臺灣博碩士論文加值系統

人類上皮生長因子接受器2 (HER2) 為一個跨膜醣蛋白，已被證實與人類的乳癌有關係。20-30% 之乳癌患者有HER2過度表現的現象，且HER2基因已被發現有exon 16缺失和codon 655多型性，而codon 655之胺基酸可為valine 或isoleucine。此外乳癌患者的HER2過度表現也代表不好的預後。犬HER2基因位於染色體1q13.1。前人的研究已經發現犬的乳腺腫瘤有HER2基因增幅和HER2蛋白過度表現，但是目前還未有研究同時分析犬惡性乳腺腫瘤HER2蛋白的過度表現和手術後存活的關係，也沒有完整HER2核酸序列分析之研究。本實驗目的為偵測犬惡性乳腺腫瘤之HER2蛋白表現和分析犬HER2之核苷酸序列。

本實驗收集91個犬惡性乳腺腫瘤組織，使用免疫組織化學法偵測HER2蛋白表現。如同人類的結果犬惡性乳腺腫瘤之HER2蛋白表現為29.7% (27/91)。經過2年術後的存活分析發現HER2陽性的患犬存活時間較HER2陰性的患犬長。接著我們挑選部分樣本進行HER2 exon14-18和全長cDNA的序列分析。所有的序列皆分別地與正常犬之HER2 genomic DNA (GenBank NC006591) 和complementary DNA (GenBank AB008451) 比對。結果發現犬惡性乳腺腫瘤的HER2基因序列不像人一樣有exon 16缺失和codon 655多型性的現象，只有核苷酸1725有單一核苷酸多型性的現象但是胺基酸並沒有改變皆為cysteine。因此，我們推測HER2蛋白的過度表現在犬惡性乳腺腫瘤的發育起始點扮演一個重要的角色，但是還不足以可以當成一個獨立的預後因子。

Human epidermal growth factor receptor 2 (HER2), a transmembrane glycoprotein receptor, has been shown to be involved in carcinogenesis of human breast cancer and is associated with poor prognosis. It has been shown that HER2 overexpression contributes to 20-30% of human breast carcinoma. HER2 lacking exon 16 is represented in some cases of human breast carcinoma, while a polymorphism at codon 655 (GTC/valine or ATC /isoleucine) has been identified in other cases. The canine HER2 gene has been mapped to chromosome 1q13.1. Previous studies have shown that either HER2 gene amplification or HER2 protein overexpression are present in canine mammary tumors, but none has addressed the simultaneous analysis of HER2 protein expression and the relationship between post-surgery survival time in canine malignant mammary tumors. Besides, there are no studies exploring the contribution of the HER2 gene to canine mammary tumors. The aims of this study are to exam HER2 protein expression and analyze the HER2 nucleotide acid sequence in canine malignant mammary tumors.

In this study, tissue specimens collected from a series of 91 dogs with malignant mammary tumors were analyzed for expression of HER2 by immunohistochemistry. HER2 protein overexpression was detected in 29.7% (27/91) of the canine malignant mammary tumors. After 2 years survival analysis, we found that HER2 positive dogs with malignant mammary tumors tended to have longer survival time than those that did not overexpress HER2. Subsequently, samples were chosen for sequence analysis of the cDNA region from exon14 to 18 and whole cDNA in HER2 gene. All samples were compared with normal HER2 gDNA (GenBank NC006591) and with cDNA (GenBank AB008451), respectively. Neither exon 16 deletion nor codon 655 polymorphism was found in the HER2 gene of canine malignant mammary tumors. However, 3 out of 19 (15.8 %) cases had single nucleotide polymorphism (SNP) in HER2 gene, T1725C, but the amino acid was not changed (Cys-Cys). Hence, our results suggested that overexpression of HER2 may play a role in the development of canine malignant mammary tumors, but it is not adequate for serving as an independent prognostic factor.

