the detection of micrometastasis
指導教授:簡一治
學位類別:碩士
校院名稱:國立彰化師範大學
系所名稱:生物學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:80
中文關鍵詞:血漿DNA微衛星標記對偶基因異結合型缺失微轉移癌症
外文關鍵詞:plasma DNAmicrosatellite DNA markersloss of heterozygosity(LOH)micrometastasiscancer
相關次數:
  • 被引用被引用:1
  • 點閱點閱:345
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
摘要
對於已接受治療的癌症病患而言,如何有效地在早期診斷出病患是否有癌細胞的轉移,一直是生物醫學研究中所重視的課題。而對偶基因異結合型的缺失(loss of heterozygosity, LOH)與微衛星不穩定性(microsatellite instability, MSI)均代表癌細胞內累積了許多遺傳變異而造成基因組不穩定的現象。本研究的目的是要在癌症病患的血漿DNA中,尋找和腫瘤組織DNA中LOH或MSI變異形式相符的微衛星標記(microsatellite markers)作為偵測癌細胞微轉移的生物標記,在本實驗中共使用18組微衛星標記分別針對肝癌、胃癌和乳癌病患取得其腫瘤組織、正常組織、血球或血漿DNA,利用螢光PCR-毛細管電泳的方法進行微衛星DNA的分析。而依據各個微衛星標記的異結合型比例、在腫瘤組織中發生LOH的比例及在血漿DNA中偵測到LOH的比例,分別篩選出在肝癌、胃癌和乳癌中的三種等級之微衛星標記。比較三種等級的標記在癌症病患腫瘤組織及血漿DNA中可偵測到LOH現象的比例,結果在乳癌中已找到第一等級的微衛星標記LPL、D16S413和TP53適合作為偵測乳癌癌細胞微轉移的生物標記,而在肝癌及胃癌中則需要再尋找更相關的微衛星標記。此外,比較乳癌病患的病理資料顯示在血漿DNA中能否偵測到LOH現象與腫瘤相關的病理資料均無顯著差異。而利用血漿進行LOH分析的偵測方法與臨床的淋巴腺病理切片方法相比較,發現利用血漿DNA進行微衛星分析的方法具有較高的偵測能力。所以,從本實驗結果中,提供了一個較無侵襲性又兼具專一性的檢驗方法,可應用於早期偵測癌細胞及作為癌症病患預後追蹤的參考依據。

Abstract
Early diagnosis of micrometastasis of cancer patients is always the major topic in cancer clinic. It is reasonable to assume that if we can detect cancer cells in peripheral blood of cancer patients, implying that they have high probability to have cancer metastasis. The phenomena of loss of heterozygosity (LOH) and microsatellite instability (MSI) in cancer cells are derived from the accumulation of genetic alterations. Both have been widely accepted as biomarkers of cancer cells. For the purpose of identifying the biomarkers for detecting micrometastasis of cancer cells, we designed the present study to determine the presence of tumor DNA in the plasma of patients with hepatocellular carcinomas (HCC), gastric cancer (GC), and breast cancer (BC), characterized by LOH in 18 microsatellite markers. DNA extracted from tumor tissues, neighboring normal tissues, blood cells and plasma of cancer patients was amplified with fluorescent primers specific for the microsatellite markers. PCR amplicons were separated on the ABI-3100 16 capillary array electrophoresis instrument and analyzed with GeneScan analysis software v.3.7. According to the ratios of heterozygosity, LOH in tumor and plasma DNA, microsatellite markers were classified as four grades. Markers with heterozygosity≧80 %, LOH in tumor DNA≧50 %, and LOH in plasma DNA≧30 % were classified as the first grade; those with heterozygosity≧80 %, LOH in tumor DNA≧50 %, and LOH in plasma DNA≦30 % were classified as the second grade; those with heterozygosity≧80 % and LOH in tumor DNA≦50 % were classified as the third grade; while those with heterozygosity≦80 % were classified as the fourth grade, which were considered as unsuitable markers. Three markers (LPL, D16S413 and TP53) were classified as the first grade markers in BC, which together detected 85.3 % (29/34) of tumor DNA with LOH, and 41.2 % (14/34) of plasma DNA with LOH. In HCC, TP53 were the only first grade marker, which detected 44.9 % (22/49) of tumor DNA with LOH, and 24.5 % (12/49) of plasma DNA. None was qualified as the first and second grade markers in GC. No association was found between the presence of LOH in plasma and clinicopathological parameters, such as tumor size, tumor stage, tumor grade, the expression of estrogen receptor, progesterone receptor, c-erbB-2 oncoprotein and p53. However, our method was significantly better than dissected lymph node histochemical method in detecting micrometastasis. As a conclusion, we have provided a specific and noninvasive method, which can be applied as a diagnostic technique for early diagnosis and prognostic follow-up tests for cancer.

