Role Of Vascular Endothelial Growth Factor C/D Mediated Lymphangiogenesis On Metastasis Of Gastric Cancer

Gastric cancer is the forth most common malignancy in the world,and is the second leading cause of cancer death after lung cancer.Lymph node metastasis is the strongest prognostic factor,and the major cause of treatment failure and death of patients with gastric cancer.About 60%of resecable gastric cancers have lymph node metastasis at the time of diagnosis,and so,the 5-year overall survival of advanced gastric cancer is lower than 20%.However,little is known about the molecular mechanisms of lymph node metastasis in gastric cancer.As we now known,lymph node metastasis is a multistep process that is initiated by cancer cell invasion into lymph vessels.Proliferation of lymphatic vessels is a common finding in gastric cancer tissue,and this may favors of the permeation of cancer cells into the lymphatic channels.Accordingly,lymphangiogenesis around cancer cells may play a crucial role during the course of lymphatic tumor spread.Vascular endothelial growth factor(VEGF)-C is well known to induce lymphangiogenesis by activating VEGF receptor(VEGFR)-3, which is expressed on lymphatic endothelial cells.VEGF-D is a new member of the VEGFs,and its amino-acid sequence shows 23.3%identity with that of VEGF-C.VEGF-D shares two receptors VEGFR-2,VEGFR-3 with VEGF-C, however,Physiological functions of VEGF-D remains unknown.Despite these well-established principles,many key questions regarding the mechanisms of lymphatic tumor spread still remain under resolved:1.Is there functional lymphatics within malignant tumor of human? 2.Does the de novo formation of lymphatic capillaries and/or lymphatic enlargement dilatation increase the probability of lymphatic tumor dissemination beyond that which would occur exclusively through preexisting lymph vessels? 3.Does increased VEGF-C or D expression promote an increase in lymphatic vessel density (lymphangiogenesis)and/or size? If so,is this sufficient to increase the rate of metastasis to lymph nodes? Are there other functions of VEGF-C and D that account for the increase in lymph node metastasis? 4.How to predict lymph node metastasis accurately?In the present study,using transmission electron microscope,immunohistochemistry and RT-PCR,we examined the morphological features of lymph vessels and the expression of VEGF-C and D in 70 human gastric cancers,and investigated the correlation between the expression of VEGF-C/D,lymphangiogenesis, lymph vessel enlargement,lymph vessel invasion,preexisting lymph vessel and lymph node metastasis and other established clinicopathological features,expect to find an accurate indicator of lymph node metastasis.1.Lymph vessels in normal,peripheral,and central region of 70 human gastric cancers were investigated by transmission electron microscope and D2-40 immunostaining,and the relationship between lymphangiogenesis,lymph vessel enlargement,lymph vessel invasion and lymph node metastasis were analysed.2.We detected the expression of VEGF-C/D in 70 HGCs by RT-PCR and IHC,and analysed their role on lymphangiogenesis,lymph vessel enlargement, lymph vessel invasion,and lymph node metastasis.3.We observed lymph vessels in every layer of normal gastric wall by D2-40 immunostaining,and analysed whether they were related with lymph node metastasis. 4.Using HE,IHC,and RT-PCR,we detected carcinomatous metastasis in SLNs of 57 HGCs which were stained by blue dye,and analysed the feasibility of lymph vessel invasion and SLNB for the prediction of lymph node metastasis in gastric cancer.Our results showed that:1.Distribution and morphological features of lymph vessels in human gastric cancerThere is no lymph vessel in central region of gastric cancer;In normal region of gastric cancer,lymph vessel is larger and intact;Compared with the normal region of gastric cancer,lymph vessels in peripheral region is smaller, irregular,and confined to dilate,and LVI is a common finding in periphery of gastric cancer.2.Comparision of lymph vessel in peripheral and normal region of gastric cancerCompared with the normal region of gastric cancer,the LVD of peripheral region of gastric cancer is significantly high(41.32±15.62 vs 30.06±11.86,P= 0.000),and there was a significant decrease in LVMA(802.12±387.09μm² vs 1511.28±701.21μm²,P=0.000),LVMP(132.35±65.76μm vs 196.19±93.72μm, P=0.000),LVMD(4.23±2.19 vs 5.08±2.33,P=0.000),and LVTA(33139±16352μm² vs 45424±21824μm²,P=0.008);the LVTP in peripheral and normal region of gastric cancer(5467±2040μm vs 5896±2166μm,P=0.229)are equal.3.Morphological features of lymph vessels in periphery of gastric cancer correlate with lymph node metastasisThe LVD(43.67±16.42 vs 33.38±9.02,P=0.019),LVTA(35866.71±16678.53μm² vs 23933.34±10405.2μm²,P=0.029),LVTP(5851.28±1897.13μm vs 4173.56±2028.18μm,P=0.003),LVMA(1050.31±465.47μm² vs 723.76±334.75μm²,P=0.000),LVMP(161.90±77.13μm vs 123.01±60.88μm,P=0.000),and LVMD(4.50±2.09 vs 4.19±1.96,P=0.004)of lymphatics in peripheral region of gastric cancer with lymph node metastasis was significantly increased than that of gastric cancer without lymph node metastasis.4.Lymph vessel invasion in periphery of gastric cancer correlates with lymph node metastasisIn 70 HGCs,we found that 58 patients have lymph vessel invasion in peripheral region in which 54 were confirmed with lymph node metastasis;and lymph