The Detection Of Breast Cancer Serum Markers With Suspension Array And High Performance Liquid Chromatogram

BackgroundBreast cancer is one of the most common malignancies among women around the world, seriously threatening women's physical and mental health. The incidence of breast cancer around the world has shown a steady upward trend, in the late 20th century the world's annual statistics has show that about 1.15 million people are diagnosed with breast cancer,410 thousand people die of this disease, and the patients are younger and younger. Breast cancer has became the leading cause of death among female patients. However, the etiology of breast cancer is not yet clear, its pathogenesis is very complex and many factors affect the incidence. In recent years, with the deepening of research in tumor molecular pathology and molecular biology, it is recognized that the occurrence and development of cancer is a gradual process involving multi-stage and multi-gene changes. A number of oncogene proteins encoded by the abnormal activation, over-expression and deletion of tumor suppressor genes, and malignant transformation caused by inactivating mutations and uncontrolled cell proliferation may be the molecular biological basis of tumor. New ideas of treatment put forward and the implementation of comprehensive treatment, the treatment of breast cancer has improved continuously. The overall survival rate is gradually increased, but recurrence and metastasis of breast cancer are still the main causes for treatment failure and death. Therefore, the study of biological behavior in breast cancer, and discovering the relevant biological markers of sensitivity will contribute to clinical work. HIF-1αand Her-2ECD are two independent prognostic factors of breast cancer, which have significant influence on the incidence of, development and metastasis of breast cancer. Due to their biological roles in breast cancer, they are implications of prognostic factors of breast cancer and have important functions in specific monoclonal antibodies to treat breast cancer, HIF-1αand Her-2ECD have increasingly become hot research issues in recent years. In the past domestic and foreign researches prevalent in the immunohistochemical study of breast cancer, immunohistochemical study of operation is relatively more complex, time-consuming, laborious, and can not detect the real-time and dynamic changes in patients, and there is no relevant reports about co-quantitative detection of HIF-1αand Her-2ECD in the serum of breast cancer patients. Glycine and alanine, regulated by HIF-1αchannel, which are important synthetic raw materials of tumor cells and metabolic products of tumor, play very pivotal roles in the occurrence, development and metastasis of the cancer, but no related research has ever been reported. So it is very important to look for a new detection method, quantitatively analyzing the tumor markers of related metabolic pathways, to increase the detection rate to the maximum.This research subject is to apply floating-chip technology combining with high-performance liquid chromatography with quantitative detection of change in content of HIF-1α, Her-2ECD, Ala and Gly content in breast carcinoma patients, analzing its relevance and its relationship with the internal breast cancer pathology, and the relationship between the biological characteristics, and approach the role of HIF-la, Her-2ECD, Ala and Gly in breast cancer diagnosis and monitoring of illness, as well as to assess its diagnostic value of breast cancer.ObjectivesThis study is based on chip technology combined with high-performance liquid chromatography suspended from the body's metabolic pathways of breast cancer patients to find the dynamic tumor markers to explore the intrinsic correlation between them, to analyze HIF-1α, Her-2ECD, glycine and alanine acid changes and clinical pathological factors and prognosis and study the prognosis of breast cancer. And to further explore the value and significance of her-2ECD, HIF-la, Ala and Gly in the diagnosis of breast cancer.Methods1. Cases from Breast surgery of Ningbo University School of Medicine Affiliated Hospital from 2006 to 2008, Primary breast infiltrating ductal carcinoma:117 cases. Postoperative recurrence of breast infiltrating ductal carcinoma:10 cases. Benign breast tumor:20 cases. breast cosmetic surgery of the health:31 cases(The normal control group). Primary invasive ductal breast cancer were not treated by preoperative radiotherapy, chemotherapy, endocrine therapy and immunotherapy.2. Serum specimen collection:with the patient's own consent, each selected object is collected blood 10ml in median cubital vein in the early morning. Put them aside for 120 minutes, then take serum after 3000r/min decentralization for 20 minutes, number, and preserve in -80℃refrigerator, screening serum of the breast invasive ductal carcinoma and benign tumor and breast cosmetic surgery of the health patients for inspection according to surgery pathological findings. 