目次
英文摘要………………………………………………………………VII
中文摘要………………………………………………………………IX
導論…………………………………………………………………… 1
材料與方法……………………………………………………………18
結果……………………………………………………………………23
討論……………………………………………………………………34
參考文獻………………………………………………………………48
圖………………………………………………………………………57
表………………………………………………………………………70

參考文獻
Anker, P., Mulcahy, H., Chen, X.Q., and Stroun, M. (1999) Detection of circulating tumour DNA in the blood (plasma/serum) of cancer patients. Cancer Metastasis Rev. 18: 65-73.
Anker, P., Lyautey, J., Lederrey, C., and Stroun, M. (2001) Circulating nucleic acids in plasma or serum. Clin. Chim. Acta 313: 143-146.
Atkin, N.B. (2001) Microsatellite instability. Cytogenet. Cell Genet. 92: 177-181.
Bennett, P. (2000) Demystified…Microsatellites. Mol. Pathol. 53: 177-183.
Berg, K.D., Glaser, C.L., Thompson, R.E., Hamilton, S.R., Griffin, C.A., and Eshleman, J.R. (2000) Detection of microsatellite instability by fluorescence multiplex polymerase chain reaction. J. Mol. Diag. 2: 20-28.
Bevilacqua, R.A.U., Nunes, D.N., Stroun, M., and Anker, P. (1998) The use of genetic instability as a clinical tool for cancer diagnosis. Seminars in Cancer Biology 8: 447-453.
Böckmann, B., Grill, H.J., and Giesing, M. (2001) Molecular characterization of minimal residual cancer cells in patients with solid tumors. Biomolecular Engineering 17: 95-111.
Boland, C.R., Thibodeau, S.N., Hamilton, S.R., Sidransky, D., Eshleman, J.R., Burt, R.W., Meltzer, S.J., Rodriguez-Bigas, M.A., Fodde, R., Ranzani, G.N., and Srivastava, S. (1998) A National Cancer Institute workshop on microsatellite instability for cancer detection and familial predisposition:development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res. 58: 5248-5257.
Canzian, F., Salovaara, R., Hemminki, A., Kristo, P., Chadwick, R.B., Aaltonen, L.A., and Chapelle A.D.L. (1996) Semiautomated assessment of loss of heterozygosity and replication error in tumors. Cancer Res. 56: 3331-3337.
Chen, X.Q., Stroun, M., Magnenat, J.L., Nicod, L.P., Kurt, A.M., Lyautey, J., Lederrey, C., and Anker, P. (1996) Microsatellite alterations in plasma DNA of small cell lung cancer patients. Nat. Med. 2: 1033-1035.
Chou Wu, Y.H., Chung, K.C., Jeng, L.B., Chen, T.C., and Liaw, Y.F. (1998) Frequent allelic loss on chromosomes 4q and 16q associated with human hepatocellular carcinoma in Taiwan. Cancer Lett. 123: 1-6.
Claij, N. and Riele, H.T. (1999) Microsatellite instability in human cancer:A prognostic marker for chemotherapy?Exp. Cell Res. 246: 1-10.
Coulet, F., Blons, H., Cabelguenne, A., Lecomte, T., Laccourreye, O., Brasnu, D., Beaune, P., Zucman, J., and Laurent-Puig, P. (2000) Detection of plasma tumor DNA in head and neck squamous cell carcinoma by microsatellite typing and p53 mutation analysis. Cancer Res. 60: 707-711.
Deng, G., Lu, Y., Zlotnikov, G., Thor, A.D., and Smith, H.S. (1996) Loss of heterozygosity in normal tissue adjacent to breast carcinomas. Science 274: 2057-2059.
Devilee, P., Cleton-Jansen, A.M., and Cornelisse, C.J. (2001) Ever since Knudson. Trends Genet. 17: 569-573.
Dietmaier, W., Riedlinger, W., Köhler, A., Wegele, P., Beyser, K., Sagner, G., Wartbichler, R., and Rüschoff, J. (1999) Detection of microsatellite instability (MSI) and loss of heterozygosity (LOH) in colorectal tumors by fluorescence-based multiplex microsatellite PCR. Roche Mol. Biochem. 2: 42-45.
