| Objectives:1.To observe the expression of stromal tumor infiltrating lymphocytes(s TILs)and programmed death receptor ligand-1(PD-L1)before and after neoadjuvant chemotherapy for breast cancer,and examine its relationship with clinical efficacy and clinicopathological features.2.To observe the changes of the infiltration of s TILs(CD4~+s TILs,CD8~+s TILs,FOXP3~+s TILs)before and after neoadjuvant chemotherapy in breast cancer,and to analyse its relationship with clinical outcomes and clinicopathological features.Methods:1.106 cases of breast cancer after neoadjuvant chemotherapy were retrospectively collected from January 2015 to October 2019 from the Department of Pathology,Affiliated Hospital of Zunyi Medical University,all patients had a core needle aspiration biopsy of the breast before treatment and were diagnosed with breast cancer by histopathology,and radical breast cancer surgery was performed at the end of neoadjuvant chemotherapy,based on grading according to the Miller-Payne(MP)system of these,26 cases were graded MP 5,i.e.pathologic complete response(p CR)after neoadjuvant chemotherapy,and 80 cases did not achieve pathologic complete remission(non-p CR).2.The s TILs was assessed on HE stained sections and the expression of PD-L1(Ventana SP142),the infiltration of CD8~+s TILs,CD4~+s TILs and FOXP3~+s TILs in the tumor tissues were detected by IHC staining.3.Patients were followed to observe the changes in PD-L1,s TILs,CD8~+s TILs,CD4~+s TILs and FOXP3~+s TILs before and after neoadjuvant chemotherapy,and aim to explore its relationship with clinicopathological characteristics and prognosis.4.SPSS 18.00 was used for statistical analysis of data,chi-square test and Fisher’s exact probability method for correlation analysis of two categorical variables,non-parametric U-test and W-test for comparison of non-normal continuous variables,binary logistic regression analysis for univariate and multi-factor analysis,Kappa test for concordance and difference analysis of paired categorical data,respectively,and The Kaplan-Meier method was used to compare the disease-free survival and overall survival of the groincreases,and the Graphpad Prism 8.0 software was used for graphing,with the difference considered statistically significant at P<0.05.Results:1.Basic clinical characteristics of patients and its relationship with neoadjuvant treatment efficacy(p CR rate):The mean age of the 106 women with breast cancer was(47.3±9.4)years(29-72years),and the molecular typing included 37 cases(34.9%)of Luminal A,29 cases(27.4%)of Luminal B,24 cases(22.6%)of HER2(3+),and 16 cases(15.1%)of triple negative breast cancer(TNBC)in 16 cases(15.1%).Among them,26 cases(24.5%)achieved p CR after neoadjuvant chemotherapy,with p CR rates of 8.1%,20.7%,41.7%and 43.8%for patients with Luminal A,Luminal B,HER2(3+)and TNBC respectively.The p CR rate after neoadjuvant chemotherapy was associated with small tumour volume(P=0.04),estrogen receptor(ER)negative(P=0.003),HER2 positive(P=0.022),PD-L1 expression(P=0.015)and high levels of s TILs before neoadjuvant chemotherapy(P=0.001)were positively correlated and the differences were statistically significant.2.Expression of PD-L1 before and after neoadjuvant chemotherapy and its changes in relationship with clinicopathological features:PD-L1 expression before neoadjuvant treatment was positively correlated with ER negativity(P=0.012),no lymph node metastasis(P=0.021),and high water s TILs(P=0.003);neoadjuvant PD-L1 expression after treatment was positively correlated with no lymph node metastasis(P=0.007)and high levels of s TILs(P=0.007).The PD-L1 positivity rate before and after neoadjuvant chemotherapy was 18.8%and32.5%.10 cases of positive to negative conversion after neoadjuvant chemotherapy,49 cases of unchanged before and after,and 21 cases of negative to positive conversion.3.The relationship between infiltration level of s TILs and its changes with clinicopathological features before and after neoadjuvant chemotherapy:Twenty-nine of the 80 breast cancer patients who did not achieve p CR had elevated s TILs and eight had reduced s TILs,while pre-neoadjuvant s TILs levels were associated with lower histological grade(P=0.002),ER expression(P=0.002),molecular type(P=0.004)and absence of vascular invasion(P=0.005).