| Background and objectiveBreast cancer is the most frequently diagnosed cancer and the leading cause of cancer death among females worldwide. Risk factors for breast cancer include reproductive and hormonal factors such as a long menstrual history, recent use of oral contraceptives, and never having children. Potentially modifiable risk factors include weight gain, being overweight or obese (especially in postmenopausal obese), use of menopausal hormone therapy, physical inactivity, and alcohol consumption. Breast cancer is now the most frequently diagnosed cancer and is the sixth leading cause of cancer-related death in Chinese women. Since the 1990s its incidence has increased more than twice as fast as have global rates, particularly in urban areas. With the continuous growth of our economy, more and more breast cancer treatment methods are introduced and applied to clinical treatment, and the prognosis of breast cancer patients is significantly improved. And with the development of breast cancer research, a variety of molecular marker for the diagnosis and treatment of breast cancer are found, which make the treatment of breast cancer individual and accurate. But the wide application of neoadjuvant chemotherapy and neoadjuvant endocrine therapy for breast cancer may cause the molecular markers change after treatment, and the changes of markers may lead to the loss of accurate judgment on the prognosis of the patients after the operation, and affect the decision of treatment strategy. In addition, the combined detection of markers is expensive, it is difficult to achieve in the most of the less developed areas in China, which will lead to the screening of high-risk patients difficult and the lack of pertinence of the treatment or the follow-up after surgery. Therefore, we need to identify a simple, instructive marker to screen high-risk patients and to detect the risk of postoperative recurrence and metastasis in patients undergoing neoadjuvant chemotherapy.Fibrinogen, a 340-kDa glycoprotein that is synthesized by liver and is converted to fibrin by activated thrombin, is an important coagulation factor. A growing number of reports have confirmed a wide range of biological activities of fibrinogen and its degradation products. The physiological activity of these molecules includes not only participation in the coagulation process, but also repair of damaged vessels, stimulation of migration, and proliferation of various cells, angiogenesis and wound healing. Furthermore, fibrinogen and its derivatives affect the contraction and relaxation of blood vessels, stimulate the permeability of capillaries and modulate the activation of blood platelets. Most of these properties of fibrinogen, fibrin and their fragments are particularly important in the biology of developing tumors and actively participate in the progression of cancer.In this study, first of all, breast cancer patients were divided into two groups, operative group and neoadjuvant chemotherapy group, the correlation between the preoperative plasma fibrinogen levels, fibrinogen level changes before and after neoadjuvant chemotherapy, clinicopathological parameters, the disease-free and overall survival rates in patients were investigated respectively. And again the differences deposition of fibrinogen in breast cancer tissue and normal breast tissues adjacent to cancer, the correlation between the different deposition and clinicopathological parameters, the correlation between the deposition of fibrinogen in breast cancer tissue and the plasma fibrinogen levels, and the expression of fibrinogen in breast cancer tissue and their metastatic axillary lymph nodes were discussed respectively. Finally, through the analysis of the above conclusions, the relationship between fibrinogen and breast cancer, and its application prospects in the treatment of breast cancer could be demonstrated.MethodsThis research was divided into three parts. First, four hundred and eighty-five operable patients with invasive breast cancer treated at Qilu Hospital of Shandong University from January 2009 to January 2011 were prospectively enrolled in this study. Patients were included if they met the admission criteria and exclusion criteria. In total, two hundred and twenty-three patients were left to be evaluated for the study. All of medical records were reviewed retrospectively. The end of the follow-up was the time of the last follow-up encompassed by this study (March, 2015) or death. Then we investigate the correlation between the preoperative plasma fibrinogen levels, clinicopathological parameters, the disease-free and overall survival rates in patients, respectively. Second, ninety-two advanced breast cancer patients received neoadjuvant chemotherapy treated at Qilu Hospital of Shandong University from January 2010 to January 2013 were prospectively enrolled in this study. Patients were included if they met the admission criteria and exclusion criteria. In total, sixty-seven patients were left to be evaluated for the study. All of medical records were reviewed retrospectively. The end of the follow-up was the time of the last follow-up encompassed by this study (March,2016) or death. Plasma fibrinogen levels and its changes were analyzed along with patient clinicopathological parameters, disease-free survival (DFS) and overall survival (OS). Both univariate and multivariate analyses were performed to identify the clinicopathological parameters associated with DFS and OS. Third, eighty-five operable patients with invasive breast cancer treated at Qilu Hospital of Shandong University in 2010 year were randomly selected in this study. Immunohistochemical techniques were applied to all the cases using rabbit polyclonal anti-fibrinogen antibody, and 10 normal breast tissues adjacent to cancer in these patients as controls. The differences of the fibrinogen expression between breast cancer and normal breast tissues adjacent to cancer were compared after immunohistochemical analyses. Associations between clinical pathological characteristics and positivity of immunohistochemical analysis, correlation between the expression of fibrinogen and plasma fibrinogen levels were investigated respectively. Then we performed immunohistochemical analyses on metastatic lymph nodes of 8 patients with lymph node metastasis in these patients to investigate the associations between fibrinogen and lymphatic metastasis. All patients were treated by modified radical mastectomy or radical mastectomy, and received systemic therapy in the adjuvant setting. Neoadjuvant chemotherapy with the combination of epirubicin 60 mg/m2 and docetaxel 75 mg/m2 was repeated twice every 3 weeks for 4-6 cycles. Patients were followed carefully after the initial treatment and reassessed every 3 months for the next 3 years and once a year thereafter. In the medical records, plasma fibrinogen concentrations of peripheral venous blood samples taken before breakfast were examined less than 7 days before neoadjuvant chemotherapy and surgery. For assaying fibrinogen, the clotting method of Clauss with the STA(?)