【Abstract】 Objective To study the the gastric lymphatic morphological characteristics of the relationship with lymph node metastasis. Methods D2 40 immunohistochemical staining in 70 cases of gastric central area, paracancer normal area of lymphatic analysis of the morphological characteristics of the lymphatic vessels and lymph node metastasis. Results of gastric cancer next District lymphatic vessel density (41.32 ± 15.62) a total area of (33 139.08 ± 19 352.37) μm2 average area (802.12 ± 728.09) μm2 average circumference (132.35 ± 65.76) μm; the normal zone lymphatic vessel density (30.06 ± 11.86) months, the total area (45 424.65 ± 33 824.64) μm2 average area (1 511.28 ± 1 301.21) μm2, average circumference (196.19 ± 103.72) μm, the differences were statistically significant (P = 0.000 , 0.009,0.000,0.000). Lymph node metastasis of gastric cancer paracancer lymphatic vessel density (43.67 ± 16.42) a total area of (35 866.71 ± 19 678.53) μm2 total perimeter (5 851.28 ± 897.13) μm, average area (1 050.31 ± 765.47) μm2 average circumference (161.90 ± 77.13) μm, density area in the next group of gastric cancer without lymph node metastasis (33.38 ± 9.02) a total area (23 ± 15 405.2) 933.34 μm2 total circumference (173.56 ± 2 028.18) μm average area (723.76 ± 534.75) μm2 average circumference (123.01 ± 60.88) μm, the differences were statistically significant (P is for 0.019,0.029,0.003,0.000,0.000); lymphatic vessels to the area next to the gastric cancer infringement predicting lymph node 0.7，P=0.000）。">no statistically significant differences in the detection rate of transfer and pathology (P = 0.125), and the goodness of fit (κ = 0.822> 0.7, P = 0.000). Conclusion gastric lymphatic new survival lies paracancer lymphangiogenesis tube cavity; gastric cancer cell destruction paracancer lymphatics into the lymphatic circulation and the formation of lymph node metastasis; paracancer lymphatic vessel invasion and lymphatic vessel density with lymph node metastasis predict lymph node metastasis and to determine the surgical approach is expected to become an important factor.
Key words gastric tumor lymphatic lymph node metastasis
Lymph node metastasis in gastric cancer is the most important transfer and independent prognostic factors, lymph node status is the decision to stage the key factor for prognosis and treatment . Lymphangiogenesis and tumor lymph node metastasis (Lymph node metastasis, LM) relationship has attracted the attention of many scholars, lymphatic tumor cell interactions is the key link. D2 40 in the embryonic testis and testicular germ cell tumors in a molecular weight of 40KD glycoprotein antibody reaction with lymphatic endothelial detect lymphatic the ideal antibody . This study the lymphatic endothelial marker D2 40 immunohistochemical staining, pathological image analyzer normal gastric wall of the gastric cancer Center District, paracancer the the far cancer District lymphatic density (lymphatic vessel density, LVD), the area and circumference quantitative measurement and analysis of its relationship with lymph node metastasis to explore the mechanisms of lymph node metastasis.
1 Materials and Methods
1.1 The object of study
Select from January 2006 to October 2006, the First Affiliated Hospital of Guangxi Medical University, the gastrointestinal glands surgical line gastrectomy fresh gastric specimens 70 cases. 54 cases were male and 16 females; aged 32 to 76 years, with a median age of 58 years old; lesions: the upper 12 cases, 26 cases in central and lower part of the 32 cases; the the high differentiated six cases of poorly differentiated 64 cases; lesions ≤ 5cm 32 5cm 38例；Ⅰ、Ⅱ期22例，Ⅲ、Ⅳ期48例；有淋巴结转移54例，无淋巴结转移16例。">cases,> 5cm 38 cases; Ⅰ, Ⅱ 22 cases, Ⅲ, Ⅳ 48 cases; lymph node metastasis, 54 patients without lymph node metastasis in 16 cases. Inclusion criteria were: pathologically confirmed adenocarcinoma; did not receive preoperative radiotherapy and chemotherapy and immunotherapy; to exclude associated with autoimmune diseases and severe trauma inflammation.
