||The Correlation of Expression of Kruppel-like factors (KLF) and the Clinical Manifestations of the Breast Cancer
||Graduate Institute of Clinical Medicine
Kruppel-like factors (KLFs)是一群鋅指樣轉錄因子（Zinc finger like transcription factors），最初被發現和細胞生長的調控有關，目前已知共有超過20種KLFs。最近幾年有越來越多探討KLF在乳腺癌表現的研究，多數研究主要著重於KLF涉及的致癌機轉，而且有數篇研究認為KLF為乳腺癌新的預後因子。在乳腺癌最重要和最被廣泛探討的有KLF4和KLF5，但研究主要局限於西方國家。為了對本地乳腺癌提供更好的治療策略，本研究的目的是探討KLF4和KLF5在台灣婦女乳腺癌的表現情形和與乳腺癌的生物行為相關性。
本研究共收集了60名乳腺癌患者，其平均年齡為47歲，平均腫瘤大小為2.7公分。臨床表現為第一期的有30%；第二期有43.3%；第三期有21.7%；第四期有5%。經診斷後的平均追蹤時間為27個月（範圍從8至59個月），其中只有一名患者是死於乳腺癌。本研究中90%病例的乳腺癌的組織型態為浸潤性管道癌（Invasive ductal carcinoma, IDC），且66.7%為中度分化。於病灶旁邊管道原位癌（Ductal carcinoma in situ, DCIS）有60%的分化程度為最差等級的。將KLF4和KLF5的免疫組織染色結果與這些病例的臨床表現作關聯性探討時，發現KLF4表現以細胞質和核為主，其中43.3%的病人為腫瘤部分的表現強度比非腫瘤部分強。若腫瘤細胞的KLF4核表現傾向≧25%的病例，有較高的癌症分期（p=0.006），並有較大的腫瘤（最大徑超過2公分，p=0.035）。KLF4的表現也有年齡的相關性，即年齡超過50歲的病例，浸潤癌或原位癌的表現比非腫瘤部分來的更強（p= 0.007），而且，其浸潤性癌的分化也較差（p=0.033）。此外，我們還發現同一病例的浸潤性癌和原位癌之表現有一致性：若原位癌的表現越強，其浸潤性癌的表現也強（p=0.002）；核表現的傾向也有一致性（p<0.001）。KLF5表現以細胞質為主，其中58.3%的病人為腫瘤部分（含浸潤癌或原位癌）的表現強度比非腫瘤部分強。就KLF5方面，浸潤性癌若為陰性或弱的細胞質染色時，比較強細胞質表現的乳癌有較好的組織學分化（p=0.035）。另外，同一腫瘤內KLF5的表現在浸潤性癌和原位癌有一致性：若原位癌的表現越強，其浸潤性癌的表現也強（p<0.001）；細胞質表現的傾向也具有一致性（p<0.001）。此外，KLF4的表現強度和型態分別和以下因子的表現無相關：動情素接受器（p=0.271和p=0.925），黃體激素接受器（p=0.191和p=0.448），HER-2/neu（p=0.136和p=0.454），p53（p=1.000和p=0.925）和p21 （p=0.572和p=0.367）。KLF5的表現強度和型態分別和以下因子的表現無相關：動情素接受器（p=1.000和p=0.512），黃體激素接受器（p=1.000）和HER-2/neu（p=0.520和p=0.443）。
||Breast cancer is the fourth cause of female cancer deaths in Taiwan with increased incidence and young age tendency (age?T40 years old, 29.3%). In recent years, the distinct age distribution and more aggressive clinical behavior in the young patient are noted in Taiwanese women and this phenomenon is different from that in the Western countries. Besides, due to the heterogeneity of breast cancer, designation of an ideal treatment protocol for breast cancer could not only be based on the traditionally histological, clinical, and biological markers (such as ER, PR and HER-2/neu) but also some new prognostic factors. Therefore, the specific study of breast cancer in Taiwan women becomes an important issue.
Kruppel-like factors (KLF) belong to a group of zinc finger like transcription factors and are involved in regulating cell proliferation. KLFs have more than twenty subtypes. The studies of Kruppel-like factors in breast cancer are increased recently and are mainly focused on their roles in tumorigenesis. The KLFs are considered as new prognostic factors in breast cancers in some studies. Among them, KLF4 and KLF5 are most important and are broadly studied, but most studies are mainly in Western countries. In order to provide better treatment strategies for native breast cancers, the aim of this study is to evaluate the correlation of KLF4 and KLF5 expression with pathologic changes and clinical behaviors of breast cancers in Taiwanese women.
In the literatures, KLF4 has both tumor suppressor gene and oncogene functions. KLF4 can promote the proliferation of cancer cells and also can regulate production of extracellular matrix. More aggressive clinical manifestations may be associated with the cellular location of KLF4 in cancer cells. The patients have poor prognosis when nuclear localization of KLF4 in cancer cells. KLF5 also has both tumor suppressor gene and oncogene functions. KLF5 can facilitate the proliferation and transformation of cancer cells. Increased expression of KLF5 is a poor prognostic factor and is positively correlated with the expression of HER-2/neu and Ki-67 in breast cancer. KLF5 also has increased expression in breast cancer patients younger than 50 years old.
In this study, we used immunohistochemistry method to evaluate both staining intensity and staining pattern of expression of KLF4 and KLF5 in non-tumor and tumor parts (including invasive and in situ cancers) of breast tissues. We also analyzed the associations of expression status of KLF4 and KLF5 with histological features, clinical presentation and other prognostic factors of breast cancer.
