Study On The Tumor Vessel Characteristic And Ultrastructure In Renal Cell Carcinoma

Background and objective: Although the relationship between growth, infiltrating, metastasis and prognosis of RCC AND the occurrence and densities of neovascularization is close, there is no related research report on RCC hemodynamics, MVD, as well as ultramicropathological of RCC microvascular. This research attempt to investigate blood flow distribution feature, Resistive index, MVD, the expression of VEGF as well as the changing of tumor capillary vessel ultrastructural among RCC diversity clinical stage by chromatic color Doppler ultrasonography, immunohistochemistry and transmission electron microscopy, to approach the following concern: (1) the relationship between the blood flow distribution and MVD of RCC AND carcinoma clinical stages and pathology grades; (2) the dependablity on RI and MVD between peritumor and intratumor zone; (3) the dependablity on express of MVD and VEGF of RCC; (4) the ultrastructural changing of capillary vessel and cancer cell of intrammor RCC as well as peritumor part; (5) attempt to set up a simple, fast and noninvasive assessment system on RCC imaging research by comparative study on RCC hemodynamics, MVD, the expression of VEGF and RCC Ultrastructural cell, as well as investigation on blood flow feature of RCC Doppler Ultrasound and RCC clinical stage, malignant biological behaviors and prognosis. This research supply new idea in ultrasound imaging of RCC.Methods:35 cases with Renal cell carcinoma, 22 men and 13 women, median age 56.71 years, SD 9.33,range 40-77.The tumor clinic stage was defined by Robson stage, wasⅠin 7 (20.0%) ,Ⅱin 18 (51.4%) andⅢin 10 (28.6%)cases. The pathologic grade was defined according to Fuhrman grade, wasⅠin 3 (8.6%),Ⅱin 20 (57.1%),Ⅲin 12(34.3%), which undergo histopathology verification. 18 clear cell carcinomas, 8 Granular cell carcinoma and 9 Combining cell carcinoma were included in this study. In 35 cases renal cell carcinoma, the blood flow signals were detected by GE Logic-9 Color Doppler Flow Imaging Ultrasound, and were graded into (0-Ⅳ)according to the blood flow distribution and degree of blood supply richness. RI and PSV were recorded by Pulsed Wave Doppler in region of interestperitumor and intratumor before operation. There are four specimens were taken from peritumor and intratumor parts for CD34 and VEGF immunohistochemical staining. Red-brown granular cytoplasmic staining for VEGF was considered as positive staining. The immunohistochemical studies were scored as follows: the VEGF was expressed as percentages of the VEGF positive cells by counting tumor cells in three areas at×200 magnification. For VEGF, staining was determined semiquantitatively according to a two-grade scale: low expression was cytoplasmic immunostaining of tumor cells(＜25%);high expression was cytoplasmic immunostaining of tumor cells(≥25%). MVD was quantitatively assessed at×400 magnigication (×40 objective lens and×10 ocular lens). For each tumor, microvessels were counted in five areas at×400 magnification and microvessel count per×200 magnification area was recorded as the MVD. The tissue samples of transmission electron microscopy the same as immunohistochemistry. The tissue sample was 1 mm width, 1 mm thick and 1 mm length; then quickly immerging into fixing solution after 0.5-2 min ex vivo. The specimens was cut 50 nanometer sections by LKB-V type ultramicrotome and was urany acetate stained 30-45 min, Lead Citrate counterstained 30 min after refixation. The ultrastructure change of tumor vessel in different parts in RCC by JEOL-100CX type transmission electron microscopy. The result were analysed statically using the SSPS 13.0 software. P＜0.05 was considered to indicate statistical significance in all tests.Result:1. The blood flow characteristic:The morphologies and distributions of microvessels in 35 renal cell carcinomas various. PSV=(110.16±69.96) cm/s versus (96.23±58.87) cm/s in peri-tumor and intra-tumor, the different was not statistically significant (P＞0.05).RI=0.69±0.08 versus 0.55±0.09 in peri-tumor and intra-tumor, and the difference of Rl was also significant (P＜0.05). RI of peri-tumor was higher than that of intra-tumor. The blood flow classification was found to significantly correlate with clinical stage and pathological grades.