乳癌為世界上女性最常見的惡性腫瘤；其在亞洲的發生率不僅為各大癌症中的第一名，發生的比例更是逐年攀升，包括台灣。賀爾蒙受體陽性除了作為管腔型乳癌分子分型的指標外，更是決定是否使用內分泌輔助型療法的關鍵，但是此類療法的困境是，在持續給藥的5至10年後，大多數病人都會有抗藥性，所以找到新的生物標記去界定乳癌的進程或是找尋新的治療標的是非常急需的。高度表現兩種代謝標記與乳癌的惡性息息相關。在王雯靜教授實驗室先前的回顧性研究中，發現此二種代謝標記與較短的無疾病存活期（DFS）的相關性，並且許多文獻指出淋巴轉移與DFS有關，但是他們在台灣管腔型乳癌的臨床相關性卻尚未明瞭。因此，我們與林口長庚醫院合作，取得27個管腔型乳癌病人，以一個前導實驗去探討它們彼此的關聯。我們看到相較於原位癌細胞，其中一種代謝標記在轉移至淋巴的癌細胞，其細胞核中有較顯著的表現，並且淋巴轉移對淋巴的巨噬細胞聚集與M2極化有相當的影響，最後我們進一步探討代謝標記與巨噬細胞M2極化的相關性；這些結果說明了此兩種重要的代謝標記成為這類病人治療途徑或生物標記的潛力。

Breast cancer (BC) is the most prevalent malignancy in women worldwide. Incidence of BC is not only the highest but also on rising in East Asia including Taiwan. Hormone-receptor-positive (HR+) is the signature for luminal type BC as well as a marker for endocrine adjuvant therapy. Despite an effective treatment, a significant proportion of patients acquire resistance and suffer from a relapse and the recurrence rate increased at a constant rate for more than 5-10 years. It is thus important to find new prognostic biomarkers. High expressions of two important metabolic markers were associated with BC malignancy. Our lab has previously found that these metabolic markers serve as an independent prognostic predictor for luminal BC in a retrospective study. However, their role in lymph node metastasis remains unclear. In this investigation, we aim to characterize the correlation of these markers with M2-marcrophage infiltration in primary breast tumors and in metastatic lymph nodes and to evaluate the relationship with clinicopathologic parameters. In total, 27 patients (lymph node positive, n = 14; lymph node negative, n = 13) collected at Chang-Gung Memorial Hospital were enrolled. A higher degree of nucleus metabolic marker expression was found in the lymph node metastatic site than in the primary site. Significant differences in M2-marker expression were found between the lymph node-positive and –negative groups at the sites of metastatic lymph nodes. The relationship of two vital metabolic markers on macrophage M2 polarization and lymph node metastasis is discussed.

中文摘要----------------------------------------------------------i
Abstract--------------------------------------------------------ii
誌謝------------------------------------------------------------iii
CONTENTS--------------------------------------------------------iv
List of figures-------------------------------------------------vi
List of tables-------------------------------------------------vii
Abbreviation--------------------------------------------------viii
CHAPTER 1 Introduction-------------------------------------------1
1.1 Breast cancer------------------------------------------------1
1.1.1 Epidemiology of Breast cancer------------------------------1
1.1.2 Molecular classification-----------------------------------2
1.1.3 Treatment guideline for luminal type breast cancer---------3
1.2 O-GlcNAc and its role in Breast cancer-----------------------4
1.3 Pyruvate kinase----------------------------------------------5
1.3.1 Pyruvate kinase family- M1&M2------------------------------5
1.3.2 Two states of pyruvate kinase M2---------------------------6
1.4 Macrophage---------------------------------------------------7
1.4.1 M1 & M2 macrophages----------------------------------------7
1.4.2 Tumor-associated macrophages in tumor-microenvironment-----8
1.5 The purpose of this research---------------------------------9
CHAPTER 2 Material and method-----------------------------------10
2.1 Antibodies--------------------------------------------------10
2.2 Study subjects----------------------------------------------10
2.3 Immunohistochemistry (IHC) staining-------------------------11
2.4 IHC scoring and macrophage counting method------------------11
2.5 Statistical Analysis----------------------------------------12
CHAPTER 3 Results-----------------------------------------------13
3.1 Characterization of the infiltrating immune cell profiles in Taiwanese primary BC--------------------------------------------13
3.2 Clinical relevance of PKM2/O-GlcNAc-------------------------14
3.3 Patient collection for the pilot study----------------------15
3.4 Higher nucleus O-GlcNAc status in metastatic lymph node site than primary tumors.--------------------------------------------16
3.5 Lymph node metastasis and M2 macrophage polarization--------17
CHAPTER 4 Conclusion and Discussion-----------------------------19
4.1 Macrophages took a large proportion in infiltrating immune cells in primary BC---------------------------------------------19
4.2 PKM2/O-GlcNAc were associated to DFS and nucleus O-GlcNAc were significantly increased in tumors at lymph node site------------20
4.3 Significantly fewer densities of CD68+ and CD163+ macrophages but higher M2 proportions in N+ cases than in N- cases----------22
4.4 The connection between PKM2/O-GlcNAc and M2 polarization----23
4.5 The significance and the future work of the study-----------24
CHAPTER 5 Figures-----------------------------------------------26
CHAPTER 6 Table-------------------------------------------------33
Reference-------------------------------------------------------43

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