乳腺癌患者血清中纤维蛋白原水平及其在癌组织中表达的临床研究

发布时间：2018-05-11 12:29

本文选题：纤维蛋白原 + 乳腺癌　；参考：《山东大学》2016年博士论文

【摘要】：研究背景与目的乳腺癌是全球女性中最常见的恶性肿瘤,亦是全球女性癌症相关死亡的主要原因。乳腺癌的发病危险因素包括生育及激素因素,如长月经史、口服避孕药及不生育,潜在可改变的危险因素包括肥胖(尤其是绝经后肥胖)、绝经后激素替代治疗、运动减少及酗酒。在我国,尤其是在城市地区,乳腺癌也是女性中最常见的恶性肿瘤,自上世纪90年代,其发病率每年便以全球同期发病率的两倍水平在上升,是中国女性癌症相关死亡的第六位因素。随着我国经济的不断增长,越来越多的乳腺癌相关治疗手段被引进并应用于临床,使乳腺癌患者的预后得到明显改善。并且随着对乳腺癌的研究深入,各种用于乳腺癌诊断及治疗的分子标记物被发现,使乳腺癌的治疗趋向于个体化的精准治疗。但乳腺癌新辅助化疗、新辅助内分泌治疗等的广泛应用,有可能造成上述分子标记物在治疗后出现变化,从而在手术后因为指标改变失去对患者预后的准确判断,影响治疗策略的决定；此外,多种指标的联合检测费用昂贵,在我国大部分经济欠发达地区难以实现,这将导致对高危患者的筛选出现困难,使术后的治疗及随访失去针对性。因而,我们需要确定一种简单的、具有指导意义的标记物,来筛选高危患者并且能检测接受新辅助化疗患者术后复发及转移的风险。纤维蛋白原是一种糖蛋白,分子量为340kDa,经肝脏产生。在凝血系统中,纤维蛋白原由活化的凝血酶作用而转化为纤维蛋白,是一种重要的凝血因子。越来越多的报道证实纤维蛋白原及其降解产物具有广泛的生物活性,不仅参与凝血过程,还在血管损伤修复、刺激迁移、各种细胞增殖、血管形成及创伤修复中起重要作用。此外,纤维蛋白原及其衍生物还影响血管收缩和舒张,刺激毛细血管的通透性和调节血小板活化,这些在恶性肿瘤的发生及进展中起着极其重要的作用。本研究中,首先将乳腺癌患者分为可直接手术组与新辅助化疗组,分别探讨血清中纤维蛋白原水平或化疗前后纤维蛋白原水平的变化与各组患者临床病理学指标及预后的关系,然后对乳腺癌组织中纤维蛋白原的沉积与癌旁正常乳腺组织的差别及其与临床病理学指标的关系进行探讨,并研究血清中纤维蛋白原的水平与组织中纤维蛋白原沉积的相关性以及乳腺癌和其转移淋巴结纤维蛋白原的表达情况。最后通过对以上实验结论的分析总结,初步阐明纤维蛋白原与乳腺癌的关系及其在乳腺癌治疗中的应用前景。研究方法本研究共分为三部分,第一部分回顾性分析山东大学齐鲁医院2009年1月—2011年1月共485例可手术乳腺癌患者的临床资料,经过入组条件筛选及去除资料不完整患者,最终入组223例可手术乳腺癌患者,并随访患者至2015年3月,将临床资料及随访资料进行统计学分析。探讨可手术乳腺癌患者血清中纤维蛋白原水平与患者临床病理学指标之间的关系,并对该样本进行生存分析,阐述血清中纤维蛋白原水平对可手术乳腺癌患者预后的意义。第二部分回顾性分析山东大学齐鲁医院2010年1月—2013年1月共92例行术前新辅助化疗的进展期乳腺癌患者,经过入组条件筛选及去除资料不完整患者,最终入组67例乳腺癌患者,并随访患者至2016年3月,将所得临床资料及随访资料进行统计学分析,探讨行术前新辅助化疗的乳腺癌患者血清中化疗前纤维蛋白原水平、手术前纤维蛋白原水平以及新辅助化疗前后血清纤维蛋白原水平变化与患者各临床病理学指标之间的关系,尤其与新辅助化疗疗效之间的关系；并对该样本进行生存分析,阐述血清中新辅助化疗前、新辅助化疗后手术前纤维蛋白原水平及纤维蛋白原水平的变化对新辅助化疗乳腺癌患者预后的意义。第三部分随机选取山东大学齐鲁医院2010年度85例可手术乳腺癌患者的病理切片标本,并同时随机选取其中10例乳腺癌患者癌旁正常乳腺组织病理切片标本作对照,使用兔多克隆抗纤维蛋白原抗体进行免疫组化检测,对所得结果进行计分并分级处理,比较乳腺癌组织、癌旁正常乳腺组织中纤维蛋白原表达的差异,回顾性分析患者的临床资料,探讨纤维蛋白原在乳腺癌组织中的表达水平与患者临床病理学指标之间的关系,组织中纤维蛋白原水平与血清中纤维蛋白原水平的相关性；另随机选取其中8例乳腺癌合并腋淋巴结转移患者的转移淋巴结的病理切片行免疫组化检测,初步探讨纤维蛋白原与乳腺癌淋巴转移之间的关系。可手术乳腺癌患者及新辅助化疗后的乳腺癌患者均接受乳腺癌改良根治术或乳腺癌根治术,术后辅以系统治疗。新辅助化疗方案采用表柔比星60 mg/m2搭配多西他赛75 mg/m2的化疗方案4-6周期,每21天为1周期。随访策略为三年内每3个月随访一次,三年后每年随访一次。临床资料中患者血清纤维蛋白原水平测定均为化疗前或手术前7天内清晨空腹抽取外周静脉血后,利用克劳斯法于自动血凝分析仪进行检测所得。血清纤维蛋白原水平的参考值范围为2-4g/L。利用PASS 14.0.2、CRAN网站的R程序及SAS 9.4对所得数据进行统计学分析。结果1.第一部分研究结果显示：术前可手术乳腺癌患者血清纤维蛋白原水平的升高与发病年龄、绝经与否、肿瘤大小、肿瘤TNM分期和淋巴结转移数目相关,而与组织学分级、分子分型及其他免疫组化指标无关；其中因血清纤维蛋白原水平受年龄增高的影响,可将发病年龄及绝经与否的影响舍弃。在单因素COX回归分析中,肿瘤TNM分期、肿瘤大小、腋窝淋巴结转移数目及血清纤维蛋白原水平与无病生存率及总生存率均相关,而在多因素分析中仅腋窝淋巴结转移数目及血清纤维蛋白原水平与预后相关。2.第二部分研究结果显示：行术前新辅助化疗的患者,其血清纤维蛋白原水平在新辅助化疗前后的变化与患者绝经与否、新辅助化疗的临床疗效以及术后转移有关。血清纤维蛋白原水平在新辅助化疗过程中出现降低的患者表现出更好的对新辅助化疗的疗效反应,具有更高的临床缓解率,并且术后出现远处脏器转移的几率减小。在对新辅助化疗的进展期乳腺癌患者的单因素及多因素COX回归分析中,我们发现,乳腺癌的分子分型、新辅助化疗的临床疗效及新辅助化疗过程中血清纤维蛋白原水平的变化与患者的无病生存率及总生存率均相关,HER2过表达型及三阴型乳腺癌患者、新辅助化疗反应差的患者以及纤维蛋白原水平在新辅助化疗前后稳定或出现升高的患者的预后较差,并且三者均为行新辅助化疗的进展期乳腺癌患者的独立预后因子。3.第三部分研究结果显示：纤维蛋白原在乳腺癌组织中呈现不同程度的表达,而癌旁正常乳腺组织中未见表达；癌组织中纤维蛋白原的表达与年龄、绝经与否、组织学分级、ER、PR及Ki-67有关,而与临床常见指标肿瘤长径、淋巴结转移及肿瘤TNM分期无关,年龄越大、绝经、组织学分级升高、细胞增长迅速及ER、PR表达减少均会导致纤维蛋白原表达升高。乳腺癌组织中的纤维蛋白原水平与血清中纤维蛋白原水平无相关性；乳腺癌组织及其转移的腋窝淋巴结均见纤维蛋白原表达。结论1.可手术乳腺癌患者血清中纤维蛋白原水平的升高与肿瘤大小、肿瘤TNM分期及腋窝淋巴结转移数目有关,其升高导致患者无病生存率及总生存率下降,是独立预后影响因子,这说明纤维蛋白原水平与肿瘤进展及转移密切相关。2.行新辅助化疗的乳腺癌患者,化疗前后出现血清中纤维蛋白原水平升高的患者新辅助化疗的临床缓解率、术后无病生存率及总生存率均下降,使其可以作为新辅助化疗疗效及预后判断的可行标记物。3.纤维蛋白原在乳腺癌组织中沉积并且其表达与血清中纤维蛋白原水平无相关性证实乳腺癌细胞可自行产生纤维蛋白原,并且癌细胞自行产生的纤维蛋白原与血清中纤维蛋白原在癌的发展及转移过程中作用可能不同；其表达水平与乳腺癌组织学分级相关,证实其与乳腺癌细胞周围空间结构稳定性、乳腺癌细胞周围血管形成及细胞迁移有关；癌组织及腋窝转移淋巴结中同步表达证实其参与乳腺癌的淋巴转移。意义多年来,有关凝血系统与癌症关系的探讨从基础实验到临床研究不断完善,凝血蛋白不仅在机体凝血过程中起重要作用,其在恶性肿瘤的进展过程中同样扮演重要角色。不仅参与肿瘤细胞周围空间结构的构建、新生血管的形成,而且使血管通透性增加并帮助肿瘤细胞跨膜迁移,并在其转移过程中通过形成血栓栓子来帮助其逃避免疫监视。我们的研究在基础实验方面验证了纤维蛋白原在肿瘤周围的沉积与肿瘤恶性程度的关系,并在对不同分期、不同处理的乳腺癌患者的分组回顾性生存分析中,发现血清中纤维蛋白原水平与乳腺癌淋巴转移、血行转移的相关性及对预后判断的指导意义。由于血清纤维蛋白原水平检测方便、快捷、价格低廉以及可重复性,使其成为极具临床应用价值的判断预后的血清标记物,并且因为其在肿瘤转移过程中的重要作用,使其有望成为新的抗肿瘤转移治疗的靶点。最近已有研究证实抗凝治疗及低分子肝素的应用可延长癌症患者的生存期,虽然抗凝剂应用的安全性及有效性尚需更多临床研究的支持,但此种治疗手段在提高癌症患者预后上的表现还是值得期待的。
