Abstract Objective To investigate esophageal uPA and uPAR protein expression with clinicopathological factors and prognosis radiotherapy alone. Methods Immunohistochemical SP method detected 59 patients with esophageal cancer and 41 patients with esophageal cancer adjacent tissues uPA and uPAR protein expression analysis of the relationship between protein expression and clinicopathological factors and the impact of radiotherapy on prognosis. Results of uPA in 59 cases of esophageal carcinoma the uPAR protein positive expression rate was 76.27%, 77.97%, next to the 41 cases of esophageal cancer positive expression rates were 48.78%, 43.90%, next to the cancer tissue and cancer organizations in the two proteins The positive expression rate differences were statistically significant (P <0.01). Chest CT scan showed strong positive expression of the the triangular gap esophageal lesions level prevertebral disappeared uPA protein was 70.37%, significantly higher than 43.75% of the presence of the triangular gap, the difference was statistically significant (P = 0.040). Distant transfer of uPA protein expression was also significantly higher than distant metastasis, 100% and 68.89%, respectively, and the difference was statistically significant (P = 0.042). Further analysis revealed that of uPA, uPAR protein expression and prognosis of esophageal cancer is no significant correlation of uPA, uPAR expression of the two proteins showed significant positive correlation. Conclusion esophageal cancer the uPA protein related to the occurrence of high expression may be related to local tumor invasion and distant metastasis.

Key words esophageal squamous cell carcinoma; uPA; uPAR; immunohistochemistry; radiation therapy

Study of uPA and uPAR Protein Expression and Prognostic Factors in Esophageal Squamous Cell Carcinoma Treated by Radiotherapy

    ZHU Shu  chai, WANG Ya  fei, SHEN Wen  bin, LI Juan, SU Jing  wei, WANG Yu  xiang

    Department of Radiation Oncology, The Fourth Hospital of Hebei Medical University, Shijiazhuang 050011, ChinaAbstract: Objective To explore the relationship between the uPA, uPAR protein expression and the prognostic with clinical and pathologic factors in esophageal squamous cell carcinoma treated by radiotherapy.Methods We measured uPA, uPAR protein expression in 59 specimens of esophageal carcinoma and 41 peri  carcinoma tissues with immunohistochemistry, and analyzed the relationship between these protein expression and the clinicopathological parameters, and explored the prognostic factors in esophageal squamous cell carcinoma treated by radiotherapy alone.Results The positive rates of expression of uPA in specimens of esophageal carcinoma and the peri  carcinoma tissues were 76.27% and 48.78% respectively, and the difference had statistical significance (P <0.01). The rates of positive expression of uPAR in specimens of esophageal carcinoma and the peri  carcinoma tissues were 77.97% and 43.90% respectively, and the difference had statistical significance (P <0.01). The expression of uPA was significantly correlated with the status of fat interspace between the esophageal lesion and vertebrae in the CT scanning.The rates of strong positive expression was 43.75% in which the fat interspace existed, while it was 70.37% with the fat interspace elapsed, and there were statistical significance (P = 0.040). The rates of positive expression was 100% with distant metastasis patients, while it was 68.89% in those without distant metastasis, this difference had statistical significance (P = 0.042). The protein expression of uPA and uPAR did not significantly effect on prognosis and the short  term result of esophageal carcinoma.Conclusion Maybe the protein expression of uPA, uPAR were correlated with the local invasion and distant metastasis of the esophageal squamous cell carcinoma.

    Key words: Esophageal squamous cell carcinoma; uPA; uPAR; Immunohistochemistry; Radiotherapy

To explore the urokinase-type plasminogen activator substance (uPA), uPA receptor (uPAR) protein expression in esophageal cancer development, invasion and metastasis of this study, using immunohistochemical SP method detected 59 cases of esophageal mirror tissue next to the biting examination squamous cell carcinoma and 41 cases of cancer of uPA and uPAR protein expression, analysis of protein expression of uPA, uPAR and esophageal cancer clinicopathological factors, efficacy and prognosis after radiotherapy for esophageal cancer treatment new indicators of prognosis.

    1 Materials and Methods

    1.1 Selection of cases

    Radiation Oncology collected in our hospital from February 2002 to August 2004, the initial treatment of esophageal cancer patients before treatment line routine chest CT scan, abdominal ultrasound or CT scan, routine blood biochemistry and peripheral blood examination. All cases were in radiotherapy before gastroscopy room adjacent tissues derived cancer tissue and cancer (visual observation of normal mucosa Lugol’s solution staining is not stained area) cancer tissues were pathologically confirmed squamous cell carcinoma and adjacent tissues atypical hyperplasia . 36 males and 23 females, aged 37 to 82 years, an average of 63 years old, all patients underwent a simple radical radiotherapy. Our department [1] proposed staging clinical stage T1, 48 cases of T3 to (4) of 11 cases, N0 29 cases, N1 13 patients (both mediastinal lymph node metastasis), N2 17 cases ( The mediastinal and locked lymph node metastasis in 13 cases, only locked lymph node metastasis in 4 cases), TNM stage Ⅰ, Ⅱ of 31 cases, Ⅲ, Ⅳ 28 cases.

