【作者】 刘巍；

【导师】 张祥宏；

【作者基本信息】 河北医科大学 ， 病理学与病理生理学， 2005， 博士

【摘要】 近年来,随着免疫学和分子生物学技术的飞速发展,对乳腺癌标志物及其意义的研究也不断增加,从大分子蛋白质标志物到基因肿瘤标志物等达几十种之多,如c-erbB-2、uPA、VEGF、BRCA、muc-l、CD44、maspin、mdr1 等等,其中对c-erbB-2(HER-2)研究比较深入,研究发现c-erbB-2过度表达的乳腺癌细胞倍增速度快、侵袭性强、内分泌依赖性差、易产生化疗耐药性。但c-erbB-2 作为乳腺癌特异性诊断及预测指标其特异性及灵敏性均不够非常理想,如乳腺癌初诊病例中只有20%～30%的病人存在着c-erbB-2 的过度表达,很多其它恶性上皮肿瘤(卵巢癌、前列腺癌及鼻咽癌等等)也可见c-erbB-2 过度表达。另外,部分c-erbB-2 阴性病例预后也很差。因此,探求更敏感、更特异的乳腺癌相关指标有着非常重要的意义。maspin 基因是1994 年发现的一种肿瘤抑制基因,它是丝氨酸蛋白酶抑制剂的一种,文献报道maspin 基因变化在乳腺癌发生、发展过程中起着重要的作用。BCSG1 基因是1997 年Ji 等利用直接差异cDNA 序列分析克隆出的一组新的人类基因,是SNCs(neural protein synuclein)家族的新成员,因发现其中一种在浸润性乳腺癌中高度表达,而在正常乳腺及良性病变中几乎不表达,故命名为乳腺癌特异基因(breast cancer specific gene 1,BCSG1)。有人通过原位杂交和RT-PCR 方法研究了BCSG1 基因在乳腺癌中的表达情况,认为其表达与乳腺癌的发生、发展有关,但研究病例数比较少。文献中尚未见maspin、BCSG1 在乳腺癌中的表达情况及其临床病理意义的大宗病例的研究报道。有鉴于此,为进一步探究maspin、BCSG1 在乳腺癌的发生、发展中作用的可能机制及其在乳腺癌生物学行为、诊断及预后判定方面的意义,本研究采用SP 免疫组织化学技术分别检测了乳腺癌及其癌旁组织中maspin、BCSG1 蛋白的表达情况,同时结合临床、病理形态学资料,对比观察分析多种生物学指标的表达情况及其相关性,并就maspin、BCSG1更多还原

【Abstract】 Breast cancer is one of most common malignant tumors in woman. As the rapid development of immunological and molecular biological technologies in the past a few decades, the clinical and pathological significance of many biomarkers such as c-erbB-2, uPA, VEGF, BRCA, muc-1, CD44, mdr1, etc, have been studied in breast cancer at cellular and molecular levels. Among all these biomarkers, c-erbB-2 (HER-2) is widely studied and considered to be of important prognostic values. Studies have shown that the breast cancer overexpressing c-erbB-2 have shorter cell doubling time, stronger invasiveness, poorer endocrine-dependency and more vulnerable to drug resistance. But using c-erbB-2 as a diagnosis and prognostic marker is still not satisfactory enough considering its specificity and sensitivity because only 20%～30% newly diagnosed breast cancer patients overexpress c-erbB-2 and overexpression of c-erbB-2 could be found in many other malignant epithelial tumor (such as ovarian carcinoma, prostate carcinoma and nasopharyngeal carcinoma, etc). Up to now, searching for better diagnosis and prognostic indicators for breast cancer is still the challenge the oncologists are facing. maspin is a tumor suppressive gene found in 1994. It is a member of the serpin family of protease inhibitors. Studies have shown that the change of maspin may play an important role in carcinogenesis and progression of breast cancer. BCSG1 gene is identified and cloned by direct-differential cDNA sequencing approach and defined as a new family member of human brain SNCs (neural protein synuclein). It has been found that this gene expressed highly in invasive breast cancer, while almost no positive expression in normal breast and benign lesions was found. Thus, it was named as breast cancer specific gene 1 (BCSG1). Though In situ hybridization and RT-PCR study suggested BCSG1 might play an important role in the carcinogenesis and progression of breast cancer. Up to now, immunohistochemical and molecular biological studies on the expression of maspin and BCSG1 in large number of breast cancer cases have not been seen. In order to further explore the putative roles of maspin and BCSG1 in carcinogenesis and progression of breast cancer, and to evaluate their possible values in the diagnosis and prognostic prediction of breast cancer, the expression of maspin and BCSG1 and their clinical pathological significance in breast cancer tissues and tissues adjacent to breast cancer were studied with immunohistochemical method. At the same time, the relationship between the expressions of BCSG1, c-erbB-2 and the expressions of several biological markers were analyzed. On the basis of the afore-mentioned work, the effects and the putative significance of neoadjuvant chemotherapy on the expression of maspin and BCSG1 in breast cancer were evaluated with immunohistochemical staining and RT-PCR methods. PART Ⅰ The Expression of maspin in Breast Cancer and Tissues Adjacent to Breast Cancer Objective: To explore the significance of expression of maspin in breast cancer and tissues adjacent to breast cancer. Methods: SP immunohistochemical method was used to