表次 iv
圖次 v
中文摘要 vi
英文摘要 vii
第一章 文獻探討 1
第一節 犬乳腺腫瘤之探討 1
一、犬乳腺腫瘤之流行病學 1
二、犬乳腺腫瘤之病因學 2
三、犬乳腺腫瘤之臨床表現 5
四、犬乳腺腫瘤之組織病理學分類 6
五、犬乳腺腫瘤之臨床分期 10
六、犬乳腺腫瘤之外科治療方式 11
七、犬乳腺腫瘤之預後因子 13
第二節 HER2 在犬乳腺腫瘤之探討 14
一、HER2之介紹 14
二、HER2蛋白之結構及作用機制 15
三、HER2在腫瘤之表現 16
四、HER2在乳腺腫瘤之表現 17
五、HER2基因突變於乳腺腫瘤 18
六、HER2過度表現之化療藥物 19
第三節 研究目的 21
第二章 材料與方法 22
第一節 犬正常乳腺與乳腺腫瘤之採樣與處理 22
一、樣本來源 22
二、犬正常乳腺與乳腺腫瘤的處理 22
三、組織切片之製作與判讀 23
四、患犬之術後追蹤 24
第二節 利用免疫組織化學法偵測HER2蛋白 24
一、染色前的準備 24
二、組織之脫臘與復水 24
三、HER2蛋白之免疫組織化學染色 25
四、HER2蛋白過度表現之評估標準 26
第三節 統計學分析 27
第四節 犬HER2基因exon 14-18之序列分析 28
一、實驗樣本 28
二、Total RNA 的萃取 28
三、偵測Total RNA 的濃度 28
四、純化Total RNA 29
五、逆轉錄聚合酶連鎖反應 29
六、聚合酶連鎖反應 30
七、DNA之純化 31
第五節 犬HER2 CDNA之全長序列分析 32
一、實驗樣本 32
二、逆轉錄聚合酶連鎖反應 33
三、聚合酶連鎖反應 33
第三章 結果 35
第一節 犬惡性乳腺腫瘤HER2蛋白之免疫組織化學染色分析 35
一、樣本資料統計 35
二、HER2蛋白免疫組織化學染色之結果 37
三、HER2蛋白過度表現之存活分析 41
第二節 犬HER2 exon 14-18基因序列分析 45
一、樣本資料 45
二、利用光電比色計偵測RNA濃度 45
三、分析犬HER2 exon14-18基因序列 45
第三節 犬HER2 CDNA之序列分析 47
一、樣本資料 47
二、利用光電比色計偵測RNA濃度 47
三、分析犬HER2 全長基因序列 47
第四章 討論 49
第一節 犬乳腺腫瘤HER2蛋白表現之探討 49
第二節 犬乳腺腫瘤HER2蛋白表現與術後存活時間之探討 50
第三節 犬HER2 核苷酸序列分析之探討 52
第四節 結論 54
參考文獻 55
附錄 62

1. Harvey HJ. Mammary glands. In: Gay S, ed. Current techniques in small animal surgery. 4th ed. Williams & Wilkins Co, Pennsylvania, 579-584, 1998.
2.Misdorp W. Tumors of the mammary gland. In: Meuten DJ, ed. Tumor in domestic animals. 4th ed. Blackwell Publishing Co, Iowa State Press, 575-606, 2002.
3.Rutteman GR, Withrow SJ, MacEwen EG. Tumors of the mammary gland. In : Withrow SJ, MacEwen EG, eds. Small animal clinical oncology. 3rd ed. WB Saunders Co, Philadelphia, 455-477, 2001.
4.Almasri NM, Al Hamad M. Immunohistochemical evaluation of human epidermal growth factor receptor 2 and estrogen and progesterone receptors in breast carcinoma in Jordan. Breast Cancer Res 7: 598-604, 2005.
5.Ahern TE, Bird RC, Bird AE, Wolfe LG.. Expression of the oncogene c-erbB-2 in canine mammary cancers and tumor-derived cell lines. Am J Vet Res 57: 693-696, 1996.