We enrolled 60 breast cancer patients with the mean age 47 years old and the mean tumor size was 2.7 cm. The clinical presentation was stage I: 30.0%; stage II: 43.3%; stage III: 21.7%; and stage IV: 5.0%. The follow-up period of these patients ranged from 8 to 59 months (mean 27 months) and only one patient died of disease. Pathologically, most of them were invasive ductal carcinoma (IDC) (90.0%) and showed moderately differentiation (66.7%). The accompanied ductal carcinoma in situ (DCIS), if present, was predominantly highest grade (60.0%). The immunohistochemical study of KLF4 in cancer cells showed cytoplasmic and nuclear expression. The intensity of tumor part was stronger than non-tumor part in 43.3% patients. We evaluated the association of the immunohistochemical results of KLF4 and KLF5 and clinical manifestations of these patients. We found that more KLF4 nuclear expression in tumor cells positively correlated with more advanced stage (p=0.006) and larger size of the tumor (size more than 2 cm in maximal diameter, p=0.035). KLF4 expression was also age-related. KLF4 intensity was stronger in tumor part than non-tumor part in patients older than 50 years old (p=0.007) and, in this setting, the invasive cancer tended to be poorly differentiated (p=0.033). Besides, consistent expression of KLF4 between DCIS and invasive cancers was also found: stronger intensity in DCIS accompanied with stronger intensity in invasive cancers (p=0.002), more predominant nuclear expression in DCIS with more predominant nuclear expression in invasive cancers (p<0.001). The expression of KLF5 in cancer cells was mainly cytoplasmic. The intensity of tumor part was stronger than non- tumor part in 58.3% patients. For KLF5, invasive breast cancers with negative or weak cytoplasmic expression showed better differentiation compared with strong cytoplasmic expression (p=0.035). Consistent expression of KLF5 between DCIS and invasive cancers was also found: stronger intensity in DCIS with stronger intensity in invasive cancers (p<0.001) and more predominant cytoplasmic expression in DCIS with more predominant cytoplasmic expression in invasive cancers (p<0.001). Moreover, there was no association between the following factors and the KLF4 expression intensity and pattern, respectively: ER (p=0.271 and p=0.925), PR (p=0.191 and p=0.448), HER-2/neu (p=0.136 and p=0.454), p53 (p=1.000 and p=0.925), and p21 (p=0.572 and p=0.367). There was also no correlation between the following factors and the KLF5 expression intensity and staining pattern, respectively: ER (p=1.000 and p=0.512), PR (p=1.000 and p=1.000), and HER-2/neu (p=0.520 and p=0.443).
Our study found that KLF4 expression is positive association with tumor stage, tumor size, and age but could not conduct the conclusion that nuclear KLF4 expression was an adverse prognostic factor proposed in the literatures. In the other hand, KLF5 expression was associated with the differentiation of invasive cancers. We also found that KLF5 nuclear localization was mainly restrictedly in non-tumor breast ducts and lobules (16.7%) and loss of nuclear expression in DCIS and invasive cancers, the finding not mentioned in literatures before. Although we didn’t study the biologic function of KLF5, it maybe presented a possible tumor suppressor gene-like function of KLF5. We found that there were associations of KLF4 and KLF5 expressions and clinical manifestations in breast cancers but the expressions of KLF4 and KLF5 were not enough to predict the prognosis and survival rate. The major cause was due to too short follow up period of our patients to exactly evaluate the association of survival rate and expressions of KLF4 and KLF5. Therefore, well-designed retrospective studies with adequate follow up period for studying correlation of expressions of KLF4 and KLF5 and prognosis and survival rate of breast cancers are necessary.
縮寫表 (Abbreviations) IV
第一章 中文摘要 (Abstract in Chinese) 1
第二章 英文摘要 (Abstract in English) 4
第三章 緒論 (Introduction) 8
3.1 Breast Cancer in Taiwan 8
3.2 Kruppel-like Factors and Breast Cancer 14
3.3 Kruppel-like Factor 4 (KLF4) and Breast Cancer 16
3.4 Kruppel-like Factor 5 (KLF5) and Breast Cancer 21
3.5 Aims of Our Study 22
第四章 研究方法與材料 (Materials and Methods) 24
4.1 Study Design and Patient Selection 24
4.2 Pathological Evaluation 24
4.3 Immunohistochemistry 25
4.3.1 Immunohistochemistry of KLF4 in Breast
4.3.2 Immunohistochemistry of KLF5 in Breast
4.3.3 Immunohistochemistry of p53 and p21 in
Breast Cancers 28
4.3.4 Immunohistochemistry of ER, PR, and
HER-2/neu in Breast Cancers 28
4.4 Statistical Analysis 30
第五章 實驗結果 (Results) 31
5.1 Clinical Characteristics of the Patients 31
5.2 Pathological Evaluation of Breast Cancers 31
5.3 Immunohistochemical Results of KLF4, KLF5, p53,
and p21 in Breast Cancers 32
5.3.1 Immunohistochemical Results of KLF4 in
Breast Cancers 33
5.3.2 Immunohistochemical Results of KLF5 in
Breast Cancers 34
5.3.3 Immunohistochemical Results of p53 and p21
in Breast Cancers 35
5.4 Correlation of Clinical Features, Histological
Pictures, and Immunohistochemical Results of
KLF4, KLF5, p53, p21, ER, PR, and HER2/neu in
Breast Cancers 36
第六章 討論 (Discussion) 39
第七章 結論與展望 (Conclusion and Perspectives) 49
第八章 參考文獻 (References) 50
第九章 圖表 (Tables and Figures) 56
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