[Abstract]:Background and objective breast cancer is the most common malignancy among women in the world. It is also the main cause of cancer related deaths in women worldwide. The risk factors for breast cancer include reproductive and hormonal factors such as long menstrual history, oral contraceptives and childbearing, and potentially possible changes in risk factors including obesity (especially postmenopausal obesity). In China, especially in urban areas, breast cancer is the most common malignant tumor in China, especially in urban areas. Since 90s, the incidence of breast cancer was two times as high as the global incidence of the same period. It was the sixth factor of cancer related death in Chinese women. More and more breast cancer related treatments have been introduced and applied to the clinic to improve the prognosis of breast cancer patients. And with the study of breast cancer, various molecular markers used for the diagnosis and treatment of breast cancer have been found, and the treatment of breast cancer tends to be individualized and accurate. The extensive application of neoadjuvant chemotherapy and neoadjuvant endocrine therapy may result in the changes in the molecular markers after treatment, thereby losing the accurate judgment of the prognosis of the patients after the operation, and affecting the decision of the treatment strategy. In addition, the combined detection of various indicators is expensive and most of the economy is in our country. The difficult implementation of developed areas will lead to difficulties in screening high-risk patients and the loss of post-operative treatment and follow-up. Therefore, we need to identify a simple, instructive marker to screen high-risk patients and to be able to detect the risk of postoperative recurrence and metastasis in patients with neoadjuvant therapy. It is a glycoprotein whose molecular weight is 340kDa and is produced by the liver. In the coagulation system, fibrin is converted into fibrin by activated thrombin action. It is an important coagulation factor. More and more reports have confirmed that fibrinogen and its degradation products have extensive biological activity, not only in coagulation process, but also in vascular damage. Injury repair, stimulation of migration, various cell proliferation, angiogenesis and trauma repair play an important role. In addition, fibrinogen and its derivatives also affect vasoconstriction and relaxation, stimulate capillary permeability and regulate platelet activation. These play an important role in the development and progression of malignant tumors. First, the breast cancer patients were divided into a direct operation group and a new adjuvant chemotherapy group. The relationship between the changes of fibrinogen level in serum and the changes of fibrinogen level before and after chemotherapy and the relationship between the clinicopathological indexes and prognosis of each group were discussed, and the difference between fibrinogen deposition in breast cancer and normal breast tissue adjacent to the cancer was also