Prognostic Significance Of Tumor-Infiltrating Lymphocyte Density,Subsets And Pd-L1 Expression In Breast Ductal Carcinoma In Situ

Objective:To investigate the correlation of tumor-infiltrating lymphocyte(TILs)density,subsets and their expression of programmed cell death ligand 1(PD-L1)in ductal carcinoma in situ(DCIS)with clinicopathological characteristics of the patients,and to analyze their prognostic significance as biological indicators.Methods:The clinicopathological data of 100 breast DCIS patients diagnosed between January2015 and December 2017 in the Department of Pathology,Affiliated Hospital of Qingdao University were retrospectively analyzed,including age,menopausal status,tumor size,necrosis,calcification,histological grade,estrogen receptor(ER),progesterone receptor(PR),human epidermal growth factor receptor 2(HER2),Ki-67 index,surgical type and postoperative therapy.The density of tumor stromal TILs was assessed on hematoxylineosin staining(HE)sections,and immunohistochemistry(IHC)was used to detect subsets of TILs(CD4+T,CD8+T,FOXP3+T cells)and PD-L1(SP142)expression.Chi-square test was used to analyze the relationship between TILs density,subsets,PD-L1 expression and clinicopathological characteristics,Kaplan-Meier method was used for survival analysis,and univariate and multivariate Cox regression analysis was used to analyze the influencing factors of prognosis of DCIS patients.SPSS 26.0 software was used for statistical analysis of the data.Results:(1)100 cases of DCIS,with a median age of onset of 51.5 years(26-84 years);43patients were postmenopausal;the median tumor diameter was 1.8 cm(0.2-9 cm);33lesions were accompanied by necrosis,and 18 lesions were accompanied by calcification;low grade in 17 cases(17.0%),intermediate grade in 48 cases(48.0%),and high grade in35 cases(35.0%);Luminal A in 38(38.0%),Luminal B in 25(25.0%)%),HER2 overexpression in 23(23.0%)and triple negative breast cancer(TNBC)in 14(14.0%).Of all patients,79(79.0%)underwent breast-conserving surgery,and the other 21(21.0%)underwent mastectomy;75(75.0%)received postoperative radiation therapy,and 60(60.0%)received Postoperative endocrine therapy.As of the last follow-up time on October 7,2021,the median follow-up time was 57.5 months(7-81 months),20(20.0%)patients developed ipsilateral recurrence,including 8 cases of DCIS(40.0%);invasive ductal carcinoma(IDC)12 cases(60%),no distant metastasis and no deaths.(2)High-density stroma TILs(≥20%)were associated with high-grade(P=0.008),ER negativity(P=0.003),PR negativity(P=0.009),HER2 positivity(P=0.018),postoperative endocrine therapy(P=0.015).(3)High-density TILs subsets(CD4+T,CD8+T,FOXP3+T cells)were associated with tumor necrosis,high-grade,ER negativity,PR negativity,HER2 positivity and postoperative endocrine therapy(all P<0.05);high density of FOXP3+ T cells was also associated with tumor size(P=0.002),Ki-67 index(P < 0.001)and surgical type(P=0.048).However,no statistical significance was found between TILs subsets and age,menopausal status,calcification,and postoperative radiotherapy(all P>0.05).In addition,we found that CD4/CD8+T>1 was associated with ER negativity(P=0.001),PR negativity(P=0.002),HER2 positivity(P=0.009),high Ki-67 proliferation index(P=0.018)and postoperative endocrine therapy(P=0.003).(4)Our study found that PD-L1(SP142)was almost not expressed in DCIS tumor cells,and 18 cases(18.0%)of DCIS stroma TILs expressed PD-L1(SP142)to varying degrees,and its positive expression was related to tumor necrosis(P =0.025),high grade(P=0.004),ER negativity(P=0.001),PR negativity(P=0.001),Ki-67 high proliferative index(P=0.001)and high density of stroma TILs(P<0.001).No correlation was found between PD-L1(SP142)expression and HER2 status(P=0.080).(5)Further analysis based on molecular subtypes of DCIS patients found that 52.2%of HER2 overexpression subtypes and 64.3% of TNBC had stroma TILs density≥20%.Meanwhile,the expression of PD-L1(SP142)in HER2 overexpression(34.8%)and TNBC(35.7%)subtypes was significantly higher than that in Luminal A(2.6%)and Luminal B(16.0%).High-density TILs subsets(CD4+T,CD8+T,FOXP3+T cells)were significantly correlated with HER2 overexpression subtypes and TNBC(all P<0.05).(6)The correlation analysis between the clinicopathological features and recurrence of DCIS found that recurrence was associated with younger age(P=0.044),premenopausal(P=0.006),tumor necrosis(P=0.004),high grade(P=0.009),high density of stroma TILs(P<0.001),high density of CD4+ T cells(P<0.001),high density of CD8+ T cells(P<0.001)and PD-L1+ in stroma TILs(P<0.001).(7)The average disease-free survival time of 100 DCIS patients was 70.816±2.103(95% CI: 66.694-74.937)months.Kaplan-Meier survival curve analysis showed that high-density stroma TILs(P<0.001),CD4+ T cells(P<0.001),CD8+ T cells(P<0.001)and PD-L1+ in the stroma TILs(P < 0.001)was associated with worse disease-free survival(DFS)in DCIS patients.Univariate analysis showed that the factors affecting the prognosis of DFS in DCIS patients were: necrosis(P=0.006),histological grade(P=0.011),stroma TILs density(P<0.001),CD4+T cell density(P<0.001)),CD8+ T cell density(P < 0.001)and PD-L1+ in stroma TILs(P < 0.001).Multivariate Cox proportional hazards regression model analysis showed that stroma TILs density(P=0.005,HR: 47.12;95% CI: 3.26-681.11)was an independent prognostic factor for DFS in DCIS patients.Conclusion:High-density Stroma TILs,subsets(CD4+T,CD8+T)and expression of PD-L1 are associated with some poor prognosis clinicopathological features and ipsilateral recurrence in DCIS patients;Stroma TILs density,subsets,and PD-L1 expression are different among the molecular subtypes of DCIS.High-density stroma TILs and subsets are more common in HER2 overexpression subtypes and TNBC,and higher proportion of PD-L1+TILs in TNBC,suggesting that HER2 overexpressing subtypes and TNBC may have higher immunogenicity and TNBC tumor immunosuppression is more obvious;Stroma TILs density is an independent prognostic factor for DCIS patients,suggesting that it can be used as a biological indicator for evaluating prognosis.