6.Allred DC, Clark GM, Elledge R, Fuqua SA, Brown RW, Chamness GC, Osborne CK, McGuire WL. Association of p53 protein expression with tumor cell proliferation rate and clinical outcome in node-negative breast cancer. J Natl Cancer Inst 85:200-206, 1993.
7.Bozionellou V, Mavroudis D, Perraki M, et al. Trastuzumab administration can effectively target chemotherapy-resistant cytokeratin-19 messenger RNA-positive tumor cells in the peripheral blood and bone marrow of patients with breast cancer. Clin Cancer Res 10: 8185-8194, 2004.
8.Borresen-Dale AL. TP53 and breast cancer. Hum Mutat 21:292–300, 2003.
9.Benjamin SA, Lee AC, Saunders WJ. Classification and behavior of canine mammary epithelial neoplasms based on life-span observations in beagles. Vet Pathol 36:423-36, 1999.
10.Bostock DE, Moriarty J, Crocker J. Correlation between histologic diagnosis mean nucleolar organizer region count and prognosis in canine mammary tumors. Vet Pathol 29:381-385, 1992.
11.Bargmann CI, Weinberg RA. Oncogenic activation of the neu-encoded receptor protein by point mutation and deletion. EMBO J 7:2043-2052, 1988.
12.Bargmann CI, Hung MC, Weinberg RA. Multiple independent activations of the neu oncogene by a point mutation altering the transmembrane domain of p185. Cell 45:649-657, 1986.
13.Chang SC, Chang CC, Chang TJ, Wong ML. Prognostic factors associated with survival two years after surgery in dogs with malignant mammary tumors: 79 cases (1998-2002). J Am Vet Med Assoc 227:1625-1629, 2005.
14.Cohen P, Clemmons DR, Rosenfeld RG. Does the GH-IGF axis play a role in cancer pathogenesis? Growth Horm IGF Res 10:297-305, 2000.
15.Carraway KL 3rd, Cantley LC. A neu acquaintance for erbB3 and erbB4: a role for receptor heterodimerization in growth signaling. Cell 78:5-8, 1994.
16.De Las Mulas JM, Millan Y, Dios R. A prospective analysis of immunohistochemically determined estrogen receptor alpha and progesterone receptor expression and host and tumor factors as predictors of disease-free period in mammary tumors of the dog. Vet Pathol 42:200-212, 2005.
17.Dorn CR, Taylor DO, Schneider R, Hibbard HH, Klauber MR. Survey of animal neoplasms in Alameda and Contra Costa Counties, California. II. Cancer morbidity in dogs and cats from Alameda County. J Natl Cancer Inst 40:307-318, 1968.
18.Earp HS, Dawson TL, Li X, Yu H. Heterodimerization and functional interaction between EGF receptor family members: a new signaling paradigm with implications for breast cancer research. Breast Cancer Res Treat 35:115-132, 1995.
19.El Etreby MF, Muller-Peddinghaus R, Bhargava AS, El Bab MR, Graf KJ, Trautwein G. The role of the pituitary gland in spontaneous canine mammary tumorigenesis. Vet Pathol 17:2-16, 1980.
20.Flint AF, U''Ren L, Legare ME, Withrow SJ, Dernell W, Hanneman WH. Overexpression of the erbB-2 proto-oncogene in canine osteosarcoma cell lines and tumors. Vet Pathol 41:291-296, 2004.
21.Ford D, Easton DF, Stratton M, Narod S, Goldgar D, Devilee P, Bishop DT, Weber B, Lenoir G, Chang-Claude J, Sobol H, Teare MD, Struewing J, Arason A, Scherneck S, Peto J, Rebbeck TR, Tonin P, Neuhausen S, Barkardottir R, Eyfjord J, Lynch H, Ponder BA, Gayther SA, Zelada-Hedman M, et al. Genetic heterogeneity and penetrance analysis of the BRCA1 and BRCA2 genes in breast cancer families. The Breast Cancer Linkage Consortium. Am J Hum Genet 62:676-689, 1998.
