| Background and objectiveBreast cancer in women in the world within the scope of women malignant tumor incidence. Female breast cancer incidence of a disease urban and rural areas in China were rising trend year by year, but its occurrence and development of many link is currently still not entirely clear. Including breast infiltrating ductal carcinoma are the main types of breast cancer, about80%of all breast cancer. Breast infiltrating ductal carcinoma metastasis is main way for lymph node metastasis, lymph nodes effectively transfer, may prolong survival and to reduce mortality, or even cure the tumor type. Studies have found that hypoxia is the initiating factors of malignant transformation and tumor metastasis, spent nuclear factor-1alpha (HIF-1α) is anoxic conditions exist widely in nurturing a transcription factor, animal and human body to maintain its activity of tumor cells energy metabolism, angiogenesis, promoting new plays an important role in tumor proliferation and metastasis. Twist1protein is one of its key regulatory role in embryonic development of transcription factors, through influence the apoptosis of tumor cells, inhibition of differentiation, inducing epithelial-mesenchymal transition (EMT), participate in the drug resistance of tumor, tumor angiogenesis, play an important role in invasion and metastasis of malignant tumor. This topic in cases of primary tumors infiltrating ductal breast carcinoma and corresponding normal breast tissue adjacent to carcinoma and local axillary lymph node as the research object, using immunohistochemical techniques detect alpha and Twist1, HIF-1α protein expression of the two indicators, discusses HIF-1α, Twistl protein expression in breast invasive ductal carcinoma and explore the clinical significance of its relationship with lymph node metastasis of gastric cancer.Method1. Specimen collection and handling, nanfang hospital, southern medical university were randomly selected, shenzhen maternity and child care, the eighth people’s hospital of shenzhen in2010～2012by surgical removal of the archive with complete medical records of61cases of breast invasive ductal carcinoma specimens of paraffin embedding. Each case including primary tumors,2cm away from the tumor, above the normal breast tissue adjacent to carcinoma and local axillary lymph nodes. According to the situation of lymph node metastasis in lymph node metastasis groups all cases with lymph node metastasis group and31cases of lymph node metastasis group, lymph node metastasis group of30cases. Lymph nodes metastasis lymph nodes and lymph node metastasis group than normal lymph nodes transfer group. Sex in all specimens by formaldehyde solution fixed, conventional paraffin embedding,4microns thin serial section.2. Clinical and pathological data:61cases breast infiltrating ductal carcinoma of early cure patients, preoperative without put, chemotherapy for cancer treatment. All61cases for women, ages26to73years old, average45.79±1.42years old.31cases with lymph node metastasis group, lymph node metastasis group of30cases. TNM stages:stage Ⅰ8cases, stage Ⅱ23cases, stage Ⅲ30cases. Histologic classification: G11case, G229cases, G331cases.3.Reagents:HIF-1α rabbit anti human monoclonal antibody from Epitomics companies in the United States, Twistl mouse anti human monoclonal antibody from Abcam British company, ready-to-use immunohistochemical ultra sensitive SP/rabbit (rat) kit, enhanced DAB kit, rapid dyeing liquid wood blank cells were purchased from fuzhou new biotechnology development co., LTD.4. Immunohistochemical staining, immunohistochemical ultra sensitive SP three-step method is used to detect primary tumors and normal breast tissue adjacent to carcinoma and local axillary lymph nodes HIF-1α, Twist1protein expression changes, concrete steps to consult detection kit instructions.5. Results judgement:HIF-1α and Twist1protein expression by being future-proof-CH2optical microscope for testing. With PBS instead of a resistance as negative control, with a known HIF-1α and Twist1protein positive breast infiltrating ductal carcinoma biopsy as a positive control. HIF-1α and nucleus of Twist1proteins expression said biologically active form of transcription factors. HIF-1α, Twist protein positive signals inside the cell nucleus is brown or tan, alpha, Twistl and HIF-1α protein expression levels by shading percentage of tumor cells and normal mammary gland cells and staining intensity of scoring points.6. Quality control:pathological diagnosis by two senior