discussed. The relationship between the serum levels of fibrinogen and fibrinogen deposition in the tissue and the expression of fibrinogen in breast cancer and its metastatic lymph nodes were investigated. Finally, the relationship between fibrinogen and breast cancer was preliminarily clarified by the analysis of the above conclusions. Department and its application in the treatment of breast cancer. The research method is divided into three parts. The first part is a retrospective analysis of the clinical data of 485 patients with surgical breast cancer in the Qilu Hospital of Shandong University from January 2009 to January 2011. Through the screening and removal of incomplete data, 223 cases of surgical breast can be included. The patients were followed up to March 2015, and the clinical data and follow-up data were statistically analyzed. The relationship between the serum fibrinogen level and the clinicopathological indexes of the patients with operable breast cancer was investigated, and the survival analysis of the sample was carried out. The serum fibrinogen level was predefined for the patients with surgical breast cancer. The second part of the second part retrospective analysis of the 92 cases of advanced breast cancer patients in Qilu Hospital of Shandong University from January 2010 to January 2013. After screening and removing the incomplete data, 67 cases of breast cancer patients were finally enrolled and followed up to March 2016, the clinical data and follow-up were followed up. The data were statistically analyzed to explore the level of fibrinogen before chemotherapy in the serum of breast cancer patients with neoadjuvant chemotherapy, the level of fibrinogen before and after the operation and the relationship between the changes of serum fibrinogen level before and after the neoadjuvant chemotherapy and the relationship between the clinical and pathological indexes of the patients, especially with the therapeutic effect of neoadjuvant chemotherapy. The survival analysis of the sample was carried out, and the significance of the changes of fibrinogen level and fibrinogen level before neoadjuvant chemotherapy before neoadjuvant chemotherapy in sera before neoadjuvant chemotherapy on the prognosis of neoadjuvant chemotherapy for breast cancer patients. The third part randomly selected 85 cases of surgical breast cancer patients in Qilu Hospital of Shandong University in 2010. The specimens of 10 patients with breast cancer were randomly selected as control, and the rabbit polyclonal anti fibrinogen antibody was used for immunohistochemical detection. The results were graded and graded to compare the expression of fibrinogen in the breast cancer tissue and the normal breast tissue near the cancer. The difference, retrospective analysis of the patient's clinical data, the relationship between the expression of fibrinogen in the breast cancer tissue and the clinicopathological indexes, the correlation between fibrinogen level and serum fibrinogen level in the tissue, and the random selection of 8 cases of breast cancer with axillary lymph node metastasis. The pathological sections of the lymph nodes were detected by immunohistochemistry. The relationship between fibrinogen and lymphatic metastasis of breast cancer was preliminarily investigated. All patients with breast cancer and breast cancer after neoadjuvant