Correlation Between Tumor Immune Microenvironment And Molecular Subtype And Prognosis Of Breast Cancer

Chapter 1 Tumor immune microenvironmental characteristics and their clinical significance analysis of hormone receptor positive breast cancerPurpose Tumor-infiltrating lymphocytes(TILs)have strong prognostic values in triple-negative and HER2-enriched breast cancer,but their prognostic roles in luminal breast cancer(LBC)are less clear.Here,we assessed the overall TIL levels and CD8+T-cells in relation to the prognosis of LBC patients from China.Meanwhile,the immunological microenvironmental characteristics of the hormone receptor positive breast cancer were further investigated by analyzing the immunorelated gene expression spectrum.Methods A total of 596 patients with LBC who were premenopausal and treated with adjuvant endocrine therapy were included.Among them,160 cases were evaluated for CD8 by immunohistochcmical(IHC)staining.Wholc-scction hematoxilyn and eosin and IHC staining were visually assessed for stromal TILs(sTILs),stromal CD8+T-cclls(sCD8)and intratumoral CD8+T-cclls(iCD8).Multivariable analyses were used to test the associations between TILs and discase-free survival(DFS)and overall survival(OS)with the adjustment of clinicopathologic characteristics and treatment.In the collected cases,62 samples were successful in RNA sequencing,and the differences in the correlation between the recurrence group(21 cases)and the non-recurrent group(41 cases),different molecular subtypes and different TILs levels expressed immunorelated genes and immune cell levels were compared.Results High TILs(≥10%)were associated with high histologic grade(P<0.001),luminal B/HER2-(P<0.001),luminal B/IIER2+subtype(P=0.002),and high Ki67 expression(≥25%;P=0.014).Similar associations were observed for sCD8 but not for iCD8.While TILs and sCD8 were not associated with cither DFS or OS,the presence of iCD8(≥1%)was associated with better DFS in both univariate(HR=0.51,95%CI 0.26-0.96,P=0.042)and multivariate(HR=0.48,95%CI 0.25-0.92,P=0.027)analyses.Similar but less significant associations were found for iCD8 and OS(adjusted HR=0.35,95%C1 0.11-1.10,P=0.073).RNA sequencing results showed that the immune correlation gene expression,immune regulating signal pathway and immune cell distribution were slightly different,and the partial results were not statistically significant,but the analysis showed that there was a certain heterogeneity in luminal breast cancer,especially the expression of B lymphocyte receptor and its relevant signaling pathway.Conclusions Among premenopausal patients with LBC,iCD8 demonstrated suggestive associations with favorable outcomes.In contrast,although TILs and sCD8 were associated with more aggressive tumor features,they did not appear to be associated with clinical outcomes.There was a certain heterogeneity in the TILs,CD8+T cells and B-cell related receptor expression and its signaling pathway in luminal breast cancer.In addition,luminal breast cancers were mainly in the state of immunosuppression.Chapter 2 Correlation of tumor infiltrating lymphocytes with molecular subtypes and prognosis of breast cancerPurpose Tumor-infiltrating lymphocytes(TILs)are different in the value of prognosis prediction for different molecular subtypes of breast cancer,and its prognostic role in HR+positive breast cancer is not clear.We included a large sample of breast cancer cases to explore the distribution of TILs level in different molecular subtypes of breast cancer and its correlation with clinicopathological factors and prognosis.Methods A total of 1032 cases with pathological diagnosis of breast cancer after surgical resection were collected and stromal TILs were evaluated visually in the whole HE section.The correlation of TILs level and molecular classi fication and clinical pathologic factors were analysed,and univariate and multivariate Cox proportional hazards regression model were used to test the associations between TILs and disease-free survival(DFS)and overall survival(OS)with the adjustment of clinicopathologic characteristics and treatment.At the same time,stratified analysis was performed according to the molecular subtypes to further evaluate the relationship of TILs with DFS and OS in different molecular subtypes breast cancers.Results Higher level TILs(≥10%)were significantly correlated with the ER,PR,and HER2 