Esteller, M., Sanchez-Cespedes, M., Rosell, R., Sidransky, D., Baylin, S.B., and Herman, J.G. (1999) Detection of aberrant promoter hypermethylation of tumor suppressor genes in serum DNA from non-small cell lung cancer patients. Cancer Res. 59: 67-70.
Försti, A., Louhelainen, J., Söderberg, M., Wijkström, H., and Hemminki, K. (2001) Loss of heterozygosity in tumour-adjacent normal tissue of breast and bladder cancer. Eur. J. Cancer 37: 1372-1380.
Fournié, G.J., Courtin, J.P., Laval, F., Chalé, J.J., Pourrat, J.P., Pujazon, M.C., Lauque, D., and Carles, P. (1995) Plasma DNA as a marker of cancerous cell death. Investigations in patients suffering from lung cancer and in nude mice bearing human tumours. Cancer Lett. 91: 221-227.
Fujiwara, Y., Chi, D.D.J., Wang, H.J., Keleman, P., Morton, D.L., Turner, R., and Hoon, D.S.B. (1999) Plasma DNA microsatellites as tumor-specific markers and indicators of tumor progression in melanoma patients. Cancer Res. 59: 1567-1571.
GDB(TM)Human Genome Database [database online]. Johns Hopkins University, Baltimore, Maryland, USA. 1990-, updated daily. Available from Internet: URL http://www.gdb.org/.
Goessl, C., Heicappell, R., Münker, R., Anker, P., Stroun, M., Krause, H., Müller, M., and Miller, K. (1998) Microsatellite analysis of plasma DNA from patients with clear cell renal carcinoma. Cancer Res. 58: 4728-4732.
Goessl, C. (2000) Laser-fluorescence microsatellite analysis and new results in microsatellite analysis of plasma/serum DNA of cancer patients. Ann. N. Y. Acad. Sci. 906: 63-66.
Grundei, T., Vogelsang, Holger, Ott, K., Mueller, J., Scholz, M., Becker, K., Fink, U., Siewert, J.R., Höfler, H., and Keller, G. (2000) Loss of heterozygosity and microsatellite instability as predictive markers for neoadjuvant treatment in gastric carcinoma. Clin. Cancer Res. 6: 4782-4788.
Harn, H.J., Fan, H.C., Chen, C.J., Tsai, N.M., Yen, C.Y., and Huang, S.C. (2002) Microsatellite alteration at chromosome 11 in primary human nasopharyngeal carcinoma in Taiwan. Oral Oncology 38: 23-29.
Hayden, J.D., Cawkwell, L., Quirke, P., Dixon, M.F., Goldstone, A.r., Sue-Ling, H., Johnston, D., and Martin, I.G. (1997) Prognostic significance of microsatellite instability in patients with gastric carcinoma. Eur. J. Cancer 33: 2342-2346.
Hibi, K., Robinson, C.R., Booker, S., Wu, L., Hamilton, S.R., Sidransky, D., and Jen, J. (1998) Molecular detection of genetic alterations in the serum of colorectal cancer patients. Cancer Res. 58: 1405-1407.
Jahr, S., Hentze, H., Englisch, S., Hardt, D., Fackelmayer, F.O., Hesch, R.D., and Knippers, R. (2001) DNA fragment in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells. Cancer Res. 61: 1659-1665.
Jiricny, J. (1998) Replication errors: cha(lle)nging the genome. EMBO J. 17: 6427-6436.
Kiaris, H., and Spandidos, D.A. (1996) Quantitation of the allelic imbalance provides evidence on tumour heterogeneity: a hypothesis. Mutat. Res. 354: 35-39.
Kim, H.S., Woo, D.K., Bae, S.I., Kim, Y.I., and Kim, W.H. (2001) Allelotype of the adenoma-carcinoma sequence of the stomach. Cancer Detect Prev. 25: 237-244.
Kinoshita, M., Nakamura, J., Kusaka, H., Hadama, T., Bago, K., Kitajima, M., and Baba, S. (1999) Automated and simultaneous identification of microsatellite instability by fluorescence-based polymerase chain reaction (PCR) in four loci. Clin. Chim. Acta 279: 15-23.
Ko, J.M.Y., Wong, C.P.S., Tang, C.M.C., Lau, K.W., and Lung, M.L. (2001) Frequent loss of heterozygosity on multiple chromosomes in Chinese esophageal squamous cell carcinomas. Cancer Lett. 170: 131-138.
