Expression And Clinical Significance Of Twist,vimentin,vasculogenic Mimicry In Triple-negative Breast Cancer

BackgroundTriple-negative breast cancer（TNBC）accounts for about 15.0%～20.0%of all breast cancers.Compared with non-triple-negative breast cancer（N-TNBC）,TNBC tends to be younger^[1],more aggressive,and more prone to liver and brain metastasis.Up to now,the treatment for TNBC is still limited and often resistant^[2].The median survival time of TNBC patients with metastasis is only 1 year,much lower than that of N-TNBC patients^[3].Twist is an important driver of epithelial-mesenchymal transformation（EMT）process^[4],and Vimentin is an iconic molecule in EMT process.During EMT,the adhesion between epithelial cells was weakened,polarity was lost,basement membrane was degraded,extracellular matrix was remodeled,cytoskeleton,morphology and motor ability were changed,and the migration and invasion ability of tumor cells were increased.Vasculogenic mimicry（VM）forms a vascular-like structure through the deformation of tumor cells themselves and the remodment of extracellular matrix.The tumor cells covering VM channels express some endothelial cell markers^[5],suggesting that the process of VM is similar to that of EMT,which involves the transformation from epithelial characteristics to endothelial phenotype.By detecting the expression of Twist,Vimentin and VM in TNBC,and combining the clinicopathological features and the correlation between the Twist、Vimentin and VM,this paper aims to preliminarily explore the relationship between EMT and VM in TNBC,aiming to find new targets and prognostic predictors for TNBC therapy.ObjectiveBy detecting the expression of Twist、Vimentin and VM in TNBC and the corresponding benign breast disease tissues,the relationship between Twist,Vimentin and VM was analyzed with the clinicopathological characteristics of the patients and the correlation of the expression of the three.And the regulatory relationship between Twist,Vimentin and VM was preliminarily discussed.The relationship between the expression of Twist,Vimentin and VM and the prognosis of TNBC patients was analyzed by following up the prognosis of TNBC patients.To lay a foundation for finding new effective targets and prognostic predictors for TNBC therapy.Methods1.120 cases of TNBC and 30 cases of benign breast disease were included,and complete clinical data were collected.Immunohistochemical staining（IHC）was used to detect the expression characteristics of Twist and Vimentin in TNBC and the corresponding benign breast disease tissues.Platelet colorectal cancer cell adhesion molecule-1（PECAM-1/CD31）and periodic acid-Schiff stain（PAS）were used to detect the expression of VM in the two groups.The positive expression rates of Twist,Vimentin and VM in patients with different pathological characteristics were compared.Spearman rank correlation analysis was used to analyze the correlation between Twist,Vimentin and VM expressions.2.Kaplan-Meier method and Log-rank test were used to analyze the follow-up data of120 TNBC patients,and the relationship between the expression of Twist,Vimentin and VM and the prognosis of patients was analyzed.SPSS 22.0 software was used for all statistical data analysis.The statistical results were based on P<0.05 was a statistically significant difference.Results1.Expression of Twist,Vimentin and VM in TNBC and benign breast diseasesIn TNBC tissues,brownish yellow or tan particles in the nucleus were positive cells for Twist expression,and brownish yellow or tan particles in the cytoplasm were positive cells for Vimentin expression.In VM,cherry red ducts were observed in section staining,with no vascular endothelial cells lining the inner wall and red blood cells in the ducts,as positive expression;otherwise,it was negative expression.1.1 Expression of Twist in TNBC and benign breast diseasesImmunohistochemical staining was performed on paraffin tissue sections of 120TNBC and 30 benign breast diseases,and the expression of Twist was observed under microscope.Among the 120 TNBC tissues,Twist was positively expressed in 73 cases,with a positive expression rate of 60.8%（73/120）.In 30 cases of benign breast disease,4cases of Twist were positive,the positive expression rate was 13.3%（4/30）,the difference was statistically significant（χ²=21.675,P<0.05）.1.2 Expression of Vimentin in TNBC and benign breast diseasesImmunohistochemical staining was performed on paraffin tissue sections of 120TNBC cases and 30 benign breast diseases,and the expression of Vimentin was observed under microscope.Among the 120 TNBC cases,30 cases showed positive expression of Vimentin,with a positive expression rate of 25.0%（30/120）.Vimentin was positive in 2 of the 30 benign breast diseases（6.7%,2/30）,and the difference was statistically significant（χ²=4.807,P<0.05）.1.3 Expression of VM in TNBC and benign breast diseasesThe expression of VM was detected by CD31 and PAS staining in paraffin tissue sections of 120 TNBC and 30 benign breast diseases.The positive expression of VM in120 TNBC tissues was 34 cases（28.3%,34/120）.No expression of VM was detected in 30benign breast diseases.There was a significant difference between them（χ²=10.991,P<0.05）.2 Relationship between expression of Twist,Vimentin and VM and clinicopathological features of TNBCThe expression of Twist in TNBC tissue was correlated with lymph node metastasis and clinical stage,but not with tumor size,age and menstrual status.The expression of Vimentin was correlated with tumor size,lymph node metastasis and clinical stage,but not with age or menopause.The expression of VM was correlated with tumor size,lymph node metastasis and clinical stage,but not with patient age and menstrual status.3 The correlation of Twist,Vimentin and VM expression in TNBCIn TNBC,the expression of Twist and Vimentin was positively correlated with the occurrence of VM phenomenon（r=0.277,0.363,P<0.05）,and there was a significant correlation between Twist and Vimentin（r=0.384,P<0.05）.4 Relationship between the expression of Twist,Vimentin and VM in TNBC and patient prognosisTNBC patients were followed up for 9 to 83 months,with an average follow-up time of 55 months.A total of 35 patients died during the follow-up,with a survival rate of70.8%（85/120）.The survival rate of patients with Twist positive expression was 61.6%（45/73）,and that of patients with Twist negative expression was 85.1%（40/47）.The survival rate of patients with positive Vimentin expression was 43.3%（13/30）,and that of patients with negative Vimentin expression was 80%（72/90）.The survival rate of patients with positive VM expression was 55.88%（19/34）,and the survival rate of patients with negative VM expression was 76.7%（66/86）.There was a significant difference between them（P<0.05）.The survival rate of patients with positive Twist,Vimentin and VM was29.4%（5/17）,significantly lower than that of patients with negative Twist,Vimentin and VM,which was 77.7%（80/103）.Log-rank univariate survival analysis showed that the expression of Twist,Vimentin and VM was significantly correlated with the survival time of TNBC patients（χ²=9.490、22.940、10.820,P<0.05）.Conclusion1.Twist,Vimentin and VM were highly expressed in TNBC tissue compared with benign breast disease tissue.2.In TNBC tissue,the positive expression of Twist was significantly correlated with lymph node metastasis and clinical stage;The tumor diameter,lymph node metastasis and clinical stage were correlated with the expression of Vimentin.The tumor diameter,lymph node metastasis,and clinical stage were correlated with the presence of VM.3.The expression of Twist,Vimentin and VM in TNBC tissue was significantly positively correlated,suggesting that Twist and Vimentin participate in the formation of VM in TNBC.4.The results of survival analysis indicated that the positive expression of Twist,Vimentin and VM in TNBC indicated a poor prognosis,and Twist,Vimentin and VM were potential survival predictors of TNBC.