    1.2 specimen detection method

    The specimens were fixed in 10% neutral formalin solution, embedded in paraffin, 4μm slices, immunohistochemistry. The main reagents rabbit anti-human polyclonal antibody the uPA (Boster Biological Engineering Co., Ltd.); rabbit anti-human polyclonal antibody the uPAR (Boster Biological Engineering Co., Ltd.); biotinylated universal secondary antibody working solution (Beijing Zhongshan Biotechnology Company) ; horseradish peroxidase-labeled streptomycin avidin working solution (Beijing Zhongshan Biotechnology Company); DAB chromogenic reagent (Beijing Zhongshan Biotechnology Company). UPA, uPAR-positive stained sections with PBS instead of primary antibody as a blank control, known as the positive control.

    The 1.3 radiation treatment method

    First a thermoplastic membrane fixed position, marked the three-dimensional laser reference point to ensure the treatment of repetitive. CT scan analog positioning, application American Focus 3.0 CMS three-dimensional conformal radiation therapy planning system for the digital transmission of the scanned image, three-dimensional reconstruction, outlined the target of visible lesions (including the primary tumor and metastatic lymph nodes of the GTV), the upper and lower ends, respectively, external expansion 2.0 to 2.5cm, around the axially outer expansion of 1.0 ~ 1.5cm as the clinical target volume (CTV), and then evenly spread 0.3 ~ 0.5cm planned target volume (PTV), usually given three to four coplanar irradiation wild calculate the target volume and critical organ dose distribution, tumor prescription dose while ensuring that by the amount of normal tissue around the tolerated dose range. The whole group using conventional fractionation the irradiation 2Gy / d, 5F / W-, the total dose of 50 ~~ 70Gy, median dose 64Gy.

    1.4 uPA, uPAR staining results of determination method

    uPA protein positive brown particles in the cytoplasm of tumor cells, and its coloring strength higher than background nonspecific staining; brown particles appeared the uPAR protein positive for the tumor cell membrane, and its coloring strength higher than background nonspecific staining. Standard reference Iseki K [2], comprehensive judgments based on two aspects of the staining intensity and number of positive cells and transformed into a positive index: a staining intensity (0 = no, 1 = weak, 2 = medium, 3 = strong); positive cells b Number (0 = 0 to 5% positive stained cells, 1 = 5% to 50% of positive staining cells, 2 = 50% to 100% of positive cells), A + B score of 0 to 1, then the positive index 0; a + b is a score of 2, then the positive index of 1; a + b a score of 3, then the positive index is 2; a + b score of 4,5, then the positive index of 3. Comprehensive scoring <2 by negative expression ≥ 2 positive expression, comprehensive scoring ≥ 3 strong positive expression.

    1.5 statistical methods

    Statistics using SPSS 11.5 statistical analysis software package. Count data using the χ2 test or Fisher’s exact test, survival curves were plotted using the Kaplan  Meier method, and Log  rank test, Cox’s proportional hazards model to compare the impact of prognostic factors for survival, with P <0.05 as statistically significance.

    2 Results

    2.1 uPA, uPAR protein expression

    Esophageal squamous cell carcinoma, the positive expression rate of uPA protein adjacent tissues were 76.27% and 48.78%, respectively, and the difference was statistically significant (χ 2 = 8.036, P = 0.005); strongly positive expression rates were 55.93% and 12.20%, respectively. difference was also statistically significant (χ 2 = 19.641, P = 0.000), as shown in Figure 1, Table 1. Table 1 esophageal cancer and adjacent tissues of uPA and uPAR protein expression grouping total

    Esophageal cancer tissues, adjacent tissues in uPAR protein positive expression rate were 77.97% and 43.90%, respectively, between the two was statistically significant (χ2 = 12.182, P = 0.000); strongly positive expression rates were 47.46% and 9.76% , the difference was also statistically significant (χ 2 = 15.801, P = 0.000), as shown in Figure 2, Table 1.

    2.2 of uPA, uPAR protein expression with clinicopathological factors

    Chest CT scan shows prevertebral triangular gap exists uPA protein levels esophageal lesions strongly positive expression rate was 43.75%, while the triangular gap disappears strong positive expression rate of 70.37%, and the two groups was statistically significant (χ 2 = 4.210, P = 0.040). The occurrence of 14 cases of patients with distant metastasis, uPA protein positive expression rate was 100%, significantly higher than 68.89% of distant metastasis, the two groups was statistically significant (χ 2 = 4.121, P = 0.042), see Table 2. 0.05)。">UPAR protein expression was 92.86% (13/14), also higher than without distant metastasis was 73.33% (33/45), but did not reach statistical significance (P> 0.05) in patients with distant metastases. 0.05)。">Further studies showed that uPA, uPAR protein expression and patient age, gender, mediastinal lymph node metastasis after radiotherapy, the short-term effect, T stage, N stage, clinical stage and so no significant correlation (P> 0.05). UPA, uPAR expression of two proteins showed a significant positive correlation, the correlation coefficient was 0.526, P = 0.000. Table 2 esophageal uPA protein expression prevertebral the triangular gap and distant metastasis relations

