| Breast cancer is one of the most common malignant tumors and threatens the physical and mental health seriously.The incidence rate has the tendency to increase, which would be the second principal cause of cancer death in women worldwide. Lymphatic metastasis is the most common way of transfer in breast cancer,and the most important factors to determine breast cancer stage,prognosis and therapeutic scheme,generally,using for evaluing the prognosis and selecting the therapeutic scheme.Cancer cells through the blood vessels or lymphatic vessels may eventually transfer to the regional lymph nodes,and lymphatic invasion and vascular invasion in the process of lymph node metastases of tumor play an important role.However,only recently there is no in-depth research about the lymph node metastasis of breast cancer because of a lack of specific lymphatic endothelial markers until the specific marker of lymphatic endothelium was developed on the previous decade. Furthermore,research on angiogenesis and intravascular invasion is better for understanding of the stages and grades of breast cancer,many surgeons and pathogists believe that intravascular invasion is clearly related to lymph node metastasis.After application of D2-40 and CD34 to mark lymphatic and vascular endothelium respectively,the enclosed cancer cells in vessels can be easily identified, and then we analyzed the clinicopathological features associated with lymphatic invasion and vascular invasion of cancer cells combined with the significance in the process of lymphatic metastasis of breast cancer.Intravascular invasive cancer cells could be a feasible factor for clinical staging and prognosis for breast cancer.OBJECTIVETo observe the staining results of D2-40 and CD34 in breast carcinoma tissue, and explore the feasibility of D2-40 and CD34 respectively as the lymphatic and vascular markerscell.Investigate the pathological characteristics of lymphatic and vascular which were invaded by cancer cells,then analyze the relationship between LVI,BVI and various clinicopathological parameters,and explore its value and clinical significance in lymphatic metastasis of breast cancer.METHEDSWe retrieved 191 cases with breast cancer who were operated in Nangfang Hospital from April,2007 to December,2008 using the medical record database.And only the cases with invasive ductal carcinoma were selected for study.We delete the cases who were treated with chemotherapy or radiotherapy before operation,or who were recurred or had small number of dissected axillary lymph nodes(less than 10). Therefrom,we selected the cases whose clinical data and pathologic diagnosis data was complete.At last,we got the final result of 72 cases randomly.At the same time, a random sample of 10 cases with benign breast disease served as controls.We got the corresponding paraffin blocks from the department of pathology,then made the immunohistochemical sections by the two-step way of shortcut immuno -chemistry.Firstly,deparaffinaged the paraffin section;secondly,repaired the antigen in citrate buffer solution by microwave 25 minutes;thirdly,used the solution of peroxidase to quench the activity of endogenous peroxidase about 3 minutes in microwave,then washed the sections three times separately by Double distilled water and PBS;fourthly,the solution of antibodies to D2-40 and CD34 was applied and the sections were incubated at 37℃in Electric constant temperature incubator about 1 hours,then washed the sections three times by PBS;fifthly,the solution of Goat anti-rabbit HRP polymer LgG was applied and the sections were incubated at 37℃in Electric constant temperature incubator about about 30 minutes,then washed the sections three times by PBS;sixthly,used the solution of DAB to colourate about 5minutes;At last,mild Staininged with Hematoxylin after washed with water, Hydrochloride- Alcohol differentiation about 6 seconds,after dehydration by alcohol and transparent by xylene,mounted by neutral gum.We kept the solution and reaction condition identical,to ensure the unity and comparability.If the color is doubtful,we would carry out the experiment again.We got PBS instead of antibody for negative control and got the known positive reaction sections for positive control.We made sure that there were no false positive and false negative according to the color situation of Positive and negative control.On the premise of this,D2-40 immunohistochemical staining in the cytoplasm and(or) membrane showed a clear positive brown particles expression,including the brown-yellow stained-tubulin,a single endothelial cell or cell cluster;CD34-positive staining in the standard:tumor cells and the lumen of the endothelial cells or endothelial cell cluster with a brown dye distinguished from the surrounding blood vessels,tumor cells and other connective tissue.Regardless of lumen and red blood cells,all were regarded as single blood vessels can be counted.D2-40 and CD34-positive lymphatic vessels and blood vessels in which cancer cells were observed were determined as lymphatic vessel invasion(LVI) or blood vessel invasion(BVI).Data analyses were carried out with SPSS13.0 statistical software package. Comparison of inter-group differences were analyzed with the fourfold table or list of chi-square test line;Spearman correlation analysis was used to analyzed the relationship between the two factors.P values＜0.05 were considered significantRESULTS1.There were positive expressions of D2-40 and CD34 in all breast