3. The collection of tissue samples:with the consent of selected cancer patients, in which surgery would be surgical resection of malignant or benign tumor specimens were cut without necrosis of tumor tissue, each about 1g, and the removal of breast cosmetic surgery at the same time normal tissue of about 1g, and then rinse off bloody with ice saline, immediately placed in liquid nitrogen and stored at -80℃low temperature refrigerators. Before test, take some tumor tissues, respectively, and uonicate to 10% homogenatation under the ice bath after weighing with the ice-cold 0.9% saline by the ratio of 150mg:1.5ml,4000r/min (centrifugal radius 8cm) centrifuge for 10min.The supernatant stored frozen for testing.4. Sample detection:Serum HIF-1αand Her-2ECD content by suspension chip detection kit (detection range>1pg), and enzyme-linked immunosorbent assay (ELISA) is determinated as controls (HIF-la detection range:10000-156pg; Her-2ECD detection range:> 0.39 ng/ml). Serum and tissues of glycine and alanine are tested by high-performance liquid chromatography, using software packages to calculate concentration.5. Statistical analysis:SPSS13.0 data were used, all data are mean±standard deviation (x±s). Use analysis of variance (F test)to compare between the two groups. T test was used to compare among two groups, P<0.05 for the difference, there were statistical significances. On the liquid-phase ELISA or HPLC-chip method and the results of tests were using Spearson rank correlation analysis. Test level,α=0.05.Results1. Clinical Case Data:178 cases in this group were female, age distribution from 27 to 91 years old, with a median age of 49.2 years old. Primary invasive ductal breast carcinoma of 117 patients, aged from 27 to 91 years old (mean 51.56 years); postoperative recurrence of breast invasive ductal carcinoma of 10 cases, from 35 to 57 years old (mean 47.6 years); 20 cases of benign breast tumor, age 40 to 63 years old (mean 50.38 years); breast cosmetic surgery of 31 cases(The normal control group), aged from 27 to 65 years old (mean 48.77 years); Four groups selected for the patient's age were not statistically significant (P>0.05), All women.Primary invasive ductal breast cancer were not associated preoperative radiotherapy, chemotherapy, endocrine therapy and immunotherapy, clinical pathological stage:Ⅰperiod of 25 cases (21.5%),Ⅱperiod of 61 cases (52.1%),Ⅲperiod of 19 cases (16.2%);Ⅳ,12 cases (10.3%) histopathological grading:G1 phase 12 cases (10.3%), G2 phase 41 cases (35.0%), G3 period of 64 cases (54.7%); tumor size: T1 group≤2cm 41 Li (35.0%), T2 Group> 2cm,≤5cm 61 Li (52.1%), T3 group> 5cm15 cases (12.8%); lymph node metastasis:69 cases without metastasis (59.0%), the transfer of 48 patients (41%).2. Results of suspension micro-array test2.1 Primary invasive ductal carcinoma (166.18±59.15) and recurrent invasive ductal carcinoma (231.72±38.39) of serum levels of HIF-1αwere higher than benign tumor group (47.77±15.26) and normal control group (47.84±11.36) (P<0.01); Recurrent infiltrating ductal carcinoma of HIF-1αserum levels were significantly higher than those in primary invasive ductal carcinoma of (P<0.01和P<0.05); There were not statistically significant between the benign tumor group and normal control group serum and tissue HIF-1αlevels. Primary invasive ductal carcinoma of serum levels of HIF-1 levels in clinical and pathological staging of one (150.96±38.85), between the two groups (149.52±52.96) there were no statistical significance(P>0.05), but there were a significant difference between three (189.03±60.36), four stages (249.54±44.84) and one, two stages (P<0.01); and with reduce histological grade (well-differentiated, moderately differentiated and poorly differentiated), its content gradually elevated (119.80±23.28 154.39±52.67,182.42±61.79), there were a significant difference (P<0.01); tumor when the diameter of T3 (diameter>5cm), the serum levels of HIF-1 were significantly higher (215.19±57.11) than the other two groups (T1,T2) (152.42±41.05,161.82±61.69), there were