vessel invasion in peripheral region was an accurate prognostic marker for lymph node metastasis(P=0.219;κ=0.734＞0.7,P=0.000),and it has a highly sensitivity(98.15%,53/54),and accuracy(91.43%,64/70).5.The expression of VEGF-C in HGCs and its clinnical significanceWe confirmed by RT-PCR and IHC that VEGF-C mRNA expression was observed more commonly(x²=33.273,P=0.000)in cancer tissues(78.57%, 55/70)than normal tissues(30%,21/70).For cancer tissue,VEGF-C protein was positive in 51 of 70 cases(72.86%),and was negative in gastric mucosae(χ⁼ 80.225,P=0.000).There is a significant increase in LVD(44.12±16.69 vs 33.89±8.99,P= 0.014),LVMA(1183.69±531.49μm² vs 885.89±359.36μm²,P=0.000),LVMP (167.63±79.18μm vs 137.67±64.06μm,P=0.000),LVMD(4.78±2.15 vs 4.49±2.07,P=0.004),LVTA(37604±12914μm² vs 20139±8705μm²,P=0.000),and LVTP(5927±1898μm vs 4021±1643μm,P=0.000)in periphery of gastric cancer with positive VEGF-C expression than that of gastric cancer with negative VEGF-C expression.Lymph node metastasis and lymph vessel invasion are more common in patients with positive VEGF-C expression than that of patients with negative VEGF-C expression.6.The expression of VEGF-D in HGCs and its clinnical significanceWe confirmed by RT-PCR and IHC that VEGF-D mRNA expression was observed more commonly(χ²=6.440,P=0.011)in cancer tissues(58.57%,41/70) than gastric mucosae(37.14%,26/70),and VEGF-D protein was more frequent (χ²=13.050,P=0.000)in cancer tissues(55.71%,39/70)than gastric mucosae (25.71%,18/70).There is a significant increase in LVD(45.38±18.43 vs 36.26±9.13,P= 0.014),LVTA(38898±16991μm² vs 25271±11496μm²,P=0.003),and LVTP (6126±1842μm vs 4508±1869μm,P=0.001)in periphery of gastric cancer with positive VEGF-D expression than that of gastric cancer with negative VEGF-D expression.But,LVMA(1110±455μm² vs 1087±411μm²,P=0.728), LVMP(160.55±68.96μm vs 157.43±62.97μm,P=0.523),and LVMD(4.70±2.23 vs 4.69±2.18,P=0.936)are equal.Lymph node metastasis and lymph vessel invasion are more common in patients with positive VEGF-D expression than that of patients with negative VEGF-D expression.7.Lymph vessels in lamina propria correlate with lymph node metastasis in gastric cancerThere is a significant increase in LVMA(649.12±315.19μm² vs 437.37±206.51μm²,P=0.000),LVMP(131.96±59.19μm vs 107.26±47.72μm,P=0.000), LVMD(3.88±1.63 vs 3.07±1.28,P=0.000),LVTA(12086±5663μm² vs 8018±4272μm²,P=0.045)of lamina propria in normal region of gastric cancer with lymph node metastasis than that of gastric cancer without lymph node metastasis.8.Sensitivity of HE,IHC,RT-PCR for the detection of metastasis in sentinel lymph nodeIn 56 out of 57 patients(98.25%),1-6 stained nodes were abtained with a mean value of 2.45,and all the 137 SLNs were detected for carcinomatous metastasis by HE,IHC,and RT-PCR.The number of positive SLN detected by HE,IHC,RT-PCR was 62,71,86,respectively.Sensitivity of RT-PCR for the detection of metastasis in sentinel lymph node is higher than that of HE(χ²= 8.463,P=0.004＜0.0125).9.Prediction of LVI and SLN status for entire lymph node status in gastric cancer44 out of 56 patients were diagnosed positive lymph node metastasis,and 46 were detected LVI in peripheral region.The number of patients with lymph node metastasis detected by HE,IHC,and RT-PCR were 36,37,41,respectively. The accuracy of SLN status detected by HE,IHC,RT-PCR,and LVI in the prediction of entire lymph node status was 85.71%,87.50%,94.64%,98.21%, respectively,and the false-negative rate was 18.18%,15.91%,6.82%,2.27%, respectively.The accuracy of prediction for entire lymph node metastasis by combination LVI with SLNB is improved to100%.10.Impact of T stage on the accuracy of SLNBThe accuracy of SLN status in diagnosis of the entire lymph node status of gastric cancer was 94.64%.The accuracy of SLNB in T_1-3stage is 100%, 95.45%,93.33%,respectively.And,the false-negative rate is 0%,6.67%,7.14%, respectively.Our results suggest that:1.Lymphangiogenesis exists in the peripheral region of HGCs,and the newly formed lymph vessels are small,irregular,and confined to dilate.It may plays a major role in lymph node metastasis of HGC that gastric cancer cells penetrate the lymphatics and get into lymphocinesia via destroyed parts of endothelium in peripheral region of gastric cancer.Lymphangiogenesis,lymph vessel enlargement,and lymph vessel invasion may favor of lymphatic tumor dissemination of gastric cancer.2.Up-regulation of VEGF-C/D in gastric cancer may play an important role in lymphatic tumor spread.VEGF-C expression in gastric cancer may induce lymphangiogenesis,lymph vessel enlargement,and lymph vessel invasion,and then favors of lymphatic tumor dissemination.VEGF-D may induce lymphangiogenesis,lymph vessel invasion,but not lymph vessel largement,and also increase the probability of lymphatic tumor spread.3.Preexisting lymph vessel may take part in lymph node metastasis of HGC,and preexisting lymph vessel in lamina propria of gastric wall may play a role in lymphatic tumor spread of early gastric cancer before lymphangiogenesis or breaking through muscularis mucosae.The variation of lymph vessel in lamina propria may results in the disparity of metastasis in early gastric cancer.4.SLNB combined with the detection of lymph vessel invasion in periphery may improve the accuracy and lower the false-negative rate of the prediction of lymph node status in gastric cancer.