cancer tissue. D2-40 expressed positively in four cases of benign breast lesions.D2-40 positive expression rate were significantly different between breast cancer and benign lesions group(x2=46.611,P=0.000).However,there were positive expressions of CD34 in all benign lesions.Vascular randomly distributed in the center and periphery of tumor in breast cancer group,and lymphatics usually distributed in the surrounding mesenchymal tissue,especially at the junction site of cancer and normal tissue. Central parts of lymphatic vessels were usually narrow,even atresia,however, peripheral lymphatic often expaned.Compared to the vascular marked with CD34, lymphatic wall marked with D2-40 was thin,often incompletely,irregular,with inconsistent lumen size,with the wall of single-layer squamous epithelium without myometrial,without red blood cell and neutrophil.Stromal components also usually expressed as positive staining with CD34 as vascular markers,required combination of morphological to determinate.The number of blood vessels and lymphatic vesels are fewer in benign lesions compared to the breast cancer group.2.LVI-positive rate was 69.4%,BVI-positive rate was 52.8%,the LVI-positive rate is higher than BVI,the differences were significant(x2=4.208,P=0.04);There was no lymphatic or vascular invasion in benign tumors.That vascular was invaded by Cancer cells could perform as single cancer cell or some cancer cells or cancer mission which even could fill the entire bureaucratic.Lymphatic infiltration and distribution of lymphatic vessels were in line,LVI was seen more in surrounding parts of cancer where lymphatic distributed intensively.Sometimes,the lymphatic infiltration was seen in normal adipose tissue and fascia cancer organizations surrounding the cancer.However,the Vascular invasion located in the center or distributed in the surrounding normal tissue.The vascular invasion in the incomplete vascular,formed by immature endothelial cells,was not uncommon, which should not determined as positive vascular invasion.3.LVI positive rate in breast cancer group with lymph node metastasis was 85.7%,it was 54.1%in group whithout lymph node metastasis,the former was higher than the latter significantly(x~2=8.496,P=0.004).There was significantly positive correlation between LVI and axillary lymph node metastasis of breast cancer (r=0.382,P=0.001).LVI positive rate was highest in those patients whose number of lymph node metastasis was more than 10,node-negative group was lowest(54.1 %).There were significant differences among the three groups(x~2=10.700, P=0.013).BVI positive rate in breast cancer group with lymph node metastasis was 68.6%,it was 37.9%in group without lymph node metastasis,the former was higher than the latter significantly(x~2=6.817,P=0.009).There was significantly positive correlation between BVI and axillary lymph node metastasis of breast cancer (r=0.332,P=0.004),with the increase in the number of lymph node metastasis,BVI positive rate was significantly increased.There were significant differences among the three groups(x~2=8.125,P=0.043).LVI and BVI had no significantly positive correlation with age(r=-0.042,P=0.729;r=0.220,P=0.854),the positive rate in each group was not significantly different(x~2=0.657,P=0.720;x~2=0.043,P=0.979), positively related to tumor size and histological grade(r=0.407,P=0.000,r=0.334, P=0.004),the positive rate in each group was not significantly different(x~ 2=13.192,P=0.001,=8.698,P=0.013).ReIation was not seen between LVI and clinical stage(r=0.102,P=0.393),however,BVI was positively related to clinical stage(r=0.543,P=0.000).LVI was positively related to ER and PR(r=-0.406, P=0.000;r=-0.341,P=0.003),negatively correlated with Her-2 and VEGF and EGFR(r=0.357,P=0.002;r=0.435,P=0.000;r=0.267,P=0.024).however,BVI was positively correlated with VEGF(r=0.405,P=0.000),had no relation to ER or PR or Her-2 or EGFR(r=-0.156,P=0.192;r=-0.143,P=0.232;r=0.074,P=0.534;r=0.160, P=0.180),the positive rate in each group was not significantly different(x~2=2.300, P=0.512;x~2=4.812,P=0.186;x~2=3.996,P=0.262;x~2=7.534,P=0.057).CONCLUSION1.D2-40 can specially mark the lymphatic vessel,get a better staining result, which makes it a reliable marker of lymph vessel,however,CD34 performs a poor specificity,is also positive in surrounding stromal staining,not only in vascular staining.2.In the process of lymphatic metastasis of breast cancer,the cancer cells may invade the lymph vessel earlier than vascular;Vascular invaded by cancer cells are probably the blood vessels which already exists in the tumor,however,the cancer cells,invading blood vessel,can induce the formation of neovascularization.3.LVI and BVI are closely related to lymph node metastasis of breast cancer, which can occur earlier than lymph node metastasis,with the most target-related poor prognosis of breast cancer,suggesting it a possible target for a poor prognosis.With those,the prognosis can be estimated,specific-targeted therapeutic regimen can be made and the efficacy of therapy can be evaluated for patients with breast cancer. Therefore,LVI and BVI,as a routine test items,should be taken seriously.Therefore,lymphatic vessel invasion in breast invasive ductal carcinoma should be as an independent clinical staging classification indicators,may be one of the poor prognosis factors.Lymphatic vessel invasion should be recommended as a regular test items in clinical pathology. |
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