22.Gouvea AP, Milanezi F, Olson SJ, Leitao D, Schmitt FC, Gobbi H. Selecting antibodies to detect HER2 overexpression by immunohistochemistry in invasive mammary carcinomas. Appl Immunohistochem Mol Morphol 14:103-108, 2006.
23.Graham JC, O''Keefe DA, Gelberg HB. Immunohistochemical assay for detecting estrogen receptors in canine mammary tumors. Am J Vet Res 60:627-630, 1999.
24.Griffey SM, Verstraete FJ, Kraegel SA, Lucroy MD, Madewell BR. Computer-assisted image analysis of intratumoral vessel density in mammary tumors from dogs. Am J Vet Res 59:1238-1242, 1998.
25.Gilbertson SR, Kurzman ID, Zachrau RE, Hurvitz AI, Black MM. Canine mammary epithelial neoplasms: biologic implications of morphologic characteristics assessed in 232 dogs.Vet Pathol 20:127-142, 1983.
26.Hadsell DL, Bonnette SG. IGF and insulin action in the mammary gland: lessons from transgenic and knockout models. J Mammary Gland Biol Neoplasia 5:19-30, 2000.
27.Hulka BS, Liu ET, Lininger RA. Steroid hormones and risk of breast cancer. Cancer 74:1111-1124, 1994.
28.Hellmen E, Bergstrom R, Holmberg L, Spangberg IB, Hansson K, Lindgren A. Prognostic factors in canine mammary tumors: a multivariate study of 202 consecutive cases. Vet Pathol 30:20-27, 1993.
29.Henderson BE, Ross R, Bernstein L. Estrogens as a cause of human cancer: the Richard and Hinda Rosenthal Foundation award lecture. Cancer Res 48:246-253, 1988.
30.Komar AA. Genetics. SNPs, silent but not invisible. Science 315:466-467, 2007.
31.Kimchi-Sarfaty C, Oh JM, Kim IW, Sauna ZE, Calcagno AM, Ambudkar SV, Gottesman MM. A "silent" polymorphism in the MDR1 gene changes substrate specificity. Science 315:525-528, 2007.
32.Kiessling R, Wei WZ, Herrmann F, Lindencrona JA, Choudhury A, Kono K, Seliger B. Cellular immunity to the Her-2/neu protooncogene. Adv Cancer Res 85:101-144, 2002.
33.Kwong KY, Hung MC. A novel splice variant of HER2 with increased transformation activity. Mol Carcinog 23:62-68, 1998.
34.Key TJ, Pike MC. The role of oestrogens and progestagens in the epidemiology and prevention of breast cancer. Eur J Cancer Clin Oncol 24:29-43, 1988.
35.Martin de las Mulas J, Millan Y, Dios R. A prospective analysis of immunohistochemically determined estrogen receptor alpha and progesterone receptor expression and host and tumor factors as predictors of disease-free period in mammary tumors of the dog. Vet Pathol 42:200-212, 2005.
36.Martin de las Mulas J, Ordas J, Millan MY, Chacon F, De Lara M, Espinosa de los Monteros A, Reymundo C, Jover A. Immunohistochemical expression of estrogen receptor beta in normal and tumoral canine mammary glands. Vet Pathol 41:269-272, 2004.
37.Martin de las Mulas J, Ordas J, Millan Y, Fernandez-Soria V, Ramon y Cajal S. Oncogene HER-2 in canine mammary gland carcinomas: an immunohistochemical and chromogenic in situ hybridization study. Breast Cancer Res Treat 80: 363-367, 2003.
38.Matsuyama S, Nakamura M, Yonezawa K, Shimada T, Ohashi F, Takamori Y, Kubo K. Expression patterns of the erbB subfamily mRNA in canine benign and malignant mammary tumors. J Vet Med Sci 63:949-954, 2001.
39.Moe L. Population-based incidence of mammary tumours in some dog breeds. J Reprod Fertil Suppl 57:439-443, 2001.
40.Murua Escobar H, Becker K, Bullerdiek J, Nolte I. The canine ERBB2 gene maps to a chromosome region frequently affected by aberrations in tumors of the dog (Canis familiaris). Cytogenet Cell Genet 94: 194-195, 2001.