a significant difference (P<0.01); Tumor diameter, T1, T2 were not statistically significant (P>0.05). Lymph node metastasis in patients with serum levels of HIF-1 were significantly higher (197.48±55.28) than that in patients with lymph node metastasis did not occur (144.40±51.80), there were a significant difference(P<0.01), progesterone receptor-negative expression (186.16±59.35) was significantly higher than positive expression(153.23±55.69), there were a significant difference (P<0.01); expression of estrogen receptor-positive (159.91±56.41) and negative expression (179.20±63.27) had no significant difference (P>0.05).2.2 Primary invasive ductal carcinoma (11.20±6.116) and invasive ductal carcinoma of recurrence (17.61±3.63) of serum Her-2ECD levels were significantly higher than benign (4.91±3.40) and normal control group (4.56±2.95) (P<0.01), Recurrent infiltrating ductal carcinoma higher than primary invasive ductal carcinoma (P<0.01); There were no statistical significance between the benign tumor group and normal control serum Her-2ECD content(P>0.05).Primary invasive ductal breast cancer patients with Her-2ECD level of content and serum levels of HIF-1 were similar, to the clinical pathological staging one(7.89±2.94), two (9.30±4.48), there were no statistically significant (P>0.05), but there is a significant difference between three (15.44±6.38), four stages (21.67±2.76) and one, two stages (P<0.01); tumor tissue with low (13.04±6.53) credit-based were higher than those higher degree of differentiation (in the differentiation and well-differentiated) (9.33±5.33,7.80±1.12), there were a significant difference (P<0.01); Her-2ECD level of concentration with the increase of tumor diameter for increased levels (T3>T2>T1) (18.02±5.57,11.17±6.03,7.98±3.35), there is a significant difference (P<0.01); Lymph node metastasis (15.75±7.06) than those without metastasis (8.04±2.15) in patients with serum Her-2ECD levels were significantly increased, there were a significant difference (P<0.01); Estrogen and progesterone receptor positive and negative expression of Her-2ECD between the serum levels were different, and negative expression was significantly higher (13.49±6.43,13.05±6.42) than positive expression (10.10±5.68,10.01±5.64), there is a significant difference (P<0.01).3. Results of high-performance liquid chromatography3.1 Analysis found that serum Ala (338.57±97.37), Gly (176.06±46.9) content in the primary, recurrent invasive ductal carcinoma (240.71±74.33,135.49±31.34) were lower than the normal benign tumor group (414.49±64.94,320.22±42.84) and control group (423.37±80.57,342.19±48.15) (P<0.01); That tissues Ala (177.14±67.99), Gly (147.92±56.74) content in the primary, recurrent invasive ductal carcinoma (224.62±44.96,204.64±40.94) were lower than the normal benign tumor group (437.44±58.10,327.32±46.93) and control group too (406.50±66.85,338.06±49.60) (P<0.01);Ala, Gly in recurrent infiltrating ductal carcinoma group were significantly higher than those in primary invasive ductal carcinoma (P<0.01和P<0.05); While the Ala, Gly in the serum of recurrent infiltrating ductal carcinoma were significantly lower than that of primary invasive ductal carcinoma (P<0.01和P<0.05); Ala, Gly levels in serum and tissues were no statistical significance between the benign tumor group and normal control group.3.2 Primary invasive ductal carcinoma of the level of serum Ala levels reduce with the clinical and pathological stage gradually increase (433.51±67.69, 342.90±74.78,272.42±60.76,210.05±82.32), there were a significant difference between the levels of different stages (P<0.01); With the histological grade (pathology classification) increased, the level of serum Ala levels reduce (389.91±76.27,362.99±92.43,313.29±97.36), there were a significant difference between different groups (P<0.01); Serum Ala levels and tumor volume is also closely related to tumor volume increased with decreasing concentration(421.19±75.45,323.10±87.08,241.02±43.87), there are significant differences. And lymph node metastases were also inter-related occurred in patients, with lymph node metastasis (297.72±96.01) in Ala serum levels were significantly lower than the transfer did not occur (366.98±88.35) in patients with the level, there were a significant difference (P<0.01); Estrogen (343.37±90.31,328.58±111.27) and progesterone (349.52±96.23 321.67±97.75) receptors and serum in Ala levels were not statistically significant (P>0.05).3.3 As to primary invasive ductal carcinoma of Gly serum levels in