Abstract Objective To investigate the lymphatic microvessel density in primary gastric adenocarcinoma clinical pathological significance. The method of application of monoclonal antibody D2  40 detection of primary gastric adenocarcinoma and the surrounding area and the central area of ​​its normal gastric tissue micro-lymphatic vessel density (LMVD) analysis with lymphatic invasion, lymph node metastasis and other clinicopathological parameters relationship. Results the gastric adenocarcinoma surrounding area LMVD significantly higher than central area LMVD in (P <0.05) and normal gastric tissue LMVD (P <0.05), and lymphatic vessels as functional. The surrounding the area LMVD with tumor Ming type, lymphatic invasion and lymph node metastasis (P <0.05), and has nothing to do with the patient’s age, sex, degree of tumor differentiation and depth of invasion (P> 0.05). Between the central area LMVD clinicopathological parameters had no correlation (P> 0.05). Conclusion primary gastric adenocarcinoma cancer weeks LMVD and lymphatic invasion, lymph node metastasis, detecting cancer the week LMVD predict an effective indicator of lymph node metastasis, and helps determine the prognosis of gastric cancer.

Key words gastric cancer; lymphatic microvessel density; D2  40; lymph node metastasis

Detection of Lymphatic Microvessel Density in Primary Gastric Adenocarcinoma and Its Clinicopathological Significance

    SUN Wei, ZHOU Xiao  jun, MA Heng  hui, ZHANG Ru  song, LU Zhen  feng

    Clinical School of Medical College, Nanjing University / Department of Pathology, Nanjing General Hospital of Nanjing Military Command, Nanjing 210002, China

    Corresponding Author: ZHOU Xiao  jun, E  mail: zhouxj1@yahoo.com.cnAbstract: Objective To investigate the clinicopathological significance of the lymphatic microvessel density (LMVD) in primary gastric adenocarcinoma.Methods Monoclonal antibody D2  40 was used immunohistochemically to detect the lymphatic microvessel density (LMVD) in the intra  and peri  tumoral areas of the primary gastric adenocarcinoma and normal gastric tissue, and analysis the relationship among the lymphatic microvessel density (LMVD), lymphatic invasion, lymph node metastasis and other clinicopathological parameters of the primary gastric adenocarcinoma.Results LMVD of the peri  tumoral area was significantly higher than that of intra  tumoral area and nomal tissue (P <0.05). LMVD of the peri  tumoral area was significantly associated with Ming Classification, lymphatic invasion and lymph node metastasis (P <0.05), while less associated with age, sex, differentiation and invasion of the tumor (P> 0.05). LMVD of the intra  tumoral area was not correlated with these clinicopathological parameters (P> 0.05). Conclusion LMVD of the peri  tumoral area was significantly associated with lymphatic invasion and lymph node metastasis, and detection of LMVD in peri  tumoral area can be used for prediction of lymph node metastasis.

    Key words: Gastric carcinoma; Lymphatic microvessel density; D2  40; Lymph node metastasis

The malignant gastric incidence and high mortality rate in the world, serious threat to human life and health. Surgical resection of the tumor tissue, the long-term survival is far from satisfactory, and its one of the important reasons is that the tumor tissue of lymph node metastasis. Due to the lack of specific markers of lymphatic endothelial cells and lymphatic metastasis research progress has been slow. The monoclonal antibody D2  40 is a newly discovered lymphatic endothelial cell-specific markers [1]. This article using immunohistochemical EnVision the application D2  40 detected in 69 patients with primary gastric adenocarcinoma lymphatic microvessel density (Lymphatic microvessel density, LMVD), to analyze the clinicopathological significance of lymphatic vessels formed in the development of gastric adenocarcinoma role.

    1 Materials and methods

    1.1 Materials

    69 patients with primary gastric adenocarcinoma specimens archived paraffin blocks from the Nanjing Military Region, Nanjing General Hospital from September 2004 to October 2006. All cases are implemented gastrectomy and lymph node peel, and who did not receive radiation therapy or chemotherapy before surgery. 50 cases were male and 19 females, aged 35 to 80 years old, with an average of 58.71 years. The pathological basis Ming typing. Lymph node metastasis in 25 cases, 44 cases of lymph node metastasis, and according to the International Union Against Cancer, the American stage and prognosis Committee (UICC / AJCC) 1997 the fifth edition of the regulations, the number of lymph nodes peel each case less than 15 a. In another 26 cases of distal gastric resection margin as a normal stomach tissue.

    1.2 Methods

    Immunohistochemistry EnVision Law. The antibody source: D2  40 (clone D2  40) is the product of the United States Dako, EnVision kit was purchased from Dako Corporation, USA. Steps are carried out in accordance with the kit instructions. Instead of primary antibody with PBS as control. Neutral gum cementing microscope.

    1.3 The results of judgment

    D2  40 located in the lymphatic endothelial cells, to the cytoplasmic brown particles like coloring positive. Lymphatic microvessel density counts using Weidner et al [2] reported first observed under low magnification (× 40) the tumor periphery District (peritumoral region), the Oncology Center District (intratumoral region) and the corresponding distal resection margin of normal tissue, select one lymphatic high-density areas (hot spots), and the number of lymphatic vessels and then counted under a high power microscope (× 200) to be dyed. Each dyed brown endothelial cells or endothelial cell clusters, as long as the surrounding vasculature, tumor cells or other connective tissue separately, i.e. as a lymphatic. Count the number of lymphatic vessels in five high power field, as the cases of LMVD in the average.

    1.4 statistical methods

    Measurement data with the mean ± standard deviation (± s) between the mean of the two samples t-test, three samples were used to compare the analysis of variance was used to compare between the number of data using SPSS11.5 software package. All hypothesis testing using a two-sided test, test level α = 0.05, P <0.05 difference was statistically significant.