expression(P<0.001),and were more common in the cases with higer level histologic grade and higher Ki67 proliferation index(P<0.001),so higher TILs level had significant correlation with molecular subtypes.Higher TILs levels were more common in Luminal B,HER2 over-expression and triple negative breast cancer subtypes(P<0.001).There was no significant correlation between TILs and patients’ age,tumor size,lymph node metastasis and vascular invasion.Univariate and multivariate Cox regression analysis of survival showed that LPBC(HR=0.17,95%CI 0.04-0.68,P=0.013)were protective factors for breast cancer recurrence or metastasis,and were less prone to recurrence or metastasis than non-LPBC patients.Stratified analysis by breast cancer molecular subtypes showed that TILs level was not significantly correlated with DFS and OS in Luminal A and luminal/HER2-breast cancer,but in Luminal B/HER2-breast cancer,TILs(continuous variable)(HR=0.97,95CI 0.95-0.99,P=0.030)was protective factors affecting patients’ DFS.And luminal B/HER2+ breast cancer with high level TILs(≥10%)had longer OS(HR=0.16,95%CI 0.04 0.74,p=0.019)than the low level TILs cases.Univariatc and multivariatc Cox proportional hazards regression model analysis showed that in whole Luminal B and HER2 positive breast cancer TILs(continuity,10%for the boundary value of binary classification variables)levels were protective prognostic factors influencing the DFS and OS.Conclusions There was heterogeneity in TILs levels in breast cancer,and TILs level in luminal A breast cancer was significantly lower than that in luminal B/HER2-and luminal B/11ER2+,HER2-enrich breast cancer and TNBC.TILs level is an independent protective factor affecting DFS in breast cancer patients,and cases with high TILs level and LPBC were less likely to have breast cancer recurrence and metastasis.In luminal breast cancer,the prognostic value of TILs is different in subtypes.TILs level has no prognostic significance in luminal A and lumianl/HER2-breast cancer,while in luminal B,regardless of HER2 status,and HER2-cnrich breast cancer,higher TILs level indicated better prognosis.Chapter 3 Comparison of PD-L1(22C3)and PD-L1(SP142)expression in triple negative breast cancer and analysis of its clinical significanceObjective To investigate the expression of PD-L1(SP142)and PD-L1(22C3)in triple-negative breast cancer(TNBC),and analyze the concordance and their correlation with the clinicopathological factors and prognosis of the patients.Methods A total of 259 patients with TNBC were included.Whole section of surgical tissue samples were collected to conduct PD-L1(SP142)and PD-L1(22C3)immunohistochemical(IHC)staining.The PD-L1 expression were visually assessed for tumor cells and tumor infiltrating immune cells respectively.Univariable and multivariable Cox proportional hazards regression models were used to test the associations between PD-L1 expression and disease-free survival(DFS)and overall survival(OS)with the adjustment of clinicopathologic characteristics.Results The positive rates of SP142(ICs≥1%)and 22C3(CPS≥1)were 42.1%and 41.3%,respectively,with the total coincidence rate of 82.33%.The Kappa value of positive expression cases was 0.571 and the distribution difference of positive expression cases of SP142 and 22C3 was statistically significant(P<0.001).The PD-L1 positive patients were less likely to have vascular invasion(P=0.024 vs P=0.003),but higher histological grade and Ki67 proliferation index were higher(P<0.001).The recurrence/metastasis rate of the patients with PD-L1(SP142)positive was significantly lower than that of patients with PD-L1(SP142)negative(P=0.016).The presence of PD-LI(SP142)expression(ICs≥1%)was associated with longer DFS in both univariate(HR=0.39,95%CI 0.77-0.85,P=0.019)and multivariate(HR=0.43,95%CI 0.20-0.96,P=0.039)analyses.Similar but less significant associations were found for PD-L1(SP 142)positive and OS(HR=0.41,95%CI 0.17-0.96,P=0.041;Log Rank P=0.035).Similar to SP142,22C3 positive expression also showed a tendency to indicate longer DFS(HR=0.63,95%CI 0.31-1.28,P=0.200)and longer OS(HR=0.75,95%CI 0.35-1.63,P=0.47),but there was no significant difference.Conclusion The expression of 22C3 was different from that of SP142 in TNBC,and the two antibodies could not be interchangeable for each other in clinical tests.DFS was significantly prolonged in patients with PD-L1(SP142)positive.