| Gastric cancers is one of the most frequent cancers and the leading causes of cancer death worldwide. In China, based on two national mortality surveys conducted in 1970s and 1990s, despite a slight increase from the 1970s to early 1990s, remarkable declines in gastric cancer mortality were noticed in almost the entire population during the last decade in China. These declines were largely due to the dramatic improvements in the social economic environment,lifestyle,nutrition,education and health care system after economic reforms started two decades ago. But, in 2005,0.3 million deaths and 0.4 million new cases from gastric carcinoma has ranked the third most common cancer .Thus,gastric cancer remains a major health hazard in China,currently.The essential package of the preventition and control strategy for gastric cancer in China would focus on controlling helicobacter pylori infection, improving educational levels, advocating health diet and anti-tobacco campaign, searching for cost-effective early detection, diagnosis and treatment programs including for curable management and palliative care.In recent years,there have been many new developments in our understanding of the molecular biology of gastric carcinoma. Many studies have clearly demonstrated that multiple genetic alterations are responsible for the development,progression and metastasis of gastric carcinoma . Alternations in specific genes that play important roles in diverse cellular functions,such as cell adhesion,signal transduction,cell differentiation,development,metastasis,DNA repair and glycosylation changes, have been identified.Thus,molecular biology is equally important for developing diagnostic metastatic and prognostic molecular markers . Therefore,the challenge is to detect stage-specific and lymph node metastasis-related genetic abnormalities that may result in early diagnosis,lymph node metastasis and even aid in selecting therapy. This study focuses on detection of vascular endothelial growth factor C and chemokine receptor CCR7 and relationship with lymph node metastasis in gastric carcinoma.The extent of regional lymph node metastasis is an important indictor of tumor aggressiveness and is a prognostic factor for patient with gastric carcinoma. It is well known that the preferential metastatic route of gastric carcinoma is initially to lymph nodes and later to distant sites such as the liver or peritomeal cavity. lymphangiongenesis, the growth of new lymphatic vessels, is believed to underlie lymph node metastasis. Many experimental studies indicated that lymphangiogenic factor(VEGF-C) could stimulate lymphangiogenesis in tumors by binding to their receptors (VEGFR-3) on the lymphatic endothelial cells and promotes lymphatic metastasis in mouse tumor models. And expression of VEGF-C by tumor cells correlates well with lymph node metastasis of gastric carcinoma in clinical research. Many studies have demonstrated that chemokine receptor CCR7 was expressed in a variety of human tumor cells. Tumor cells positive for CCR7 could preferentially metastasize towards lymph nodes highly expressed ligands of CCR7 such as CCL19 and CCL21, for providing evidence for preferential homing of cancer cells to metastasic sites. But the report on homogenous expression of VEGF-C and CCR7 in primary gastric lessions and metastatic lymph node is seldom. No investigations have been conducted regarding serum VEGF-C level in patients with gastric carcinoma, this study will also evaluated whether the combined examination of VEGF-C and CCR7 protein expression in endoscopic biopsy specimens could give additional useful information for preoperatively predicting lymph node metastasis in patients with gastric carcinoma.The objective of our study is to determine cooperation effection of VEGF-C and CCR7 in the course of development, progression and lymph node metastasis in gastric carcinoma, by determining expression of VEGF-C and CCR7 protein using immunohistochemical assay, for providing new theory basis to find new targets for the treatment of gastric carcinoma; To measure serum VEGF-C levels in patients with gastric carcinoma and the combined examination of VEGF-C and CCR7 expression in endoscopic biopsy specimens of gastric carcinoma and assessed its usefulness as a diagnostic tool for determining early gastric carcinoma and lymph node metastasis.We divide the experiment into three parts The part 1: Expression of vascular endothelial growth factor C(VEGF-C) and chemokine receptor CCR7 protein in primary gastric carcinoma lesions and