41.Moe KE, Prinz PN, Larsen LH, Vitiello MV, Reed SO, Merriam GR. Growth hormone in postmenopausal women after long-term oral estrogen replacement therapy. J Gerontol A Biol Sci Med Sci 53:117-124, 1998.
42.Misdorp W, Hart AA. Prognostic factors in canine mammary cancer. J Natl Cancer Inst. 56:779-786, 1976.
43.Nakopoulou L, Lazaris AC, Panayotopoulou EG, Giannopoulou I, Givalos N, Markaki S, Keramopoulos A. The favourable prognostic value of oestrogen receptor beta immunohistochemical expression in breast cancer. J Clin Pathol 57:523-528, 2004.
44.Nieto A, Pena L, Perez-Alenza MD, Sanchez MA, Flores JM, Castano M. Immunohistologic detection of estrogen receptor alpha in canine mammary tumors: clinical and pathologic associations and prognostic significance. Vet Pathol 37:239-247, 2000.
45.Nagai MA, Marques LA, Torloni H, Brentani MM. Genetic alterations in c-erbB-2 protooncogene as prognostic markers in human primary breast tumors. Oncology 50:412-417, 1993.
46.Osoba D, Slamon DJ, Burchmore M, Murphy M. Effects on quality of life of combined trastuzumab and chemotherapy in women with metastatic breast cancer. J Clin Oncol 20:3106-3113, 2002.
47.Ogilvie GK, Moore AS. Managing veterinary cancer pstient: a practice manual. Trenton N J, 1995.
48.Piccart-Gebhart MJ, Procter M, Leyland-Jones B, et al. Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer. N Engl J Med 353: 1659-1672, 2005.
49.Palmieri C, Lam EW, Mansi J, MacDonald C, Shousha S, Madden P, Omoto Y, Sunters A, Warner M, Gustafsson JA, Coombes RC. The expression of ER betacx in human breast cancer and the relationship to endocrine therapy and survival. Clin Cancer Res 10:2421-2428, 2004.
50.Palmieri C, Cheng GJ, Saji S, Zelada-Hedman M, Warri A, Weihua Z, Van Noorden S, Wahlstrom T, Coombes RC, Warner M, Gustafsson JA. Estrogen receptor beta in breast cancer. Endocr Relat Cancer 9:1-13, 2002.
51.Perez Alenza D, Rutteman GR, Pena L, Beynen AC, Cuesta P. Relation between habitual diet and canine mammary tumors in a case-control study. J Vet Intern Med 12:132-139, 1998.
52.Papewalis J, Nikitin AYu, Rajewsky MF. G to A polymorphism at amino acid codon 655 of the human erbB-2/HER2 gene. Nucleic Acids Res 19:5452, 1991.
53.Romond EH, Perez EA, Bryant J, et al. Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer. N Engl J Med 353: 1673-1684, 2005.
54.Ross JS, Fletcher JA, Linette GP, Stec J, Clark E, Ayers M, Symmans WF, Pusztai L, Bloom KJ. The Her-2/neu gene and protein in breast cancer 2003: biomarker and target of therapy. Oncologist 8:307-325, 2003.
55.Roh H, Pippin J, Drebin JA. Down-regulation of HER2/neu expression induces apoptosis in human cancer cells that overexpress HER2/neu. Cancer Research 60: 560–565, 2000.
56.Rungsipipat A, Tateyama S, Yamaguchi R, Uchida K, Miyoshi N, Hayashi T. Immunohistochemical analysis of c-yes and c-erbB-2 oncogene products and p53 tumor suppressor protein in canine mammary tumors. J Vet Med Sci 61: 27-32, 1999.
57.Revillion F, Bonneterre J, Peyrat JP. ERBB2 oncogene in human breast cancer and its clinical significance. Eur J Cancer 34:791-808, 1998.
58.Rutteman GR. Contraceptive steroids and the mammary gland: is there a hazard? Breast Cancer Res Treat 23:29-41, 1992.