(Spearman rank correlation, r=0.530,P＜0.001;r=0.522 P＜0.001,respectively).2. The expression of MVD and VEGF: The positive expression of MVD in both peri-tumor and intra-tumor sections of 35 cases RCC tissue, mean MVD 56.38,SD 26.74. The negative expression of MVD in 8 normal renal tissues. The MVD was higher in peri-tumor tissues as opposed to in intra-tumor tissues (MVD=45.13±20.99 versus 33.83±21.15), the different was statistically significant (P＜0.05).In our study, MVD increased with more advanced clinical stage and with higher histopathological grades. There was significant difference in each clinical stage and each histopathological grades (P＜0.001). In the normal kidney, the VEGF is weakness expressed in epithelia of collecting duct of 3 cases (37.5%);is no expressed in 5 cases. There are no expression in all glomerular epithelia. The expression levels of VEGF were different in 35 cases RCC, VEGF immunoreactivity was detected in 35 cases(100%).There was significant difference in VEGF expression between RCC and normal kidney tissue (P＜0.05). We found no different between overexpression of the VEGF and stageⅠ,Ⅱgroup or stageⅡ,Ⅲgroup in RCC (P＞0.05), however, the significant difference in VEGF overexpression in relation to stageⅠandⅢgroup (P＜0.05). There was no different in between overexpression of the VEGF and gradeⅠ,Ⅱgroup or gradeⅡ,Ⅲgroup in RCC (P＞0.05) however, the significant difference in VEGF overexpression in relation to gradeⅠandⅢgroup (P＜0.05). The high expression of VEGF was positively correlated with MVD, MVD in the high and low expression of VEGF group was 57.72±21.03 versus 32.17±17.15. Addition, there was significant difference in both group(P＜0.001).3. Observation of RCC vascular ultrastructure: to take the materials from in necrosis of intra-tumor which not detected blood vessel structure; to take the materials from marginal part of intra-tumor: TEM show that blood vessel structure not integrity, vessel lumen diameter varying from 0.5 micrometer to 10 micrometer, vessel wall is made of monolayer endothelium, non-homogeneity of endothelium, thickness of vessel wall is varying from 0.4 micrometer to 20nm. The break of non-homogeneity vessel wall endothelial cell junction emerging changed to window gap with high permeability, there is blood plasma leakage in interstitial substance, and edema fluid and small amounts of collagen fibers flooding around blood vessel. Besides, endothelial cell is cacergasia stage, vessel wall collapse and structure degradation can be detected locally. To take the materials from tissues of peri-tumor, blood capillary is abundance, tumor growth actively, cell appearance and cell organ looks good; microvessel density increased but not maturated one; Proliferation and vessel wall of blood capillary endothelial cell is non-homogeneity. Diameter of tumor microvascular is larger than normal blood capillary, meanwhile, emerging various kinds of blood vessel loop, coincidence and conjunction each other as well.Conclusion:1. Hemodynamics appearance of RCC is diversity; both carcinoma peripheral blood flow abundance and peri-carcinoma RI higher than intra-tumor suggest that angiogenic of RCC from peripheries to intra-tumor, significant reason of RI raising up is disorder of vascular structure and distribution.2. The blood flow grade of RCC is obvious positive correlation with clinical stage and pathological grade.3. It is coincidence in Doppler blood flow distribution feature that MVD of peri-tumor is higher than intra-tumor; the correlation between overexpression of VEGF gene and tumor MVD indicated that VEGF is an important gene of vascularization and growth of RCC.4. Further observation of ultrastructural tumor capillary vessel provided evidence to blood flow hemodynamics feature of RCC by Color Doppler US.