pathologists, suspicious of pathology, the first person by the expert review, diagnosis is not as a case study. Immunohistochemical results determined by experienced researchers observe judgement, and by the expert review suspicious.7. Statistical methods:the observation indexes after quantification treatment input computer, using SPSS18.0statistical software for statistical analysis, compared with paired T test measurement data, categorical data compared using the chi-square Fisher’s exact probability method, correlation analysis by Spearman rank correlation analysis, P<0.05for statistical significance.Results:HIF-laexpress positioning within the nucleus, a brown or tan. Overall breast infiltrating ductal carcinoma of the HIF-la negative expression in cancer tissue in13cases, accounted for21.3%, the positive expression of32cases, positive rate was78.7%. Which expressed weakly positive (+) in17cases, accounting for27.9%; Moderate positive expression (++) in22cases, accounting for36.1%; Strong positive expression (+++) in8cases, accounting for14.8%. In normal breast tissue adjacent to carcinoma HIF-1α negative expression of47cases, accounted for77.0%, positive expression in14cases, the positive rate is23.0%. Which expressed weakly positive (+) in7cases, accounting for11.5%; Moderate positive expression (++) in7cases, accounting for11.5%; Strong positive expression (+++) is0. HIF-1α negative expression in lymph nodes for39cases, accounted for63.9%, positive expression in22cases, the positive rate was36.1%. Which expressed weakly positive (+) in7cases, accounting for11.5%; Moderate positive expression (++) in14cases, accounting for23.0%; Strong positive expression (+++) in1cases, accounted for1.6%. Overall the paired T test statistical analysis, breast infiltrating ductal carcinoma tissues of HIF-1α positive rate is greater than the normal breast tissue adjacent to carcinoma HIF-1α positive rate, the difference was statistically significant (P<0.001); Breast infiltrating ductal carcinoma tissues of HIF-1α positive rate is greater than the lymph node HIF-1α positive rate, the difference was statistically significant (P<0.001); HIF-1α lymph node positive rate is greater than the normal breast tissue adjacent to carcinoma HIF-1α positive rate, the difference was statistically significant (P=0.034). Can therefore be concluded that on HIF-1α positive rate, breast infiltrating ductal carcinoma tissue lymph nodes>> normal breast tissue adjacent to carcinoma.Twist1protein expression is also positioning within the nucleus, the same show brown or tan. Overall breast infiltrating ductal carcinoma Twist1protein negative expression in cancer tissue for17cases, accounted for27.9%, positive expression is44cases, positive rate was72.1%. Which expressed weakly positive (+) in9cases, accounting for14.8%; Moderate positive expression (++) in26cases, accounting for42.6%; Strong positive expression (+++) in9cases, accounting for14.8%. Twistl protein negative expression in normal breast tissue adjacent to carcinoma of51cases, accounted for83.6%, the positive expression of10cases, the positive rate is16.4%. Which expressed weakly positive (+) there are4cases, account for6.6%; Moderate positive expression (++) in5cases, accounting for8.2%; Strong positive expression (+++) in1cases, accounting for1.6%. Twistl protein negative expression in lymph nodes for33cases, accounted for54.1%, the positive expression of28cases, the positive rate was45.9%. Which expressed weakly positive (+) there are4cases, account for6.6%; Moderate positive expression (++) in20cases, accounting for32.8%; Strong positive expression (+++) in4cases, accounting for6.6%. Overall the paired T test statistical analysis, breast infiltrating ductal carcinoma tissues of Twist1protein positive rate is greater than the normal breast tissue adjacent to carcinoma Twist1protein positive rate, the difference was statistically significant (P <0.001); Breast infiltrating ductal carcinoma tissues of HIF-la positive rate is greater than the lymph node Twist1protein positive rate, the difference was statistically significant (P<0.001); Twist1protein positive rate is greater than the normal breast tissue adjacent to carcinoma lymph node Twistl protein positive rate, the difference was statistically significant (P<0.001). Can be concluded, therefore, on the Twistl protein positive rate, breast infiltrating ductal carcinoma tissue lymph nodes>> normal breast tissue adjacent to carcinoma.Lymph node metastasis group and infiltrating ductal carcinoma of breast