1.2 Research Method
1.2.1 Main reagents and immunohistochemical staining methods mouse anti-human monoclonal antibody D2 40 working fluid (Mouse anti D2 40, catalog number ZM 0465), mouse anti-human cytokeratin 20 monoclonal antibody working solution (Mouse anti Cytokeratin 20, ZM 0075) were purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.. D2 40 staining immunohistochemical staining of two-step, the antigens hot fix (120 ° C, 90s; 1 mM EDTA, pH 9.5), DAB color. Positive control human tonsil tissue, non-immune serum instead of primary antibody as negative control. The CK20 also using immunohistochemical staining two-step, antigen hot fix (120 ° C, 90s; 1 mM DTA, pH 8.0), DAB color. Positive control human gastric carcinoma, non-immune serum instead of primary antibody as negative control.
1.2.2 specimens drawn and treatment of gastric cancer patients with resected specimens, the normal area paracancer, central area (normal zone normal gastric wall more than 5 cm from the tumor from the tumor and non-tumor tissue, adjacent area is confirmed by HE staining about 2mm area, the central area of the tumor is close to the edge of the section) materials, 10% neutral formalin-fixed, paraffin-embedded, 4μm thick serial sections.
1.2.3 gastric lymphatic staining judge observed under an optical microscope, lymphatic endothelial cells D2 40 positive staining for cytoplasmic membrane brownish yellow staining, cavity-free red blood cells. Lymphatic vessel density counts using Wendner , counting standards: slice first comprehensive observation under low magnification (× 100), to determine the highest point of the lymphatic vessel density. At high magnification (× 200) to count the number of D2 40 positive lymphatic. Four count each specimen within view of the number of lymphatic application multifunction pathological image analyzer (Germany LEICA Company, Model the DMR + Q 550), whichever is the total value as the specimen LVD, and measuring the area of each lymphatic and perimeter. And calculating the total area and perimeter of each case each part of lymphatic vessels.
1.2.4 gastric paracancer lymphatic invasion judge D2 40 positive lymphatic destruction of the wall at high magnification, interruption, defect, and (or) the lymphatic CK20-positive tumor cells shall LVI positive.
1.3 statistical methods
Lymphatic measurement data to ± s, McNemar χ2 test were compared using paired or independent two-sample t-test; LMVD in with LM relationship χ2 test comparing two independent sample rate; relationship between LVI and LM paired count data method and κ inspection. Application SPSS13.0 statistical package for statistical analysis. The inspection level α = 0.05.
2.1 morphological characteristics of lymphatic vessels
D2-40 immunohistochemical staining positive positioning in the cytoplasmic membrane of lymphatic endothelial cells, was pale-brown stain. Light microscopy, thin lymphatic wall composed of a single layer of endothelial cells was crack-like ring, streak or beaded, occasionally dendritic within red blood cells. Central area of gastric cancer lymphatic vessels, as shown in Figure 1a; the normal area visible hierarchy of the stomach wall is clear and complete, except epithelium layers are visible presence of lymphatic vessels, the most intensive on both sides of the muscularis mucosa, more than elongated the circular tubular or oval , was part of a crack-like morphology rules, structural integrity, non-destructive, as shown in Figure 1b; paracancer unstructured hierarchy of the stomach wall disappeared, more lymphatic showed mostly small circle, beaded, see Figure 1c , d, part lymphatic wall damage, interruption, shown in Figure 1e, and even tumor cells, Figure 1f to H.
2.2 gastric paracancer normal lymphatic comparison
Next area LVD in gastric cancer is higher than the normal area (P = 0.000), lymphatic total area of less than the normal area (P = 0.008), lymphatic total circumference no difference (P = 0.229), as shown in Table 1. The gastric paracancer the average area of lymphatic vessels the average circumference less than the normal area (P = 0.000), as shown in Table 2.
