The Expression Of Extracellular Matrix Metalloproteinase Inducer And Vascular Endothelial Growth Factor C In Nasopharyngeal Carcinoma And Their Significance

Background and objectiveNasopharyngeal carcinoma (NPC) has the highest incidence in southern China. Invasion andmetastasis are the main characters of NPC which usually spreads to cervical lymph nodeswithout any symptoms in the primary lesion. It is reported that almost 45 to 50 percent ofpatients were firstly discovered by its lymph node metastatic lesion. So it is valuable to study themechanism of hyperplasia and metastasis of NPC. Extracellular matrix metalloproteinase inducer(EMMPRIN) is a member of immunoglobulin superfamily and it is often rich in malignanttumors. EMMPRIN promotes tumor growth by stimulating hyaluronan production, inducesangiogenesis via elevating vascular endothelial cell growth factor and increases invasiveness viastimulation of matrix metalloproteinases (MMPs). Therefore, EMMPRIN plays important rolesin hyperplasia, angiogenesis and metastasis of tumor. Vascular endothelial growth factor C(VEGF-C) is the first identified regulator of lymphangiogenesis and participates inlymphangiogenesis and lymph node metastasis in many cancers. One paper says that theexpression of VEGF-C is positively related to hyperplasia and metastasis of NPC but there areonly a few articles concerning the relationship between VEGF-C and NPC up to now.Conclusively, the expressions of EMMPRIN and VEGF-C are malignant phenotypes of manytumors and they make tumors progress by stimulating their growth, angiogenesis,lymphangiogenesis and metastasis. The effects of EMMPRIN and VEGF-C maybe also existedin NPC. To make the mechanism of NPC malignance clear and provide some useful informationfor the prevention and therapy of NPC, we use immunohistochemistry, in situ hybridization andimage analysis to test the expression of EMMPRIN, VEGF-C and other relative indexes inprimary and metastatic lesion of NPC.Materials and methods 1 Tissue samples: 137 specimens were obtained from patients who underwent biopsy forchronic nasopharyngitis or NPC at Tumor Hospital of Shantou University Medical Collegeand Shantou Central Hospital from 1996 to 2005. All the specimens were divided into threegroups: 30 cases of chronic nasopharyngitis (CN group), 71 cases of primary NPC (P-NPCgroup) and 36 cases of lymph node metastatic NPC (M-NPC group). It is worthy to point outthat there were 34 paired cases both with tissues from primary tumor and its cervical lymphnode metastatic tumor (Paired group, selected from 2868 cases of NPC).2 HE stain: Cell morphology was observed by light microscope after HE stain and mitoticindex was counted at the same time.3 Image analysis: Tissue sections were stained by hematoxylin and the nucleus morphologywas quantified by Leica Qwin standard Y2.8 Image Processing and Analysis System.4 Immunohistochemistry: the Envision Labelled Peroxidase System two steps method wereperformed in the study. The expression of EMMPRIN, MMP-3, VEGF, VEGF-C, CD34 andPCNA protein were tested.5 In situ hybridization: 47 perfect samples including 31 P-NPC and 16 M-NPC were selectedfrom residual samples after IHC experiment. Expression of EMMPRIN mRNA wasexamined and its relationship with EMMPR1N protein was tested by consistency checking.Result1 Mitotic index (MI)MI in CN, P-NPC and M-NPC were 0.26±0.44, 3.00±1.66, 4.70±2.53 respectively.Comparison among these groups has significance(P＜0.01). MI in PP-NPC and PM-NPC were2.27±1.01, 4.65±2.68 respectively, comparison of them also has significance(P＜0.001).2 Image analysisThe area, perimeter, length, breadth of P-NPC and M-NPC nucleuses were significantly higher than those of CN(P＜0.01), but it is not the case to roundness. There is no significant differencebetween P-NPC and M-NPC in all morphologic parameters, so was between PP-NPC andPM-NPC.3 ImmunohistochemistryEMMPRIN protein expressed at cytoplasm or membrane of cancer cells and some epithelialcells. The positive rate of EMMPRIN expression in NPC was 67.29％, which was significantlyhigher than that of CN(P＜0.001). The positive rates of EMMPRIN expression in CN, P-NPCand M-NPC were 30.00％, 60.56％and 80.56％respectively. Significant differences were foundbetween CN and P-NPC (P＜0.01), between CN and M-NPC (P＜0.01) and between P-NPC andM-NPC (P=0.037). In PM-NPC group, it had been found that NPC cells had higher expressionrate of EMMPRIN than that in primary tumor by paired comparison (P=0.012).MMP-3 expressed in cytoplasm of cancer cells, some epithelial cells, lymph cells andfibroblast cells. The positive rates of MMP-3 expression in P-NPC and M-NPC were 35.21％,44.44％, which were significantly higher than that of CN13.33％(P=0.026和0.005), but nodifference was found between P-NPC and M-NPC, which was the same as between PP-NPC andPM-NPC.PCNA expressed in nucleuses of cancer cells and some epithelial cells. The positive rates ofPCNA expression in CN, P-NPC and M-NPC were 24.14％,59.38％,87.88％.Significantdifferences were found among these groups(P＜0.01). It was also found between PP-NPC andPM-NPC by paired comparison (P=0.039).VEGF expressed in cytoplasm of cancer cells and some epithelial cells. The positive rates ofVEGF expression in CN, P-NPC and M-NPC were 16.67％,53.62％,75.00％respectively.Significant differences were found between CN and P-NPC (P＜0.01), between CN and M-NPC(P＜0.01) and between P-NPC and M-NPC (P=0.033). It was also found between PP-NPC andPM-NPC by paired comparison (P=0.041).VEGF-C expressed in cytoplasm of cancer cells and some epithelial cells. The positive ratesof VEGF-C expression in CN, P-NPC and M-NPC were 20.00％,45.59％,66.67％. Significantdifferences were found between CN and P-NPC (P＜0.016), between CN and M-NPC (P＜0.01)and between P-NPC and M-NPC (P=0.040). It was also found between PP-NPC and PM-NPC by paired comparison (P=0.035).Microvessel Density (MVD) increased from CN, P-NPC to M-NPC. They were 7.70±4.52,13.55±7.95,15.10±5.92 respectively. MVD in P-NPC and M-NPC were remarkably more thanthat of CN(P＜0.01), but it is not the case between P-NPC and M-NPC. MVD in PP-NPC andPM-NPC were 15.20±8.93,15.03±5.96 respectively. There was also no significant differencebetween them(P＞0.05).4 In situ hybridizationEMMPRIN mRNA mainly located in tumor cytoplasm. By Consistency checking, theexpression of EMMPRIN protein using immunohistochemistry was concordance withEMMPRIN mRNA using in situ hybridization in 47 NPC samples(Kappa=0.319, P＜0.01).5 Correlation among all indexes measuredIn NPC, Spearman correlation was used to analyze the relationship between EMMPRIN andMMP-3, VEGF, VEGF-C, PCNA. It was found a linear dependence between EMMPRINexpression and the expression of VEGF, VEGF-C and PCNA. Correlation coefficient(r_s) were0.352,0.292 and 0.266 respectively (P＜0.01). It was also found a linear dependence betweenEMMPRIN expression and the expression of MMP-3(r_s=0.223, P＜0.05). Moreover, there werepositive relationship between every two indexes of MMP-3, VEGF, VEGF-C and PCNA(P＜0.05) except between MMP3 and PCNA and between VEGF-C and PCNA.In NPC, MI of EMMPRIN high expression group and low expression group were 3.99±2.35and 3.15±1.85 respectively. Significant difference was found between these two groups(P=0.047). There were also significant differences between PCNA positive and negative groupand between PCNA high expression and low expression group. With regard to VEGF-C, nodifference was found in the same condition.In NPC, there were no significant differences of MVD between EMMPRIN positive andnegative group and between EMMPRIN high expression and low expression group. With regardto VEGF and VEGF-C, there were also no differences in the same condition(P＞0.05).The positive rate of both EMMPRIN and VEGF-C expression in M-NPC was 55.56％, which was remarkably higher than that in P-NPC(P=0.032). The similar result was also fotmd betweenPM-NPC and PP-NPC (P=0.021).Conclusion1 Morphology of NPC cell nucleus doesn't alter before and after metastasis.2 EMMPRIN is an important malignant phenotype of NPC and its high expression may playsimportant roles in hyperplasia and lymph node metastasis of NPC, but not in angiogenesis.3 MMP-3 may be induced by EMMPRIN and promotes NPC metastasis.4 VEGF-C has no direct effect on hyperplasia and angiogenesis of NPC, but may takes part inthe formation and (or) development of lymph node metastatic lesion.5 EMMPRIN and VEGF-C may interact with each other in the formation and (or) developmentof lymph node metastatic lesion.