chemotherapy were treated with modified radical mastectomy or radical mastectomy with systematic treatment after operation. The new adjuvant chemotherapy regimen was treated with epirubicin 60 m G/m2 was paired with a chemotherapy regimen of 75 mg/m2 of docetaxel with a period of 1 cycles per 21 days. The follow-up strategy was followed up every 3 months in three years and one year after three years. The serum fibrinogen levels were measured before or after the early morning empty abdominal extraction in the 7 days before the operation, and the Claus method was used. The reference range of serum fibrinogen level was 2-4g/L. using PASS 14.0.2, R program of CRAN website and SAS 9.4 for statistical analysis. Results 1. the first part of the study showed that the level of blood clearing fibrinogen in patients with surgical breast cancer and the age of onset of preoperatively Menopause or not, tumor size, tumor TNM staging and number of lymph node metastases, but not related to histological classification, molecular typing and other immunohistochemical markers. The effect of age increases on serum fibrinogen levels, and the rejection of age and menopause. In the single factor COX regression analysis, the TNM staging of the tumor The size of the tumor, the number of axillary lymph node metastases, and the serum fibrinogen level were related to the disease-free survival and the total survival rate. In the multifactor analysis, the number of axillary lymph nodes and the serum fibrinogen level were related to the prognosis of the.2. second parts. The changes in the protein level before and after the neoadjuvant chemotherapy are related to the patient's menopause, the clinical efficacy of the neoadjuvant chemotherapy and the postoperative metastasis. The patients with the serum fibrinogen level in the neoadjuvant chemotherapy show a better response to the neoadjuvant chemotherapy, with a higher clinical remission rate, and after the operation. The probability of distant visceral metastasis is reduced. In the single factor and multiple factor COX regression analysis of the advanced breast cancer patients with neoadjuvant chemotherapy, we found that the molecular classification of the breast cancer, the clinical efficacy of neoadjuvant chemotherapy and the changes of serum fibrin leveling during the neoadjuvant chemotherapy and the patient's disease free survival and total survival Rates are correlated, HER2 overexpressed and three yin type breast cancer patients, patients with poor response to neoadjuvant chemotherapy and patients with stable or elevated fibrinogen levels before and after neoadjuvant chemotherapy are poor, and the three are the third part of the independent prognostic factor of the advanced breast cancer patients with neoadjuvant chemotherapy. The results of the study are the result of the third part of the study. The expression of fibrinogen in breast cancer tissues is different, but the expression of fibrinogen is not expressed in normal breast tissue. The expression of fibrinogen is related to age, menopause, histologic classification, ER, PR and Ki-67, and is not related to the common clinical indicators of tumor length, lymph node metastasis and tumor TNM staging. Large, menopause, histopathological grading, rapid cell growth and decreased expression of ER and PR could lead to increased fibrinogen expression. There