    2 Results

    The morphological characteristics of the tube 2.1 lymphatic

    D2  40 positive positioning in lymphatic endothelial cell cytoplasm, as brown particles. Light microscopy was found the D2  40 positive lymphatic expansion shape or occlusion in cords, wall simple squamous epithelium, thin, no red blood cells in the lumen. Vasculature containing erythrocytes i.e. vascular staining was negative, as shown in Figure 1a. Normal gastric tissue lymphatic often isolated and expansion-like distribution in the various layers of the stomach tissue in the submucosa up, reduced the number of cancer weeks. Primary gastric adenocarcinoma tissue surrounding area lymphatics in larger quantities, mostly expansion irregular in shape, as shown in Figure 1b, sometimes visible lymphatic tumor thrombus, as shown in Figure 1c, in the central area of ​​the cancerous tissue, lymphatic significantly reduced the number, most of them was occluded cords, as shown in Figure 1d.

    2.2 LMVD Determination Table 1 primary the gastric adenocarcinoma surrounding area LMVD and central area LMVD with clinicopathological parameters of the surrounding tissues of gastric adenocarcinoma LMVD 5.60 to 16.40, an average of 10.83 ± 3.08, a central area LMVD 1.20 to 5.20, with an average of 2.78 ± 0.99 normal gastric tissue LMVD for the 1.60 5.20, 2.94 ± 0.88 average. The the surrounding area LMVD significantly higher than that in the central area and the normal tissue, and the difference was statistically significant (t = 21.48, P <0.05; t = 12.82, P <0.05). The central area LMVD and normal tissues, the difference was not statistically significant (t =  0.703, P = 0.484).

    2.3 gastric adenocarcinoma surrounding area LMVD, the Center District LMVD clinicopathological parameters of the relationship between

    Table 1 shows that, of primary gastric adenocarcinoma the surrounding area LMVD with the tumor Ming typing, lymphatic invasion and lymph node metastasis (P <0.05), and has nothing to do with the patient’s age, sex, degree of tumor differentiation and depth of invasion (P> 0.05). The central area LMVD and clinicopathological parameters were independent (P> 0.05).

    3 Discussion

    Tumor invasion and metastasis is one of the major cause of death in cancer patients. Early gastric cancer lymph node metastasis, lymph node metastasis, the evaluation of gastric cancer staging and one of the key factors in determining the prognosis of gastric cancer. Lymph node metastasis, tumor cells must first invade the lymphatic system. But the tumor cells the existing lymphatic invasive tumor or tumor-induced lymphangiogenesis tube, and this process whether lymph node metastasis is unclear.

    Due to the lack of specific markers of lymphatic, generated in research on tumor-associated lymphoid tube has been more difficult. Previously reported to mark lymphatic endothelial cell immunohistochemical markers, such as LYVE  1 (lymphatic vessel endothelial receptor  1) [3], VEGFR  3 (vascular endothelial cell growth factor receptor  3) [4], Prox  1 (prospero  related homeobox  1) [5], etc., only applies to frozen sections, and also in the subsequent study found that these markers of poor specificity of the lymphatic affect the lymphatic vessel invasion mechanism research. Monoclonal antibody D2  40 newly discovered lymphatic endothelial cell markers, and a large number of studies have shown that lymphatic endothelial cells in normal and tumor tissue in paraffin sections can be used to detect, and with good specificity and sensitivity 1,6]. The experimental application of D2  40 monoclonal antibody immunohistochemical detection of primary gastric adenocarcinoma and normal tissue lymphatic vessels, the results show positive vessel wall is thin, irregular shape, no red blood cells in the lumen, in line with the lymphatic characteristics, while the thick wall, the lumen within the vasculature containing erythrocytes i.e. vascular staining negative, confirming the monoclonal antibody D2  40 is a current marker best marker for lymphatic endothelial cells.

    After a number of lymphatic and morphology of the 69 patients with primary gastric adenocarcinoma cancer organization Centre area, the surrounding area and its normal gastric tissue micro analysis found that the number of micro-lymphatic the cancer tissue surrounding area was significantly higher than the central area of ​​cancer tissue and normal gastric mucosa . Mostly lymphatic expansion of irregular shape, and lymphatic invasion, lymph node metastasis, in the central area of ​​cancerous tissue, lymphatic microvessel significantly reduced the number showed mostly occluded streak, and lymphatic invasion and lymph node metastasis-related sex. Speculated that tumor central area of ​​lymphatic vessels is non-functional, and the surrounding area in the expansion of lymphatic functional lymphatic vessels play an extremely important role in the lymphatic invasion and lymph node metastasis process that detect tumor surrounding area LMVD more accurately predict tumor lymph node metastasis. Leu et al [7] found that tumor tissue promote lymphangiogenesis-related factors such as VEGF C, VEGFR  3, etc., but can not form a functional lymphatic vessels, may be due to the central area of ​​the tumor lymphatic vessels in the continuous differentiation and proliferation tumor cells produced machinery collapsed under pressure atrophy due.

    Ming typing based on the growth characteristics of the gastric reaction to some extent some of the biological characteristics of the tumor [8]. The experimental results suggest that the number of micro-lymphatic expansion-type gastric cancer tissues surrounding area and infiltrating the surrounding area micro lymphatic difference was statistically significant number of tubes. The light microscope was also observed to intumescent gastric carcinoma peripheral zone of the lymphatic portion was closed shape for expansion, while the peripheral region of lymphatic infiltrating multi-shaped, in the central area there are also a small amount of expanded state lymphatic. Was expansion-like functionality lymphatic provide favorable conditions for the lymphatic metastasis of tumor cells, presumably one of the reasons why invasive gastric cancer is more prone to lymph node metastasis.