assess the expression of maspin in 104 invasive breast cancer, 26 carcinoma in situ, 63 tissues adjacent to breast cancer (including 20 epithelial atypical heperplasia, 43 simple heperplasia lesions) and 10 normal breast tissues. Meanwhile, the relationship between the expression of maspin and ER, PR, c-erbB-2, Cath-D, VEGF were analyzed. The results were evaluated on the basis of pathologicalfactors such as lymph nodes metastases, histological grading, etc. Results: The immunoreactivity of maspin was seen in the nuclei and cytoplasm. High maspin expression in nuclei was seen in all myoepithelial cells of the normal breast tissues (100%), while that for invasive breast cancer, carcinoma in situ, atypical hyperplasia and simple heperplasia lesions in tissues adjancent to cancer was 49.0%, 69.2%, 65% and 76.7% respectively. The high nuclear expression rates of maspin in breast cancer, atypical hyperplasia, simple hyperplasia lesions were all siginificantly lower than that in normal control (P<0.01), and a decreasing tendency in the nuclear expression of maspin could be found from normal control to simple hyperplasia, atypical hyperplasia, carcinoma in situ and invasive carcinoma. In the contrary to the nuclear expression, immunohistochemical analysis showed that almost no cytoplasmic expression of maspin in normal tissues could be seen, while the cytoplasmic expression of maspin was detected in 63.5% of invasive breast cancers, 69.2% of carcinoma in situ and 85.0% of atypical heperplasia lesions in tissues adjancent to cancer and 51.2% simple heperplasia lesions in tissues adjancent to cancer were also found to have positive cytoplasmic expression of maspin. The cytoplasmic expression of maspin in breast cancer, carcinoma in situ, atypical hyperplasia and simple hyperplasia lesions were all higher than that in normal control (P<0.01). Higher cytoplasmic maspin expression was detected in atyptical heperplasia lesions in tissues adjacent to cancer than in breast cancer cells. Logistic regression analysis showed that the nuclear expression of maspin in invasive breast cancer were positively correlated with ER expression, negatively correlated with the expressions of c-erbB-2 and histological grading, and no correlation could be found between nuclear expression of maspin and that of PR, Cath-D and VEGF. The expression of maspin in cytoplasma in invasive breast cancer was irrelative to pathological features, histological grading and lymph nodes metastases. Conclusion: maspin may be involved in the carcinogenesis and progression of breast cancer. Nuclear expression of maspin was closelycorrelated with histological grading and the expression of ER, c-erbB-2, while no clinical siginificance was found for cytoplasmic expression of maspin in invasive breast cancers. PART Ⅱ The Expression of BCSG1 in Breast Cancer and Tissues Adjacent to Breast Cancer Objective: To explore the significance of BCSG1 expression in breast cancer and tissues adjacent to breast cancer. Methods: SP immunohistochemical method was used to assess the expression of BCSG1 in 196 invasive breast cancer, 45 carcinoma in situ, 64 tissues adjacent to breast cancer (including 33 epithelial atypical heperplasia, 31 simple heperplasia lesions) and 10 normal breast tissues.The relationship between BCSG1 expression in invasive breast cancer and pathological grading was analyzed. Results: The high expression rate of BCSG1 in normal breast tissues was 10%, while that for invasive breast cancer, carcinoma in situ, atypical hyperplasia and simple heperplasia lesions in tissues adjancent to cancer was 65.8%, 44.4%, 45.5% and 12.9% respectively. The high expression rates of BCSG1 in breast cancer, atypical hyperplasia were all siginificantly higher than that in normal tissues (P<0.01), and a increasing tendency in the expression of BCSG1 could be found from normal control to simple hyperplasia, atypical hyperplasia, carcinoma in situ and invasive carcinoma. The expression of BCSG1 was significantly related to pathological grading of invasive breast cancer.Conclusion: BCSG1 gene may play an important role in the carcinogenesis and