The Expression Of MTDH In HER2+ Breast Cancer And Its Relationship With The Efficacy And Prognosis Of Neoadjuvant Therapy

Part Ⅰ Study on the relationship between MTDH expression in HER2+ breast cancer tissue and its clinicopathological characteristics and efficacy of neoadjuvant chemotherapy.Objective: To discuss the relationship between MTDH expression and clinicopathological characteristics and chemotherapy efficacy in patients of HER2+ breast cancer treated with neoadjuvant chemotherapy combined with Herceptin.Method: Retrospectively analyzed the data of all locally advanced breast cancer patients of positive HER2 who received trastuzumab combined with neoadjuvant chemotherapy in the Fourth Hospital of Hebei Medical University from January 2009 to July 2017.A total of 144 patients were enrolled as the treatment group.Using SPSS22 case-control matching(Case Control Matching),1:1 matching the same locally adanced HER2+ patients for control group of 144 cases who unused herceptin treated with neoadjuvant chemotherapy.All patients were confirmed to be HER2+ or amplified by immunohistochemical staining or FISH detection.The relationship between MTDH expression and clinicopathological characteristics,chemotherapy efficacy and prognosis in patients with two different treatment regimens were analyzed.Nonparametric test of two independent samples,univariate χ2 test and multivariate Logistic regression analysis showed that P<0.05 was considered statistically significant.Two-sided test was used in all statistics.Results:1.Non-parametric tests of two independent samples were conducted for clinical biological characteristics and chemotherapy regimen selection between the treatment group and the control group,and the results showed no difference in case selection between the two groups.2.There was no difference between the treatment group and the control group in the clinical efficacy evaluation,which may be caused by the different evaluation methods(P=0.154).Pathological efficacy evaluation showed that MP grade and pathological lymph node staging of the treatment group were significantly better than those of the control group(P<0.05).In terms of p CR rate,although there was no statistical significance between the two groups,however the treatment group(23.6%)showed a significantly higher trend than that of the control group(17.3%).3.Comparing the clinical pathology characteristic and the neoadjuvant chemotherapy effect,found that the higher TNM staging,higher pathological N staging,the effect of neoadjuvant chemotherapy is worse(P<0.05),Ki-67>14% achieve better effect of neoadjuvant chemotherapy(P=0.027),anthracycline-based sequential paclitaxel plus herceptin has better effect(P<0.05),higher expression of MTDH achieve the worse effect of the neoadjuvant chemotherapy(P<0.05),but age,receptor,stratification of HER2,herceptin regimen of three weeks or single week are not significantly associated with chemotherapy efficient(P > 0.05).4.MTDH expression,TNM staging,number of lymph node metastases,expression of Ki-67,choice of chemotherapy regimen and p CR rate were correlated with the effect of neoadjuvant chemotherapy(P< 0.05),while age,ER,PR,Trastuzumab regimen of three weeks or single week were not correlated with the effect of neoadjuvant chemotherapy.Single factor Logistic regression analysis found that low expression of MTDH,TNM stageⅡ,selection of AC-TH chemotherapy regimen and high expression of Ki-67 patients are more sensitive to neoadjuvant chemotherapy when Trastuzumab is added,multiariable Logistic regression analysis found that expression of MTDH,TNM stage,selection of chemotherapy regimen are the independent factors affecting the effects of chemotherapy.Part 2 Discussion of the relationship between MTDH expression and prognosis in patients with HER2+ breast cancer treated with neoadjuvant chemotherapy combined with TrastuzumabObjective: To discuss the expression of MTDH and prognostic factors affecting neoadjuvant chemotherapy combined with Herceptin and neoadjuvant chemotherapy alone in locally advanced HER2+ breast cancer patients.Method: The cases of neoadjuvant chemotherapy with Herceptin therapy from the Fourth Hospital,Hebei Medical University from2009 to 2017 were collected.Among them,144 cases were in the treatment group,and these 144 cases were followed up and the followup was successful.In the same period,144 patients underwent neoadjuvant chemotherapy alone served as the control group,and 137 of them were followed up successfully.All patients were confirmed by immunohistochemical staining or FISH testing for HER2+ or amplification,patients achieve pathological complete response(p CR)have no tumor tissue,thus eliminate p CR patients,using immunohistochemical staining MTDH protein in postoperative tissues.The relationships between MTDH expression and clinicopathological characteristics and prognosis in patients with two different treatment regimens were analyzed.SPSS22.00 was used for statistical analysis,chi-square test for correlation analysis,KaplanMeier for survival analysis,Cox univariate and multivariate regression analysis,P<0.05 was statistically significant.Results:1.The low expression of MTDH protein in 144 breast cancer patients was54.86%(79/144),and the high expression was 45.14%(65/144).The expression of MTDH was related to lymph node metastasis(X~2=71.266,P<0.001),MP grade(X~2=15.450,P<0.001),and Ki-67 expression(X~2=34.034,P=0.001),and the difference was statistically significant(P<0.05),but not with histological grade,TNM stage,ER,PR expression,the difference was not statistically significant(P>0.05).Among 144 breast cancer patients in the control group,50.00%(72/144)had low expression of MTDH protein,and 50.00%(72/144)had high expression of MTDH protein.The expression of MTDH was related to histologic grade(X~2= 9.918,P=0.005),TNM stage(X~2=13.004,P=0.001),lymph node metastasis