Detection Of Micrometastases In Bone Marrow Of Patients With Breast Cancer And The Relationships With Other Indicators

Objective: Breast cancer is a systemic malignant disease. Despite the treatment continuously renews, its overall survival , however, has no obvious exaltation in the last 20 years. The principal cause of death in breast cancer patients is metastatic spread of tumor cells. Among lymph node-negative patients about 30% of them had an unfavorable outcome at five years from primary surgery. The routine techniques of detection for the patients are not enough to monitor early metastases, to assess response to therapy during a treatment course, and to modify a particular treatment. As an occult term before clinical metastases, micrometastases is a key stage during the whole treatment of breast cancer patients. As bone marrow (BM) is one of the most common sites and often the first clinical indication of metastatic progression of breast cancer, it has more advantages than axillary lymph nodes and peripheral blood in showing the status of systemic metastases. Cytokeratin 19(CK19), a histogenetic marker of epithelial tumor cells, cannot be detected in normal BM. In this research, reverse transcriptase polymerase chain reaction (RT-PCR) was used to determine the presence of CK19 positive tumor cells in BM of 102 women with newly diagnosed stage I, II , III orⅣ breast cancer. The expression of biomarkers such as ER,PR,Cath-D,C-erbB-2 and VEGF were also detected in tumor tissue by immunohistochemical methods. These molecular markers and some conventional clinical histopathologic features were analyzed with BM micrometastases in an attempt to identify their relationships, which may indicate some regulation of metastases, provide theoretical evidence and prognosis assessment of breast cancer. Methods: All samples for the experiment were obtained from 102 patients with breast cancer and 10 patients with benign lesions of breast including fibroadenoma and fibrocystic diseases in the department of surgery of Fourth Hospital of Hebei Medical University from Oct. 2002 to Oct. 2003. All patients with breast cancer were female with median age 49.2. The pathological types of malignant breast disease patients include 53 infiltrating ductal carcinomas, 35 infiltrating lobular carcinomas, 4 classical medullary carcinomas, 2 mucinous carcinomas and 8 other carcinomas. The malignant group included various clinical stage(stage Ⅰ 12 cases, stage Ⅱ 59 cases, stage Ⅲ 29 cases, stage Ⅳ 2 cases) and different histological grade (gradeⅠ24 cases, grade Ⅱ 46 cases, grade Ⅲ 32 cases). Ten-milliliter BM aspirates sampled from anterior iliac crest of all 112 patients were centrifuged using density centrifugation Ficoll. All samples underwent RNA extraction and CK19 RT-PCR(one-step methods). All malignant tumor tissues were collected for ER,PR,Cath-D,C-erbB-2 and VEGF examination of by immunohistochemical s-p method. Statistic methods: Statistic analysis was performed with χ2. Results: 1. The positive expression of CK19 gene detected by RT-PCR: CK19 gene can not be detected in any BM aspirate samples of 10 benign lesions of breast patients, but 58 in 102 patients with breast cancer (56.9%). 2. The relationships between BM micrometastases and tumor size, metastases of axillary lymph node as well as clinical stage : The incidences of BM micrometastases were more greatly increased in patients with T2(55.9%)and T3 (77.8%) tumors than T1(25.0%)(P <0.005),and also more increased in patients with stageⅡ(52.5%)and Ⅲ～Ⅳ(74.2%)than stage I(33.3%)(P <0.05). The differences were statistically significant. Allthough the expression of CK19 gene was higher in patients with positive metastases of axillary lymph node(64.7%)than that with negative axillary lymph node(49.0%), there was no statistic difference between them(P＞0.1). 3. The relationships between BM micrometastases and hormone receptor status as well as histological grade: The expression of CK19 in the BM of breast cancer women with grade I (25.0%) was lower than that with grade II (63.0%) or III(71.9%), and the difference was...