carcinoma tissues HIF-1α negative expression in3cases, accounted for9.7%, the positive expression of28cases, the positive rate was90.3%. Which expressed weakly positive (+) in6cases, accounting for19.4%; Moderate positive expression (++),15cases (48.4%); Strong positive expression (+++) in7cases, accounting for22.6%. Without lymph node metastasis group and infiltrating ductal carcinoma of breast carcinoma tissues HIF-1α negative expression in13cases, accounted for21.3%, the positive expression of32cases, positive rate was78.7%. Which expressed weakly positive (+) in17cases, accounting for27.9%; Moderate positive expression (++) in22cases, accounting for36.1%; Strong positive expression (+++) in8cases, accounting for14.8%. Lymph node metastasis group and normal breast tissue adjacent to carcinoma of HIF-1α negative expression in21cases, accounted for67.7%, the positive expression of10cases, the positive rate is32.3%. Which expressed weakly positive (+) in5cases, accounting for16.1%; Moderate positive expression (++) in5cases, accounting for16.1%; Strong positive expression (+++) is0. Without lymph node metastasis group and normal breast tissue adjacent to carcinoma of HIF-1α negative expression of47cases, accounted for77.0%, positive expression in14cases, the positive rate is23.0%. Which expressed weakly positive (+) in7cases, accounting for11.5%; Moderate positive expression (++) in7cases, accounting for11.5%; Strong positive expression (+++) is0. HIF-1α in lymph node metastasis group and lymph node negative expression in14cases, accounted for45.2%, positive expression in22cases, the positive rate was36.1%. Which expressed weakly positive (+) in5cases, accounting for16.1%; Moderate positive expression (++) in11cases, accounting for35.5%; Strong positive expression (+++) in1cases, accounting for3.2%. HIF-1α in lymph node metastasis group and lymph node negative expression for39cases, accounted for63.9%, positive expression in22cases, the positive rate was36.1%. Which expressed weakly positive (+) in7cases, accounting for11.5%; Moderate positive expression (++) in14cases, accounting for23.0%; Strong positive expression (+++) in1cases, accounting for1.6%. The paired T test statistical analysis:lymph node metastasis group, breast infiltrating ductal carcinoma tissues of HIF-1α positive rate is greater than the normal breast tissue adjacent to carcinoma HIF-1α positive rate, the difference was statistically significant (P<0.001); Breast infiltrating ductal carcinoma tissues of HIF-1α positive rate is greater than the lymph node HIF-la positive rate, the difference was statistically significant (P<0.001); HIF-1α lymph node positive rate is greater than the normal breast tissue adjacent to carcinoma HIF-1α positive rate, the difference was statistically significant (P=0.034). Can therefore be concluded that on HIF-1α positive rate, breast infiltrating ductal carcinoma tissue lymph nodes>> normal breast tissue adjacent to carcinoma. No lymph node metastasis group, breast infiltrating ductal carcinoma tissues of HIF-1α positive rate is greater than the normal breast tissue adjacent to carcinoma HIF-1α positive rate, the difference was statistically significant (P<0.001); Breast infiltrating ductal carcinoma tissues of HIF-1α positive rate is greater than the lymph node HIF-1α positive rate, the difference was statistically significant (P<0.001); HIF-1α lymph node positive rate is greater than the normal breast tissue adjacent to carcinoma HIF-1α positive rate, the difference was statistically significant (P=0.034). Can therefore be concluded that on HIF-1α positive rate, breast infiltrating ductal carcinoma tissue lymph nodes>> normal breast tissue adjacent to carcinoma.Lymph node metastasis group and infiltrating ductal carcinoma of breast carcinoma tissues Twist1protein negative expression of17cases, accounted for27.9%, the positive expression of44cases, positive rate was72.1%. Which expressed weakly positive (+) in9cases, accounting for14.8%; Moderate positive expression (++) in26cases, accounting for42.6%; Strong positive expression (+++) in9cases, accounting for14.8%. Group without lymph node metastasis, the infiltrating ductal carcinoma of breast carcinoma tissues Twistl protein negative expression of17cases, accounted for27.9%, a positive expression for44cases, positive rate was72.1%. Which expressed weakly positive (+) in9cases, accounting for14.8%; Moderate positive expression (++) in26cases, accounting for42.6%; Strong positive expression (+++) in9cases, accounting