Kobayashi, K., Okamoto, T., Takayama, S., Akiyama, M., Ohno, T., and Yamada, H. (2000) Genetic instability in intestinal metaplasia is a frequent event leading to well-differentiated early adenocarcinoma of the stomach. Eur. J. Cancer 36: 1113-1119.
Koyama, M., Nagai, H., Bando, K., Matsumoto, S., Tajiri, T., Onda, M., Ito, M., Moriyama, Y., and Emi, M. (2000) New target region of allelic loss in hepatocellular carcinomas within a 1-cM interval on chromosome 6q23. J. Hepatol. 33: 85-90.
Kurose, K., Hoshaw-Woodard, S., Adeyinka, A., Lemeshow, S., Watson, P.H., and Eng, C. (2001) Genetic model of multi-step breast carcinogenesis involving the epithelium and stroma: clues to tumour-microenvironment interactions. Hum. Mol. Genet. 10: 1907-1913.
Leon, S. A., Shapiro, B., Sklaroff, D.M., and Yaros, M.J. (1977) Free DNA in the serum of cancer patients and the effect of therapy. Cancer Res. 37: 646-650.
Li, S.P., Wang, H.Y., Li, J.Q., Zhang, C.Q., Feng, Q.S., Huang, P., Yu, X.J., Huang, L.X., Liang, Q.W., and Zeng, Y.X. (2001) Genome-wide analyses on loss of heterozygosity in hepatocellular carcinoma in Southern China. J. Hepatol. 34: 840-849.
Maehara, Y., Oda, S., and Sugimachi, K. (2001) The instability within:problems in current analyses of microsatellite instability. Mutat. Res. 461: 249-263.
Maxwell, G.L., Risinger, J.I., Alvarez, A.A., Barrett, J.C., and Berchuck, A. (2001) Favorable survival associated with microsatellite instability in endometrioid endometrial cancers. Obstet. Gynecol. 97: 417-422.
Medintz, I.L., Lee, C.C.R., Wong, W.W., Pirkola, K., Sidransky, D., and Mathies, R.A. (2000) Loss of heterozygosity assay for molecular detection of cancer using energy-transfer primers and capillary array electrophoresis. Genome Res. 10: 1211-1218.
Momoi, H., Itoh, T., Nozaki, Y., Arima, Y., Okabe, H., Satoh, S., Toda, Y., Sakai, E., Nakagawara, K., Flemming, P., Yamamoto, M., Shimahara, Y., Yamaoka, Y., and Fukumoto, M. (2001) Microsatellite instability and alternative genetic pathway in intrahepatic cholangiocarcinoma. J. Hepatol. 35: 235-244.
Nawroz, H., Koch, W., Anker, P., Stroun, M., and Sidransky, D (1996) Microsatellite alterations in serum DNA of head and neck cancer patients. Nat. Med. 2: 1035-1037.
Oda, S., Oki, E., Maehara, Y., and Sugimachi, K. (1997) Precise assessment of microsatellite instability using high resolution fluorescent microsatellite analysis. Nucleic Acid Res. 25: 3415-3420.
Park, Y.M., Choi, J.Y., Bae, S.H., Byun, B.H., Ahn, B.M., Kim, B.S., and Shin,D.Y. (2000) Microsatellite instability and mutations of E2F-4 in hepatocellular carcinoma from Korea. Hepatology Res. 17: 102-111.
Paulson, T.G., Galipeau, P.C., and Reid, B.J. (1999) Loss of heterozygosity analysis using whole genome amplification, cell sorting and fluorescence-based PCR. Genome Res. 9: 482-491.
Peltomäki, P. (2001a) DNA mismatch repair and cancer. Mutat. Res. 488: 77-85.
Peltomäki, P. (2001b) Deficient DNA mismatch repair:a common etiologic factor for colon cancer. Hum. Mol. Genet. 10: 735-740.
Pineau, P. and Buendia, M.A. (2000) Studies of genetic defects in hepatocellular carcinoma: recent outcomes and new challenges. J. Hepatol. 32: 152-156.
Ramal, L.M., Maleno, I., Cabrera, T., Collado, A., Ferron, A., Lopez-Nevot, M.A., and Garrido, F. (2000) Molecular strategies to define HLA haplotype loss in microdissected tumor cells. Hum. Immunol. 61: 1001-1012.
Schuelke, M. (2000) An economic method for the fluorescent labeling of PCR fragments. Nat. Biotechnol. 18: 233-234.