progression of breast cancer. PART ⅢPrognostic Significance of maspin, BCSG1 and c-erbB-2 Expression in Breast Cancer Objective: To evaluate the prognostic significance of maspin、BCSG1、c-erbB-2 expression in breast cancer. Methods: The expressions of maspin, BCSG1 as well as c-erbB-2, ER, PR were detected with SP immunohistochemical method in 137 cases of invasive breast cancer cases with over 5 year follow-up data. Cox model were used to analyze the prognostic significance of maspin, BCSG1 as well as c-erbB-2 expression in the breast cancer cases. Results: The positive expression rates of maspin, BCSG1 and c-erbB-2 were 48.9%, 67.2% and 27.7% respectively. They all had significant correlation with survival time (P<0.01). The nuclear expression of maspin was negatively correlated with clinical stage and lymph nodes metastases (P<0.01、P<0.05), positively correlated with ER expression and survival time (P<0.05、P<0.01). The expression of BCSG1 was positively correlated with histological grading (P<0.05), negatively correlated with survival time (P<0.01). The expression of c-erbB-2 was positively correlated with clinical stage (P<0.05), negatively correlated with survival time (P<0.01). Conclusion: The high expression of BCSG1 and low nuclear expression of maspin significantly correlated with the shorter survival time, so they may be used as the possible predictors of prognosis as c-erbB-2 in breast cancer.PART ⅣEffect of Neoadjuvant Chemotherapy on maspin Expression in Breast Cancer Objective: To explore the effects of neoadjuvant chemotherapy of CAF (Cyclophosphamide, Adriamycin and Fluorouracil) regimen on the expression of maspin in breast cancer. Methods: Specimens were obtained from 38 breast cancer patients with neoadjuvant CAF regimen chemotherapy (CAF group) and 173 breast cancer patients without neoadjuvant chemotherapy (control group). maspin expression was detected with SP immunohistochemistry. Correlation of maspin expression and response to CAF neoadjuvant chemotherapy was analyzed. Results: Overall response rate to neoadjuvant chemotherapy in the patients with CAF group was 81.6%. Though no significant difference in expression of maspin in cytoplasm was found between CAF group and control group (93.6% vs 97.4%, P>0.05), the strong expression rate of maspin in nuclei in CAF group was significantly higher than that in control group (50.0% vs 40.5%, P<0.05). The very strong nuclear expression (+++) rates in CAF group was 7.9%, which was significantly higher than that in control group (0.6%, P<0.05). Among the patients with CAF, the strong nuclear expression of maspin in PR (partial response, grade Ⅱ) cases was significantly higher than that in NR (no response, grade Ⅲ) cases (P<0.01). Conclusion: Neoadjuvant chemotherapy of CAF regimen could increase the nuclear expression of maspin at protein level in breast cancer. PART Ⅴ Effect of Neoadjuvant Chemotherapy on the Expression of BCSG1 in Breast Cancer Objective: To explore the effects of neoadjuvant chemotherapy of CAF regimen on the expression of BCSG1 in breast cancer. Methods: Specimens were obtained from 34 breast cancer patients with neoadjuvant CAF regimen chemotherapy (CAF group) and 110 breast cancer patients without neoadjuvant chemotherapy (control group). BCSG1 expression was detected with SP immunohistochemistry and the therapeutic response of neoadjuvant chemotherapy was evaluated by pathological observation. The relationship between expression of BCSG1 and pathological response to neoadjuvant chemotherapy was analyzed. Results: In CAF group, the overall response rate to the neoadjuvant chemotherapy was 79.4%. The high expression rate of BCSG1 in CAF group was significantly lower than in control group (29.4% vs 64.5%, P<0.01). Among the cases with CAF neoadjuvant chemotherapy, the high expression rate of BCSG1 in PR (grade Ⅱ) cases was significantly lower than that in NR ( grade Ⅲ) cases (P=0.002). Conclusion: Neoadjuvant chemotherapy of CAF regimen could decrease the expression of BCSG1 at protein level in breast cancer. Key words: breast cancer; neoadjuvant chemotherapy; immunohistochemistry; BCSG1更多还原