Expression Of DLL4and VEGF In Breast Cancer And The Relationship With MVD In Tumor

Objective: Delta-like ligand4(DLL4) is one of the Notch family ligandsthat is upregulated by vascular endothelial growth factor (VEGF) and isreported to have a role in tumor angiogenesis. Evidence from xenograftstudies suggests that inhibiting DLL4-Notch signaling pathway may overcomeresistance to anti-VEGF therapy. The aim of this study was to measure theexpressions of DLL4, VEGF and calculate the microvessel density (MVD),determined using CD34labeled vascular endothelial cell in breast cancer andadjacent normal breast tissues; analyze their relationships with angiogenesis ofbreast cancer and determine if protein expression correlated with patients'clinicopathological parameters, including lymph node metastasis, histologicalgrade, age, tumor size, and clinical stage.Methods:1. In all, this study subjected sixty specimens of surgicallyresected and paraffin-embedded human breast cancer tissues and twenty casesof adjacent normal breast tissues as comparison.2. Immunohistochemistry (SP means) was performed to measure theexpressions of DLL4protein, VEGF protein and MVD (CD34protein marked)in breast cancer and adjacent normal breast tissues.3. Statistical analysis: T test, Pearson Chi-square (χ~2) test or ContinuityCorrection Chi-square (χ~2) and Spearman's rank correlation analysis wereperformed. A probability (P)-value less than0.05was considered statisticallysignificant.Result:1. By immunohistochemistry, there was a strongly positiveexpression of DLL4protein in the cytoplasm and membrane of malignant cells,VEGF protein expression was mainly observed in the cytoplasm of themalignant cells, CD34protein was stained in membrane and cytoplasm invascular endothelial cell, all of them were brownish yellow. The expression of DLL4in breast cancer tissues was remarkably higher than that in adjacentnormal breast tissues,75%(45out of60) and15%(3out of20) respectively,(χ~2=22.500, P<0.001); The positive expression rate of VEGF in breast cancertissues was70%(42out of60), evidently higher than20%(4out of20) inadjacent normal breast tissues (χ~2=15.345, P<0.001); the amount of MVD was41.57±6.588in breast cancer tissues, while in adjacent normal breast tissuesthe amount was140.827±22.390, there was a significant difference (t=30.592,P<0.001).2. In60cases of breast cancer tissues, DLL4protein expression inlymphnode metastasis positive group was higher than that in lymphnodemetastasis negative group,93.10%(27out of29) and58.06%(18out of31)respectively,(χ~2=9.811, P=0.002). DLL4protein expression in histologicalgrade Ⅲ group was100%(15out of15), which significantly higher than66.67%(30out of45) that in histological grade Ⅰ～Ⅱ group (χ~2=5.007,P=0.025). DLL4protein expression in the tumor size≤2cm group was60.87%(14out of23), while in the tumor size>2cm group was83.78%(31out of37),there was significant difference between this two groups (χ~2=3.972, P=0.046).DLL4protein expression in clinical stage Ⅱ～Ⅲ group was83.72%(36outof43), which remarkably higher than52.94%(9out of17) in clinical stage Ⅰgroup (χ~2=3.972, P=0.046). DLL4protein expression in age≤50y group was64.52%(20out of31), in age>50y group was86.21%(25out of29), therewas no statistic significance between them (χ~2=3.760, P=0.053).3. In60cases of breast cancer tissues, expressions of VEGF protein inlymphnode metastasis negative group and positive group were41.94%(13outof31), and100%(29out of29) respectively, there was statisticallysignificance between the two groups (χ~2=24.056, P<0.001). Expressions ofVEGF protein in histological grade Ⅰ～Ⅱ group and Ⅲ group were60%(27out of45), and100%(15out of15) respectively, there was statisticallysignificance between both of them (χ~2=6.772, P=0.009). Expressions of VEGFprotein in clinical stage Ⅰ group and Ⅱ～Ⅲ group were41.18%(7out of17),81.40%(35out of43) respectively, showing statistically significance between them (χ~2=9.384, P=0.002). Expression of VEGF protein in the tumor size≤2cm group and>2cm group were60.87%(14out of23),75.68%(28out of37) respectively, showing no significant difference (χ~2=1.481, P=0.224).Expression of VEGF protein in age≤50y group and>50y group were64.52%(20out of31),75.86%(22out of29) respectively, showing no significantdifference (χ~2=0.918, P=0.338).4. In60cases of breast cancer tissues, MVD in cases with DLL4-positivegroup was higher compared to the negative group (148.191±18.607vs.118.733±18.101, t=-5.345, P<0.001); MVD in cases with VEGF-positivegroup was higher compared to the negative group (149.810±17.034vs.119.867±19.386, t=-5.680, P<0.001). MVD in cases with both DLL4andVEGF-positive group was higher compared to the both of them negative andonly one of them positive group (152.822±15.141vs.121.530±18.392, t=-6.844, P<0.001).5. Under the correlation analysis, there was significant positivecorrelation between DLL4expression and VEGF, DLL4and MVD value, andso do VEGF expression and MVD value in breast cancer (rs=0.462, P=0.001vs. rs=0.553, P<0.001vs. rs=0.568, P<0.001).Conclusion:1. DLL4, VEGF and MVD expression in breast cancersignificantly higher than adjacent normal breast tissues, high expression mayindicates a poor prognosis.2. Expressions of DLL4and VEGF protein in breast cancer show asignificant correlation with lymphnode metastasis, histological grade andclinical stage, expression of DLL4show a significant correlation with thetumor size, which suggests that their expressions are closely related toinvasion and metastasis of breast cancer. The higher value of MVD in breastcancer is updated with DLL4, VEGF richer expression, which indicate that theboth of them may have some function in the form of microvascularangiogenesis in breast cancer.3. There exists a positive correlation between DLL4and VEGF, and bothof them correlate positively with MVD, which suggests that VEGF may be an important factor in inducing the up-regulation of DLL4, and both of them mayhave a synergistic effect in invasion, metastasis and angiogenesis of breastcancer.