Shao, J.Y., Huang, X.M., Yu, X.J., Huang, L.X., Wu, Q.L., Xia, J.C., Wang, H.Y., Feng, Q.S., Ren, Z.F., Ernberg, I., Hu, L.F., and Zeng, Y.X. (2001) Loss of heterozygosity and its correlation with clinical outcome and Epstein-Barr virus infection in nasopharyngeal carcinoma. Anticancer Res. 21: 3021-3030.
Shaw, J.A., Smith, B.M., Walsh, T., Johnson, S., Primrose, L., Slade, M.J., Walker, R.A., and Coombes, R.C. (2000) Microsatellite alteration in plasma DNA of primary breast cancer patients. Clin. Cancer Res. 6: 1119-1124.
Sidransky, D. (1997) Nucleic acid-based methods for the detection of cancer. Science 278: 1054-1058.
Sieben, N.L.G., Haar, N.T., Cornelisse, C.J., Fleuren, G.J., and Cleton-Jansen, A.M. (2000) PCR artifacts in LOH and MSI analysis of microdissected tumor cells. Human Pathol. 31: 1414-1419.
Silva, J.M., Gonzalez, R., Dominguez, G., Garcia, J.M., España, P., and Bonilla, F. (1999a) TP53 gene mutation in plasma DNA of cancer patients. Genes Chromosomes Cancer 24: 160-161.
Silva, J.M., Dominguez, G., Garcia, J.M., Gonzalez, R.,Villanueva, M.J., Navarro, F., Martin, S.S., España, P., and Bonilla, F. (1999b) Presence of tumor DNA in plasma of breast cancer patients: clinicopathological correlations. Cancer Res. 59: 3251-3256.
Skotheim, R.I., Diep, C.B., Kraggerud, S.M., Jakobsen, K.S., and Lothe, R.A. (2001) Evaluation of loss of heterozygosity/allelic imbalance scoring in tumor DNA. Cancer Genet. Cytogenet. 127: 64-70.
Sozzi, G., Musso, K., Ratcliffe, C., Goldstraw, P., Pierotti, M.A., and Pastorino, U. (1999) Detection of microsatellite alterations in plasma DNA of non-small cell lung cancer patients: A prospect for early diagnosis. Clin. Cancer Res. 5: 2689-2692.
Srinivas, P.R., Kramer, B.S., and Srivastava, S. (2001) Trends in biomarker research for cancer detection. Lancet Oncol. 2: 698-704.
Thiagalingam, S., Foy, R.L., Cheng, K.H., Lee, H.J., Thiagalingam, A., and Ponte, J.F. (2002) Loss of heterozygosity as a predictor to map tumor suppressor genes in cancer: molecular basis of its occurrence. Curr. Opin. Oncol. 14: 65-72.
Toh, Y., Oki, E., Oda, S., Tomoda, M., Tomisaki, S., Ichiyoshi, Y., Ohno, S., and Sugimachi, K. (1996) An integrated microsatellite length analysis using an automated fluorescent DNA sequencer. Cancer Res. 56: 2688-2691.
Wang, Y., Hung, S.C., Linn, J.F., Steiner, G., Glazer, A.N., Sidransky, D., and Mathies, R.A. (1997) Microsatellite-based cancer detection using capillary array electrophoresis and energy-transfer fluorescent primers. Electrophoresis 18: 1742-1749.
Wijnhoven, S.W.P., Kool, H.J.M., Teijlingen, C.M.M.V., Zeeland, A.A.V., and Vrieling, H. (2001) Loss of heterozygosity in somatic cells of the mouse:An important step in cancer initiation? Mutat. Res. 473: 23-36.
Zauber, N.P., Sabbath-Solitare, M., Marotta, S.P., Mcmahon, L., and Bishop, D.T. (1999) Comparison of allelic ratios from paired blood and paraffin-embedded normal tissue for use in a polymerase chain reaction to assess loss of heterozygosity. Mol. Diagn. 4: 29-35.
Zenklusen, J.C., Bièche, I., Lidereau, R., and Conti, C.J. (1994) (C-A)n microsatellite repeat D7S522 is the most commonly deleted region in human primary breast cancer. Proc. Natl. Acad. Sci. USA 91: 12155-12158.
Zheng, Y.L., Herr, A.M., Jacobson, B.A., and Ferrin, L.J. (2001) High-density allelotype of the commonly studied gastric cancer cell lines. Genes Chromosomes Cancer 32: 67-81.

QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top