ststus(X~2=30.484,P<0.001),MP grade(X~2=6.337,P=0.044),and Ki67(X~2= 28.899,P<0.001).The difference was statistically significant(P<0.05),but there was no significant difference with age,ER and PR.2.In the treatment group,there were 11 cases of death,and the 1-year,3-year and 5-year OS rates were 94.9%、93.9% and 84.0%,respectively,with a median survival time of 79 months;In the control group,28 deaths occurred in the control group,and the 1-year,3-year and 5-year survival rates were94.4%,77.9% and 71.6%,respectively.The median survival time was 54 months,and there was a significant difference between the two groups(X~2=4.204,P=0.040).TNM stage,lymph node metastasis status and MTDH expression in treatment group could affect patients’ OS(P<0.05).In the treatment group,21 patients developed recurrence or metastasis,and 1-year,3-year and 5-year DFS were 91.9%,80.1% and 77.9%,respectively.The median disease-free survival was 48 months.In the control group,55 patients developed recurrence or metastasis,and the 1-year,3-year and 5-year DFS were89.9%,64.1%and48.9%respectively.The median disease-free survival time was39 months,and there was a significant difference between the two groups(X~2=10.089,P=0.001).In treatment group,TNM stage,lymph node metastasis status,Ki67 expression status and MTDH expression could affect patients’ DFS(P < 0.05).Kaplan-Meier survival analysis was used to evaluate the effect of MTDH expression on the survival of breast cancer patients in 281 successful followup patients.The analysis results show that the OS of the MTDH-negative patients was higher than that of the MTDH-positive patients,and the OS of the MTDH-negative patients in the treatment group was higher than that of the MTDH-negative patients in the control group,and the OS of the MTDHpositive patients between two groups was same(X~2=21.711,P<0.001),the difference between the two groups was statistically significant(P<0.05).The DFS of MTDH-negative patients was higher than that of MTDH-positive patients,and the DFS of MTDH-negative patients in the treatment group was higher than that of MTDH-negative patients in the control group,and the DFS of MTDH-positive patients in the treatment group was higher than that of MTDH-positive patients in the control group,(X~2=41.742,P=0.001),the difference between the two groups was statistically significant(P<0.05).3.Univariate Cox risk ratio regression analysis showed that patients’ histological grade,TNM stage,lymph node metastasis status,MP grade,Ki67,MTDH expression had significant influence on patients’ OS,the difference was statistically significant(P<0.05).Multivariate Cox risk ratio regression analysis showed that lymph node metastasis status,TNM stage,Ki67 and MTDH expression had significant effects on OS,and the difference was statistically significant(P<0.05).Univariate Cox risk ratio regression analysis showed that histological grade,TNM stage,lymph node metastasis status,MP grade,PR,Ki67 and MTDH expression had significant effects on DFS(P<0.05).Multivariate Cox risk ratio regression analysis showed that TNM stage,lymph node metastasis status and MTDH expression had significant influence on DFS,and the difference was statistically significant(P<0.05).Part Ⅲ The effect of sensitivity on shRNA silence MTDH gene in HER2+ breast cancer SK-BR-3 cells to trastuzumab combined with paclitaxel and the tumor-forming ability of nude miceObjective: To study the effect of MTDH gene silencing on proliferation of HER2+ breast cancer SK-BR-3 cells and resistance to paclitaxel combined with herceptin,and to study the effect of MTDH silencing on proliferation of HER2+breast cancer cells in nude mice.Method:1.The HER2+ breast cancer cell line SK-BR-3 with stable low MTDH expression was constructed by lentivirus infection method.The transfection efficiency was detected by RT-qPCR and Western-blot,and the stably transfected cell line was established and named as SH-MTDH.2.MTS method was used to detect the effect of MTDH gene on the proliferation ability of breast cancer SK-BR-3 cells Which were treated with paclitaxel and herceptin.3.The breast cancer cell lines with stable and low expression of MTDH shMTDH and sh-NC were used to establish subcutaneous xenograft tumor model in nude mice.4.The growth of sh-MTDH and sh-NC cells in nude mice was detected,and the tumor size in each group was measured.5.The expression level of MTDH gene in the xenograft tumor tissues of nude mice was detected by RT-qPCR,and the expression level of MTDH protein in the xenograft tumor tissues of nude mice was detected by Westernblot.Result:1.The results of immunofluorescence and RT-qPCR showed that the HER2+breast cancer cell line sh-MTDH with stable and low expression of MTDH was successfully constructed.2.The morphological changes of stable transgenic breast cancer cells with MTDH gene and breast cancer cells treated with paclitaxel and herceptin were observed under microscope.The breast cancer cells transfected with sh NC and sh MTDH grew in good condition without significant morphological changes.After addition of 0.1ug/ml paclitaxel and 10ug/ml herceptin,the cells became round,wrinkled and showed apoptotic morphology.3.MTS method was used to detect the effect of SK-BR-3 with different MTDH expression levels on sensitivity to paclitaxel and herceptin in breast cancer cells.In breast cancer cells SK-BR-3,knockdown of MTDH significantly increased sensitivity to paclitaxel and herceptin combination therapy.4.The results of tumor formation experiments in nude mice showed that shMTDH cells reduced the volume and weight of xenograft tumors in nude mice after knockdown of MTDH gene expression(P < 0.05).5.RT-qPCR results showed that the expression of MTDH in sh MTDH xenograft tissues was decreased,and Western-blot analysis showed that the expression level of MTDH protein was decreased in xenograft tumor of nude mice(P < 0.05).