was no correlation between fibrinogen level in breast cancer tissue and serum fibrinogen level; breast cancer tissue and its metastatic axillary lymph nodes were all fibrinogen expression. Conclusion 1. can be operated in mammary gland. The increase of fibrinogen level in the serum of cancer patients is related to the size of tumor, TNM staging and the number of axillary lymph node metastasis, which leads to the decrease of disease survival and total survival rate, which is an independent prognostic factor, which indicates that the level of fibrinogen is closely related to the progression and metastasis of tumor and.2. is closely related to the breast neoadjuvant chemotherapy. In cancer patients, the clinical remission rate of neoadjuvant chemotherapy in patients with elevated serum fibrinogen levels before and after chemotherapy, the postoperative survival rate and total survival rate decreased, which could be used as a viable marker for the therapeutic effect and prognosis of neoadjuvant chemotherapy,.3. fibrinogen was deposited in the breast cancer tissue and expressed in the serum. No correlation between fibrinogen level confirms that breast cancer cells can produce fibrinogen on its own, and the role of fibrinogen and fibrinogen in cancer cells may be different in the development and metastasis of cancer, and its expression level is related to the histological grade of breast cancer, which confirms its surrounding space in breast cancer cells. Structural stability is related to angiogenesis and cell migration around breast cancer cells; synchronous expression of cancer tissue and axillary metastasis confirms its involvement in lymphatic metastasis of breast cancer. The significance of the relationship between coagulation system and cancer has been perfected from basic experiment to clinical study for many years. Coagulation protein is not only clotting in the body. It plays an important role in the progression of malignant tumor. It not only participates in the construction of the space structure around the tumor cells, the formation of the new blood vessels, but also increases the permeability of the blood vessels and helps the tumor cells to transmigrate across the membrane. In the process of metastasis, the thrombus is formed to help them escape from the immune surveillance. Our study verified the relationship between the deposition of fibrinogen around the tumor and the malignancy of the tumor, and in the retrospective subsistence analysis of patients with different stages and treatment of breast cancer, we found the correlation between serum fibrinogen level and lymph node metastasis, blood metastasis and prognosis. Because the serum fibrinogen level is convenient, fast, cheap and repeatable, it makes it a serum marker for judging prognosis of clinical value, and it is expected to be a new target for tumor metastasis treatment because of its important role in the process of tumor metastasis. Studies have shown that anticoagulant therapy and the application of low molecular weight heparin can prolong the survival of cancer patients. Although the safety and effectiveness of anticoagulants need more support from clinical research, it is still worth looking forward to improving the prognosis of cancer patients.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.9

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