clinical and pathological staging, there were no significant difference betweenⅠ(202.88±37.91),Ⅱperiod (186.18±36.57) (P>0.05), there were a significant difference betweenⅠ,Ⅱperiod andⅢ(152.54±30.75),Ⅳperiod (101.78±45.21) (P<0.01), its content reduced gradually with the classification increased; In tumor cell differentiation, there were no statistical significance between the well-differentiated group (217.50±25.89) and moderately differentiation group (199.12±39.35) (P>0.05), there were statistically significant between the well-differentiated group, moderately differentiation group and poorly differentiation group (153.520±42.089) (P<0.01), with the reduction in the degree of differentiation Gly levels were gradually reduced; Gly serum levels and tumor volume were also closely related to tumor volume increased with decreasing concentration (195.832±37.076, 177.103±45.486,130.260±43.177), there are significant differences(P<0.01). And lymph node metastases were also inter-related occurred in patients with lymph node metastasis, in the Gly serum levels (152.158±46.436) were significantly lower than the transfer did not occur in patients with the level (192.69±39.85), there were a significant difference (P<0.01); Estrogen (181.75±42.12,164.22±54.41) and progesterone receptors (182.73±45.163, 165.767±48.31) and serum of Gly levels were not statistically significant (P> 0.05).4. Correlation between HIF-1α, Her-2ECD and Ala, Gly:Spearson rank correlation analysis showed that HIF-1αwere positive correlation with Her-2ECD in serum (r=0.533,p=0.000);Ala, (r=-0.429, p=0.000) Gly (r=-0.481, p=0.000) content was negatively correlated with changes in breast cancer serum and tissue. It was closely negatively correlated between HIF-1α, Her-2ECD and Ala (r=-0.403, r=-0.413, p=0.000), Gly (r=-0.509, r=-0.481, p=0.000) in breast invasive ductal carcinoma serum, and showed a closely positive correlation with Ala (r=0.973, r=0.969, p=0.000), Gly (r=0.534, r=0.533, p=0.000) in organization. There were no statistically significance between the normal control group and benign tumors(P>0.05).5. HIF-1α(87.1%) and Gly(96.6%) is higher than Her-2CED(69.7%) and Ala(67.4%). in diagnosis on breast cancer. Early diagnosis on breast of HIF-1αis higher than Her-2CED(P<0.01); Her-2CED(84.6%) is higher than HIF-1α(67.5%), Ala(64.1%) and Gly(65.8%) in lymph node metastasis of breast cancer. Her-2CED is superior to HIF-1α(P<0.01) in lymph node metastasis of breast cancer.6. Through the correlation analysis, discriminant analysis and ROC curve analysis, suspended chip technology the ability to detect a single target goal of not less than ELISA kit, test results and contrast the results of tests kits was no significant difference, especially for the determination of the value of tumor samples, suspended Chip kit and the control kit relatively good correlation (r=0.995, r= 0.998, p=0.000)。Conclusion1. In serum, HIF-1αand Her-2ECD can be detected with high sensitivity, high-throughput and reliability by using Suspension Array Technology which had a better auxiliary value than ELISA kit in the diagnosis of breast cancer. Therefore, this theme had a viable method and a reliable result, that can be used for research project.2. The lever of HIF-1αand Her-2ECD in serum of breast invasive ductal carcinoma were overexpression, it were an indication of poor prognosis of the patients, its overexpression indicates that it played an important role in tumorigenesis and tumor induction. So dynamic detection of HIF-1αand Her-2ECD can be used as a real-time, dynamic information, to provide assistance for the development of making treatment program. Therefore, it was important for us to detect the level of HIF-1αand Her-2ECD in serum in the recurrence, early assessment of metastasis and prognosis of breast invasive ductal carcinoma.3. Ala and Gly involved in anabolic and energy metabolism of the tumor cells of the breast invasive ductal carcinoma, played an important role in the occurrence, development and progression of the breast invasive ductal carcinoma, and may become a new target of the targeted therapy.4. In serum, HIF-1α,Her-2ECD,Ala and Gly can be used as the early diagnosis of breast cancer, some more comprehensive indicators to evaluate the degree of malignancy, can reflect the biological characteristics of the breast invasive ductal carcinoma. With the basic and clinical research of HIF-1α,Her-2ECD,Ala and Gly in serum going further, it could be one of means in cancer treatment and prevention for targeted treatment.