-Fibrinogen ⑤ kit (DIAGNOSTICA STAGO Company, Ltd., Asnieres sur Seine, France) was used. The reference ranges for plasma fibrinogen levels was defined as 2.0-4.0 g/L. Data analysis was performed using PASS 14.0.2(NCSS LLC, Kaysville, Utah, USA), R from CRAN and SAS version 9.4(SAS Institute, Inc., Cary, NC, USA).Results1. Part 1:Preoperative plasma fibrinogen levels are associated with a multitude of risk factors of breast cancer, including age, menopause or not, tumor size, lymph node involvement and tumor staging, but not with histological grade, immunohistochemical parameters and molecular type. The correlation between age (or menopause) and the plasma fibrinogen levels of patients with breast cancer had no special significance because the research on human aging increased the plasma concentration of fibrinogen. In the univariate analysis, tumor stage, tumor size, lymph node involvement and plasma fibrinogen levels were associated with both DFS and OS. Then when we used the multivariate analysis to verify the potential prognostic factors, just the plasma fibrinogen levels and the lymph node involvement were found to be independently associated with DFS and OS.2. Part 2:Changes in fibrinogen plasma levels were associated with menopause, clinical response and metastasis. Among the fibrinogen levels decrease group, more patients responded to neoadjuvant chemotherapy and showed a significantly lower metastasis rate. In a univariate survival analysis, molecular type, clinical response and changes in plasma fibrinogen levels were associated with disease-free and overall survival, patients with HER2+ or Basal-like molecular type, patients with plasma fibrinogen levels increased in neoadjuvant chemotherapy treatment, and patients with poor response to chemotherapy had poor prognosis. In additional, all of them, molecular type, clinical response and changes in plasma fibrinogen levels were associated with disease-free and overall survival in a multivariate survival analysis, respectively. Part 3:Fibrinogen appeared more expressed (every intensity) among the cases compared with the controls; no significant association was found between the expression of fibrinogen in breast cancer tissue and clinicopathological parameters of the patients except age, menopause, histological grade, ER, PR and Ki-67. The expression of fibrinogen in breast cancer tissue was not related to the plasma fibrinogen levels. Fibrinogen deposited both in breast cancer tissues and their metastatic axillary lymph nodes.Conclusions1. Elevated preoperative plasma fibrinogen levels were associated with larger tumor size, TNM staging and more lymph nodes metastasis, elevated preoperative plasma fibrinogen levels could indicate earlier metastasis or death, multivariate survival analyses showed that preoperative plasma fibrinogen levels were independent prognostic factor for poor survival in patients with operative breast cancer. All of this demonstrated that plasma fibrinogen levels were closely related to tumor progression and metastasis.2. Patients received neoadjuvant chemotherapy with increased plasma fibrinogen levels after neoadjuvant chemotherapy showed significantly poor response to chemotherapy and shorter disease-free survival (DFS) and overall survival (OS). With the validity of the changes in fibrinogen levels during neoadjuvant chemotherapy, plasma fibrinogen level was found to be a possible biomarker for postoperative metastasis and death in advanced breast cancer patients who received neoadjuvant chemotherapy. Moreover, the plasma fibrinogen levels could be a simple and valuable predictive indicator for postoperative follow up.3. The deposition and expression of fibrinogen in breast cancer tissue and its’none association with the plasma fibrinogen levels showed that fibrinogen could be endogenously synthesised by tumor cells themselves, and played different roles in the progress and metastasis of breast cancer. Associations between the expression and breast cancer histological grade showed that fibrinogen contributed to the structural integrity of breast cancer tissues and participated in angiogenesis and stimulation of migration. The expression of fibrinogen in breast cancer tissue and their metastatic axillary lymph nodes showed its vital roles in lymphatic metastasis of cancer.SignificanceOver the years, the basic and clinical research about the relationship between coagulation system and cancer continues to be improved, the coagulation proteins play an important role not only in the coagulation process, but also in the progression of malignant tumor. Fibrinogen participates in the structure construction of tumor cells surrounding space and angiogenesis, makes vascular permeability increase and help tumor cell transmembrane migration, and help them escape from immune surveillance in the transfer process by thromboembolus formation. Our basis research verified the relationship between the deposition of fibrinogen around the tumor cells and its histological grade. In the retrospective survival analysis of different tumor stages, different treatment in patients with breast cancer, we found the correlations between plasma fibrinogen levels and lymph node metastasis, hematogenous metastasis of breast cancer and its guiding significance in judging the prognosis. The detection of plasma fibrinogen levels is convenience, low price and repeatability, which makes it become a serum marker of prognostic and clinical value. And because of its important role in the process of tumor metastasis, fibrinogen is expected to become a new target for the treatment of tumor metastasis. Recent studies have confirmed that the application of anticoagulation therapy and low molecular weight heparin can prolong the survival of cancer patients, although further clinical studies are needed to determine the efficacy and safety of anticoagulants, it should be noted that anticoagulation therapy is a promising treatment strategy for improving prognosis in patients with cancer. |
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