59.Rutteman GR, Misdorp W, Van den Brom WE, Rijnberk A. Anterior pituitary function in female dogs with spontaneous mammary tumors: I. Growth hormone. Anticancer Res 9:235-239, 1989.
60.Rutteman GR, Bevers MM, Misdorp W, Van den Brom WE. Anterior pituitary function in female dogs with mammary tumors: II. Prolactin. Anticancer Res 9:241-245, 1989.
61.Sato O, Wada T, Kawai A, Yamaguchi U, Makimoto A, Kokai Y, Yamashita T, Chuman H, Beppu Y, Tani Y, Hasegawa T. Expression of epidermal growth factor receptor, ERBB2 and KIT in adult soft tissue sarcomas: a clinicopathologic study of 281 cases. Cancer 103:1881-1890, 2005.
62.Sunpaweravong S, Sunpaweravong P. Recent developments in critical genes in the molecular biology of breast cancer. Asian J Surg 28:71-75, 2005.
63.Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A, Fleming T, Eiermann W, Wolter J, Pegram M, Baselga J, Norton L. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 344:783-792, 2001.
64.Stoll BA. Adiposity as a risk determinant for postmenopausal breast cancer. Int J Obes Relat Metab Disord 24:527-533, 2000.
65.Selman PJ, Mol JA, Rutteman GR, van Garderen E, Rijnberk A. Progestin-induced growth hormone excess in the dog originates in the mammary gland. Endocrinology 134:287-292, 1994.
66.Siegel PM, Dankort DL, Hardy WR, Muller WJ. Novel activating mutations in the neu proto-oncogene involved in induction of mammary tumors. Mol Cell Biol 14:7068-77, 1994.
67.Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 235:177-182, 1987.
68.Schechter AL, Stern DF, Vaidyanathan L, Decker SJ, Drebin JA, Greene MI, Weinberg RA. The neu oncogene: an erb-B-related gene encoding a 185,000-Mr tumour antigen. Nature 312:513-516, 1984.
69.Schneider R, Dorn CR, Taylor DO. Factors influencing canine mammary cancer development and postsurgical survival. J Natl Cancer Inst 43:1249-1261, 1969.
70.Venkitaraman AR. Cancer susceptibility and the functions of BRCA1 and BRCA2. Cell 108:171-182, 2002.
71.Vogel CL, Cobleigh MA, Tripathy D, et al. Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2 overexpressing metastatic breast cancer. J Clin Oncol 20: 719-726, 2002.
72.Van Leeuwen FE. Epidemiologic aspects of exogenous progestagens in relation to their role in pathogenesis of human breast cancer. Acta Endocrinol (Copenh) 1:13-26, 1991.
73.Weiner DB, Liu J, Cohen JA, Williams WV, Greene MI. A point mutation in the neu oncogene mimics ligand induction of receptor aggregation. Nature 339:230-231, 1989.
74.Xie D, Shu XO, Deng Z, Wen WQ, Creek KE, Dai Q, Gao YT, Jin F, Zheng W. Population- based, case-control study of HER2 genetic polymorphism and breast cancer risk. J Natl Cancer Inst 92:412-417, 2000.
75.Yamashita H, Yando Y, Nishio M, Zhang Z, Hamaguchi M, Mita K, Kobayashi S, Fujii Y, Iwase H. Immunohistochemical evaluation of hormone receptor status for predicting response to endocrine therapy in metastatic breast cancer. Breast Cancer 13:74-83, 2006.
76.Yoshikawa Y, Morimatsu M, Ochiai K, Nagano M, Yamane Y, Tomizawa N, Sasaki N, Hashizume K. Analysis of genetic variations in the exon 27 region of the canine BRCA2 locus. J Vet Med Sci 67:1013-1017, 2005.
77.Yamagami T, Kobayashi T, Takahashi K, Sugiyama M. Prognosis for canine malignant mammary tumors based on TNM and histologic classification. J Vet Med Sci 58: 1079-1083, 1996.