for14.8%. Lymph node metastasis group and normal breast tissue adjacent to carcinoma of Twistl protein negative expression of51cases, accounted for83.6%, the positive expression of10cases, the positive rate is16.4%. Which expressed weakly positive (+) there are4cases, account for6.6%; Moderate positive expression (++) in5cases, accounting for8.2%; Strong positive expression (+++) in1cases, accounting for1.6%. Without lymph node metastasis group and normal breast tissue adjacent to carcinoma of Twistl protein negative expression of51cases, accounted for83.6%, the positive expression of10cases, the positive rate is16.4%. Which expressed weakly positive (+) there are4cases, account for6.6%; Moderate positive expression (++) in5cases, accounting for8.2%; Strong positive expression (+++) in1cases, accounting for1.6%. Twistl protein in lymph node metastasis group and lymph node negative expression for33cases, accounted for54.1%, the positive expression of28cases, the positive rate was45.9%. Which expressed weakly positive (+) there are4cases, account for6.6%; Moderate positive expression (++) in20cases, accounting for32.8%; Strong positive expression (+++) in4cases, accounting for6.6%. Twistl protein in group without lymph node metastasis, lymph node negative expression for33cases, accounted for54.1%, the positive expression of28cases, the positive rate was45.9%. Which expressed weakly positive (+) there are4cases, account for6.6%; Moderate positive expression (++) in20cases, accounting for32.8%; Strong positive expression (+++) in4cases, accounting for6.6%. The paired T test statistical analysis:lymph node metastasis group, breast infiltrating ductal carcinoma tissues of Twistl protein positive rate is greater than the normal breast tissue adjacent to carcinoma Twistl protein positive rate, the difference was statistically significant (P<0.001); Breast infiltrating ductal carcinoma tissues of HIF-la positive rate is greater than the lymph node Twistl protein positive rate, the difference was statistically significant (P <0.001); Twistl protein positive rate is greater than the normal breast tissue adjacent to carcinoma lymph node Twistl protein positive rate, the difference was statistically significant (P<0.001). Can be concluded, therefore, on the Twistl protein positive rate, breast infiltrating ductal carcinoma tissue lymph nodes>> normal breast tissue adjacent to carcinoma. No lymph node metastasis group, breast infiltrating ductal carcinoma tissues of Twistl protein positive rate is greater than the normal breast tissue adjacent to carcinoma Twistl protein positive rate, the difference was statistically significant (P<0.001); Breast infiltrating ductal carcinoma tissues of HIF-la positive rate is greater than the lymph node Twistl protein positive rate, the difference was statistically significant (P<0.001); Twistl protein positive rate is greater than the normal breast tissue adjacent to carcinoma lymph node Twistl protein positive rate, the difference was statistically significant (P<0.001). Can be concluded, therefore, on the Twistl protein positive rate, breast infiltrating ductal carcinoma tissue lymph nodes>> normal breast tissue adjacent to carcinoma.HIF-la positive expression in cancer tissue and the patient’s age, histological grading, female progesterone receptor (ER, PR) and human epidermal growth factor receptor2(HER-2) and proliferation index (Ki-67) no correlation, positive correlation with the TNM staging (r=0.322, P=0.04); Twistl positive expression and the patient’s age, histological grading, female progesterone receptor (ER, PR) and human epidermal growth factor receptor2(HER-2) and proliferation index (Ki-67) with no correlation (P>0.05), and TNM stage had positive correlation (r=0.43, P=0.43),61cases of breast invasive ductal carcinoma tissues HIF-la and Twistl and positive expression of39cases (63.9%), while the negative expression of8cases (13.1%). HIF-1α and Twistl in cancerous tissue protein expression was positively correlation (r=0.391, P=0.391).Conclusion1. HIF-1α and Twist1in lymph node metastasis and has high expression in breast invasive ductal carcinoma, both in the lymph node metastasis plays an important role in the process, may be closely related to the prognosis of breast infiltrating ductal carcinoma.2. HIF-1α over expression could be breast infiltrating ductal carcinoma lymph node metastasis of initial factor, Twist1activation over expression could indicate cancer cell metastasis ability. |
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