Research On Lymphatic Microvessel Density And The Expression Of Endothelial Vascular Growth Factor-D In Human Laryngeal Carcinoma

Objective: The laryngeal carcinoma is a very commontype of malignant tumors in otolaryngology, metastasis ofcancer cells is a key prognostic factor for the patients's outcome.In past, being lack of lymphatic-specific markers,most studiesabout metastasis focused on the angiogenesis, whereas very fewscholars paid attention to the lymphangiogenesis, the grow ofnew lymphatic vessels.Lymphatic microvessel density(LMVD) correlates withprognosis of patients's in some tumors. Vascular endothelialgrowth factor-D promotes lymphangiogenesis and angiogenesisin tumors and promotes the metastasis of cancer cells throughlymphatic vessels.In this study, 5'-Nase-ALPase double staining method wasused to observe LMVD in laryngeal carcinoma, VEGF-Dexpression was detected by immunohistochemistry method. Theaim of this study was to dectet the relationship between LMVDand clinical parameters in patients with laryngeal carcinoma,analysis the role of VEGF-D expression in promotinglymphangiogenesis and lymphatic metastasis, and therelationship between VEGF-D expression and LMVD, offertheoretic information for choice of clinical treatment andevaluate the prognosis.Methods1 MeterialsForty-five laryngeal carcinoma fresh samples wereanalyzed and meanwhile 45 para-carcinoma tissues were alsostudied as controls.All subjects are taken from the patients whoadmitted surgically treatment in the Department ofotolaryngology-head and neck,the Fourth Hospital of HebeiMedical University, from 2003.2 to 2004.11. All samples weresnap-frozen in liquid nitron and stored at -80oC.2.1 Enzyme-histochemistryAll the samples were proceed for 5'-Nase-ALPase doublestaining, the stained sections were screened in light microscope.2.2 ImmunohistrochemistryImmunohistrochemical staining for VEGF-D wasperformed using the streptavidin-peroxidase technique,anti-VEGF-D rabbit polyclonal antibody( Santa CruzBiotech,USA) was used.3 Results judgement3.1 Enzyme-histochemistryThe stained sections were screened at 40X magnification toidentify the regions of the highest vascular density within thetumor, lymphatic vessels were counted in 4 regions of thehighest vascular density at 100X magnification. The number oflymphatic vessels was the mean of vessels in these areas.3.2 ImmunohistrochemistryThe stained sections were screened at 100Xmagnification,and 1000 tumor cells were counted at random, thedegree of immunoreactivity for VEGF-D was considered to bepositive if distinct staining of the cytoplasm was observed in atleast 10% of tumor cells, negative if less than 10% or nostaining.4 Statistic methodsAll data were collected by Excel, analysis was performedusing the SPSS 11.0 for windows software, including t or t'testand χ~2 test. P<0.05 indicating distinct statistic significance,P<0.01 indicating very distinct statistic significance.Results1 Enzyme-histochemistry1.1 MorphologyIn light microscope,lymphatic vessels were 5'-Nasestaining positively(brown), blood vessels were ALPase stainingpositively(blue). In normal mucosa, lymphatic vessels locatedon mucosa or submucosa tissues, and accompany with bloodvessels, they were usually had an irregular lumen and a thintortuous endothelial profile.Inner or at the border of tumor tissue, many lymphaticvessels were observed, the lymphatic vessels were dialated orlialated extremely at the border, but inner the tumor, littlevessels with open lumen were found.1.2 Comparsion of laryngeal carcinoma with normal mucosaAmong 45 patients, the density of lymphatic microvesselswas 26.15±2.81 in tumor tissues, 4.07±1.86 in normal mucosa(P<0.01).1.3 Relationships between LMVD and clinical parametersAmong 45 patients,there were 39 males and 6 females,LMVD was 26.25±2.95 in male group, and 25.50±1.61 infemale group(P>0.01); in group of age ≥62, LMVD was26.20±3.05, in group of age<62, LMVD was26.10±2.57(P>0.05); LMVD was 24.52±2.53 in group ofclinical stageⅠ,Ⅱ, and 28.01±1.77 in group of clinical stageⅢ,Ⅳ(P<0.01); LMVD was 26.20±2.42 in group of typesupraglottic, 26.08±3.25 in group of type glottic(P>0.05);LMVD was 28.14±1.77 in group of lymph node metastasis,25.05±2.69 in group of nonmetastasis(P<0.01). Results showsthat LMVD in lymph node was remarkable higher than that ofnonmetastasis.2 ImmunohistochemistryVEGF-D staining was observed occasionally in normalmucosa. Among 45tumor tissues, VEGF-D expressionpositively was 14, VEGF-D expression negatively was 31, of 14VEGF-D expression positively, 13 has lymph node metastasis,usingχ~2 test, the conclusion was VEGF-D expression wasasscociated with lymph node metastasis(P<0.01).3 Relationship between expression of VEGF-D and LMVD LMVD was 28.00±2.05 in group of VEGF-D expressionpositively, 25.31±2.72 in group of VEGF-D expression...