Δs TILs were associated with younger patient age(P=0.025)and absence of lymph node metastases(P=0.026).CD8~+s TILs and CD4~+s TILs infiltration increased,FOXP3~+s TILs infiltration decreased and tumour cells ki-67 decreased after neoadjuvant chemotherapy.Before neoadjuvant chemotherapy,the proportion of CD4~+s TILs,CD8~+s TILs and FOXP3~+s TILs infiltration was significantly higher in the p CR group than in the non-p CR group.The levels of CD4~+s TILs、CD8~+s TILs and FOXP3~+s TILs infiltration were significantly higher in the PD-L1(+)group than in the PD-L1(-)group(Median:50 vs 15,P<0.001;40 vs 20,P=0.002;13 vs 8,P=0.004).The changes in△CD4~+s TILs and△CD8~+s TILs in the group with increased s TILs infiltration after neoadjuvant treatment were significantly higher than those in the group with unchanged s TILs infiltration and decreased s TILs infiltration(P<0.001);△FOXP3~+s TILs in the group with increased infiltration after neoadjuvant treatment was significantly lower than those in the group with unchanged s TILs infiltration and decreased s TILs infiltration(P<0.001).s TILs decrease after neoadjuvant treatment was significantly lower than that in the s TILs infiltration unchanged group and s TILs infiltration decreased group(P<0.001).The decrease in△FOXP3~+s TILs in the PD-L1 turn positive group after neoadjuvant treatment was significantly lower than that in the PD-L1 unchanged group(P<0.01),and the PD-L1turn negative group(P<0.001).Both univariate(P=0.0001)and multivariate analyses(P=0.006)showed that the level of CD4~+s TILs infiltration prior to neoadjuvant chemotherapy was positively associated with clinical outcome(p CR rate).4.Survival analysis of different subgroups of breast cancer patients:The 106 patients with neoadjuvant breast cancer were followed up,during which5 case were lost,the remaining 101 case were followed up for a median of 29 months(14～77 months)and 14 case had an outcome event(6 recurrences and 8 deaths).Kaplan-Meier survival analysis:ER positive patients had a significantly longer DFS than ER negative patients(P=0.01),the p CR group did not show a longer DFS than the non-p CR group(P=0.194),the PD-L1(+)group did not show a longer DFS than the PD-L1(-)group after neoadjuvant chemotherapy(P=0.107),and the△FOXP3~+s TILs reduced group showed a significantly better DFS than the△FOXP3~+s TILs elevated group(P=0.024),while the DFS of the△FOXP3~+s TILs reduced group was significantly better than that of the△FOXP3~+s TILs elevated group(P=0.024).s TILs different changes group(△s TILs),PD-L1 different changes group(△PD-L1),△CD4~+s TILs group,△CD8~+s TILs group and△FOXP3~+s TILs groups did not show significant differences and advantages.Conclusion:1.Tumor immune-related indicators of breast cancer,s TILs and PD-L1,can predict neoadjuvant chemotherapy response,and higher levels of s TILs and PD-L1expression can achieve better chemotherapy efficacy;2.Neoadjuvant chemotherapy can not only promote the invasion of killing tumor immune cells(CD4~+s TILs and CD8~+s TILs)in tumor microenvironment,but also inhibit the invasion of tumor-promoting lymphocytes(FOXP3~+s TILs)in tumor microenvironment.3.The changes of PD-L1 before and after neoadjuvant chemotherapy were basically not correlated with clinicopathological parameters. |