metastatic lymph nodes .We carry out the expression of VEGF-C and CCR7 protein by using S-P immunohistochemistry in 51 cases of gastric primary carcinoma tissues and 33 cases of its corresponding lymph node metastasis lesions. We found that moderate to strong staining of VEGF-C protein was identified in the cytoplasm of gastric cancer cells. The percentage of gastric primary carcinoma tissues positive for VEGF-C was 56.9%(29/51); Moderate to strong staining of CCR7 protein was found mainly in cell membrane and\or cytoplasm of cancer cells. The percentage of patients of gastric carcinoma positive for CCR7 was 62.7%(32/51). In 33 patients of gastric carcinoma with lymph node metastasis,the positive expression rate of CCR7 was 84.8% (28 / 33);81.8%(27/33) in the primary lesions and metastasic lymph nodes, respectively . The agreed positive expression rate was 81.8%(27/33) in them. The agreed negative expression rate was 9.1%(3/33), their homogenous expression rate was also 90.9%(30/33) . It turned out that not only the gastric primary tumors but also the metastatic lymph node were positive for VEGF-C and CCR7 protein expression, the VEGF-C and CCR7 protein play an important role in the course of development ,progression and lymphatic metastasis of gastric cancer. Further analysed and found that, the agreed positive expression rates of CCR7 and VEGF-C was 66.7%(22/33) in the patients for gastric carcinoma with metaststic lymph nodes, but it was only 11.1%(2/18) in the patients for gastric carcinoma without lymph node metastasis ,(P=0.0001). It turned out that the agreed expression of VEGF-C and CCR7 was related to lymph node metastasis, it indicated the cooperation effection of VEGF-C and CCR7 may promote lymphatic metastasis in gastric cancer .The part 2: Measurement of serum VEGF-C level in patients with gastriccarcinoma Samples from 28 patients with gastric carcinoma before primary radical gastrictomy , 2 week -1 month after operation, 19 patients of which were obtained and from 21 patients of normal healthes severed as controls. Serum VEGF-A,VEGF-C level was examined by using ELISA. We found that the serum VEGF-C level in patients with gastric cancer before and after excision of tumor were 216.91±47.83 pg/ml;157.52±50.24 pg/ml, respectively. The serum VEGF-C level in healthy controls was 159.54±60.83 pg/ml. The concentrations were statistically significantly different in preoperation and postoperation, P<0.01; preoperation and health controls, P<0.01, respectively . But there is no statistically different in serum level between post -operation and healthy controls, P>0.05 . We found that the serum VEGF-A level in patients with gastric cancer before and after excision of tumor were 246.27±121.94 pg/ml; 145.88±69.61 pg/ml, respectively. The serum VEGF-A level in healthy controls was 112.38±47.23 pg/ml. The concentrations were statistically significantly different in preoperation and postoperation, P<0.01; preoperation and health controls, P<0.01, respectively . But there is no statistically different in serum level between postoperation and healthy controls, P > 0.05 . When a cut-off value of 220.37 pg/ml (the highest value of healthy controls) was applied, the percentage of patients of gastric carcinoma with lymph node metastasis positive for serum VEGF-C level was 57.8%(19/33). In group without lymph node metastasis,the percentage of patients for serum VEGF-C was 38.9%(7/18), the positive rate was no statistically different, P>0.05. It turned out that the serum VEGF-A,VEGF-C level is a reliable biologic markers in diagnosing gastric carcinoma; The change of serum VEGF-A,VEGF-C level is related to excision of tumor; There is no relationship between preoperative serum VEGF-C level and expression of VEGF-C in tissue of patients with gastric cancer.when a cut-off value of 220.37 pg/ml (the highest value of healthy controls) was applied, the percentage of serum VEGF-C in preoperational group was 57.1%(16/28), the rate of control group was 14.3%(3/21); The sensitivity, specifity, positive predictive rate, negative predictive rate and accuracy were 57.1%(16/28),85.7%(18/21),84.2%(16/19),60.0%(18/30) and 69.4%(34/49), respectively. when a cut-off value of 159.61 pg/ml (the highest value of healthy controls) was applied,the percentage of serum VEGF-A in preoperational group was 53.6%(15/28), the rate of control group was 23.8%(5/21); The sensitivity, specifity, positive predictive rate, negative predictive rate and accuracy were 53.6%(15/28),85.7%(18/21),75.0%(15/20),55.2%(16/29) and 