The relationship between the morphological characteristics and lymph node metastasis in 2.3 paracancer lymphatic
The next area of 54 patients with lymph node metastasis of gastric cancer lymphatic vessels were detected in 2198, the area next to the 16 cases without lymph node metastasis of gastric cancer were detected in 694 of the lymphatic. The paracancer lymphatic vessel density, the total area of the total circumference, the average area, average perimeter only with lymphatic metastasis, has nothing to do with other clinicopathological factors are shown in Table 3. Table 1 gastric cancer next zone and normal zone lymphatic density, total area, total perimeter Table 2 gastric cancer next area with the average area of the lymphatic vessels in the normal area table 3 gastric cancer paracancerous zone average circumference (± s) the relationship between the morphological characteristics of the lymphatic and lymph node metastasis
The 2.4 paracancer lymphatic vessel invasion relationship with lymph node metastasis
The lymphatic invasion paracancer predict the diagnosis of lymph node metastasis and pathological examination results were similar, the difference was not statistically significant (P = 0.125). 0.7，P=0.000）。">And both goodness of fit (κ = 0.822> 0.7, P = 0.000). The positive predictive value was 93.10% (54/58), and a negative predictive value of 100% (12/12), as shown in Table 4.
Role of lymphatic vessels in the evolution of human malignancies is not fully understood, even distribution of tumor lymphangiogenesis remain controversial. Leu  studies have shown that lack of functional lymphatic vessels within solid tumors, functional lymphatic vessels collapse may be due to tumor pressure. Zheng Yamin et al , Liu Hui winter  also to support gastric Center area without cream the Table 4 gastric paracancer lymphatic invasion, and lymph node metastasis in the relationship between the Palestinian tube. Study found that colorectal cancer  and cervical cancer  the presence of lymphatic vessels in the central area. The study found that gastric central area without lymphatic, the paracancer lymphatic density was significantly higher than the normal area, and paracancer existence is damaged lymphatic cancer, lymphatic The more the closer the tumor is destroyed, suggesting that gastric cancer centers District no lymphatic tumor to destroy all the results. We also observed that the massive destruction to paracancer containing tumor cells within the lymphatic proved paraneoplastic zone is the main area of cancer cells through the lymphatic metastasis. , Padera  Although the lack of functional lymphatic vessels inside the tumor, but the tumor edge functional lymphatic vessels still enough to cause lymph node metastasis.
Cancer cells into the lymphatic there are two views: One is that to enter lymphatic cancer cells through the lymphatic open connection; Another view is that cancer cells through the release of active substances such as type IV collagenase, destroy the lymphatic wall, then enter the lymphatic lymphatic wall defect. This study showed that lymphatic invasion paracancer predict lymph node metastasis in strong agreement with the routine pathological examination Sako – the  and Nakamura et al  Similar results were also obtained. These results support the latter view. The same time paracancer lymphatic vessels which will likely become a key factor for predicting lymph node metastasis violations, particularly noteworthy is its negative predictive value of 100%, will likely make paracancer lymphatic tube the patients no damage from extensive lymph node dissection. By comparison also found that the area next to the gastric cancer lymphatic tube freshmen with lymph node metastasis and peritumoral lymphatic vessel density will also become important factor for predicting lymph node metastasis.
The results of this study, measurements of the lymphatics area and perimeter gastric cancer next area average area of lymphatic vessels and the average circumference were significantly smaller than the normal area, and the total area of the lymphatic vessels in gastric cancer next area is less than the normal area. Liang et al , the results also show that colorectal cancer paraneoplastic District lymphatic maximum diameter mean significantly lower than that in normal colon lymphatic vessels. This shows the lack of lymphatic drainage of the area next to the tumor cancer tumor metabolism and tumor cell destruction caused by lymphatic embolization, local vascular permeability, the paracancer tissue caused by hydrostatic pressure and protein and metabolite penetration The pressure rise, can be well explained by tumor exists neovascularization and lymphangiogenesis remains interstitial edema.
In summary, gastric lymphatic The new survival lies paracancer lymphangiogenesis tube cavity is small enough to form a good lymphatic flow; gastric cancer cell destruction peritumoral lymphatic and then into the lymphatic circulation and the formation of lymph node metastasis; peritumoral lymphatic vessel invasion and lymphatic vessel density and lymph node metastasis, is expected to become an important factor in predicting lymph node metastasis and to determine the surgical approach.