    2.3 uPA, uPAR protein expression and survival

    Follow-up to the December 31, 2006, 2 patients were lost, lost to those lost to the date of death count, follow-up rate of 96.61%. Follow-up time of 3 to 60 months, the median follow-up time of 31 months. The term efficacy radiotherapy after complete remission rate of 54.24%, partial response rate of 45.76%, the total efficiency of 100%. uPA, uPAR protein have no significant impact on Radiotherapy recent efficacy and survival. Further analysis revealed that single factors such as patient age, X-ray lesion length, before radiotherapy sound hoarse, T stage, N stage, clinical stage esophageal recent survival related (P <0.05); Multivariate analysis showed that only T sub period, N staging prognostic factors are shown in Table 3. Whole group 1, 2 and 3-year survival rate after radiotherapy were 55.93%, 38.98%, 28.41%, a median survival of 15.0 months, as shown in Figure 3.

    3 Discussion

    The important characteristics of malignant tumors is to invade surrounding tissues and organs and form metastases in the distance, thus seriously threatening the lives of patients. Malignant tumor invasion and metastasis involving the complex relationship between the tumor cells and the host cells by a variety of gene regulation. Urokinase-type plasminogen activator (uPA), a serine protease uPA activated in addition to a direct role in the extracellular matrix, but also activation of plasminogen, so that a variety of components such as fibrin matrix, fibronectin,

    Overall survival after radiotherapy curve in Figure 3 the whole group

    Proteoglycan, laminin degradation. In addition, activation of plasmin can activate collagenase – collagen degradation, so that the main component of the basement membrane. Urokinase-type plasminogen activator receptor (uPAR) on the one hand by combination with uPA the local fibrinolytic function of cells, on the other hand uPAR can mediate uPA intracellular signal transduction, thereby affecting cell proliferation, chemotaxis adhesion, migration, and so on.

    The results of this study showed that the specimens of esophageal squamous cell carcinoma uPA, uPAR protein expression was significantly higher than its paraneoplastic tissue, as atypical hyperplasia to cancer formation, protein expression was significantly increased. Hewin DF et al [3] enzyme-linked immunosorbent assay studied 18 surgically resected esophageal cancer (10 cases of squamous cell carcinoma and adenocarcinoma) and normal tissue uPA, uPAR protein expression results showed squamous cell carcinoma and adenocarcinoma uPA and uPAR expression levels were higher than normal mucosa, which is consistent with the results of our study, suggesting that uPA may participate in the occurrence and progression of esophageal cancer.

    Tang et al [4] esophageal uPA overexpression, but with the degree of tumor differentiation and survival rate in patients with no significant correlation. The study found, the triangle between esophageal cancer staging is closely related to the factors that chest CT scan showed the esophageal lesions and vertebral body and the descending aorta space involvement, uPA strong positive rate than the triangle gap is not invaded significantly higher and statistically significant, as shown in Table 2. Shiomi et al [5] reported that uPA-positive patients with esophageal cancer tumor invasion force is strong, the overall 5-year survival rate was significantly lower. The study further observed that distant transfer of uPA positive expression rate was 100%, significantly higher than 68.89% of distant metastasis, which is consistent with the pre-basic research results that uPA can promote cell Table 3 esophageal cancer prognostic factors analysis

    Degradation of extracellular matrix proteins involved in tumor blood vessel formation, conducive to tumor cell metastasis [6], so that uPA is an accelerated local tumor invasion and metastasis of proteolytic enzymes, basement membrane and extracellular matrix degradation and destruction, to promote tumor cell invasion and metastasis.

    In recent years, the study results showed that uPA and uPAR interaction, interaction. UPAR activation of uPA binding, the original receptor complex through the accompanying effects on the cell membrane and plasminogen / plasmin close to each other, the activation of plasminogen into plasmin, while the activation of plasminogen is able to activate the metal protease involved in the hydrolysis of the extracellular protein. The results of this study show that the existence of a significant positive correlation between uPA, uPAR expression of two proteins. Prompt of uPA, uPAR protein expression between synergies may be involved in extracellular matrix degradation and tumor invasion and metastasis.

    The results of this study also showed that the protein expression of uPA, uPAR esophageal cancer radiotherapy prognosis was no significant effect, with Torzewski [7] less consistent results, he thinks the poor prognosis in patients with strong expression of uPA, while weak positive and prognosis of patients with negative expression was no significant difference. We believe that further analysis may be related to the number of cases too few cases a short follow-up time, and yet not to the 5-year survival period.

    Initial detection of uPA and uPAR protein expression help determine esophageal cancer invasion and metastasis potential, it is possible to provide a reference for the formulation and evaluation of treatment programs prognosis indicators.