63.3% (31/49), respectively. With combined determination of serum VEGF-C and VEGF-A levels, the sensitivity was 82.1%(23/28), specificity was 71.4%(15/21), positive predictive rate was 79.3%(23/29), negative predictive rate was 75.0%(15/20) and accuracy was 77.6%(38/49). Serum VEGF-C levels was closely related to lymph node metastasis(P<0.01),depth of invasion(P<0.01) and TNM stage(P<0.01). There is no relationship between preoperative serum VEGF-C and expression of VEGF-C in tissue of patients with gastric cancer.In order to investigate the relationship between serum VEGF-C and VEGF-A levels and recurrence and metastasis of gastric cancer ,we measured the serum VEGF-C and VEGF-A levels from 17 patients who had underwent operation after a half year to two years. 10 patients with no metastatic cancer ,7 patients with recurrence or liver, lung metastasis . The level of serum VEGF-C was (169.23±54.35) pg/ml;(223.17±27.89) pg/ml in group with metaststic cancer and group without it, respectively. There is a significantly stastistically different, P<0.01. There is a significantly stastistically different in serum VEGF-C levels between group with metastasic cancer and control healthes, P<0.01.The levels is no different between group without metastasic cancer and control healthes, P>0.05. The level of serum VEGF-A was (149.68±52.99) pg/ml;(406.33±103.8) pg/ml in group with metaststic cancer and group without it, respectively. There is a significantly stastistically different, P < 0.01. There is a significantly stastistically different in serum VEGF-A levels between group with metastasic cancer and control healthes, P<0.01.The levels is no different between group without metastasic cancer and control healthes, P>0.05.The part 3: Expression of VEGF-C and CCR7 protein in preoperative endoscopic biopsy in patients with gastric carcinoma Endoscopic biopsy specimens (tumor and corresponding normal mucosa) of gastric tissue from 37 patients with gastric carcinoma who later underwent surgical resection. The expression of VEGF-C and CCR7 protein was assessed by western blotting assay. We found that the positive rate of VEGF-C expression in group with lymph node metastasis and group without lymph node metastasis were 63.3% (19/30) ; 14.2 % (1/7), respectively. There is a significantly statistically different between them, P<0.05. In two groups, the positive rate of CCR7 was 76.7 % (23/30); 28.6%(2/7), respectively. There is a significantly statistically different between them, P<0.05. The sensitivity(Se), specificity(Sp), positive predictive value(PPV), negative predictive value(NPV) and accuracy(Ac) of VEGF-C and CCR7 expression in the diagnosis of lymph node metastasis for patients with gastric carcinoma were 63.3%(19/30);85.7% (6/7) ;95.0% (19/20) ;35.5% (6/17) ;67.6% (25/37) and 76.7% (23/30);71.4% (5/7) ;92.0% (23/25) ;41.7% (5/12) ;75.7% (28/37), Respectively. ROC analysis showed that the accuracy of the combined examination of VEGF-C and CCR7 expression in predicting lymph node metastasis was relatively high(area under ROC curve[Az=0.764]).From the above date and discussion we can drew the following conclusions:1. Not only the gastric primary tumors but also the metastatic lymph node were positive for VEGF-C and CCR7 protein expression, the VEGF-C and CCR7 protein play an important role in the course of development ,progression and lymphatic metastasis of gastric cancer. The agreed expression of VEGF-C and CCR7 was related to lymph node metastasis, it indicated the cooperation effection of VEGF-C and CCR7 may promote lymphatic metastasis in gastric canncer .2. The serum VEGF-A,VEGF-C level is a reliable biologic markers in diagnosing gastric carcinoma; The positive rate of combination assay of serum VEGF-A,VEGF-C level is higher than a signal of them.3. The serum level of VEGF-C is not related to the expression of VEGF-C in tissue of gastric cancer.4. There is an obviously change in serum level of VEGF-A,VEGF-C between postoperation and preoperation , the serum levels of VEGF-A,VEGF-C increase notablely in gastric cancer with recurrence, metastatic cancer,but there is no different in serum VEGF-A,VEGF-C levels in gastric carcinoma without recurrence or metastasic cancer and control healthes. It indicated that the VEGF-A,VEGF-C levels up-regulation appears to be useful biologic marker for gastric cancer metastasis or recurrence.5. The accuracy of the combined examination of VEGF-C and CCR7 expression in predicting lymph node metastasis was relatively high. |
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