Bioinformatics analysis of immune infiltration patterns in breast cancer for identification of prognostic markers

2019 ◽  
Author(s):  
Yan Yao ◽  
Tingting Zhang ◽  
Lingyu Qi ◽  
Ruijuan Liu ◽  
Gongxi Liu ◽  
...  
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
Immune Cells ◽  
Prognostic Markers ◽  
T Cell Subsets ◽  
M2 Macrophage ◽  
M2 Macrophages ◽  
Clinical Prognosis ◽  
Immune Infiltration ◽  
Significant Difference

Abstract Background/purpose Cancer immunotherapy has revolutionized the clinical treatment of several tumors. Immune infiltration has been found to be closely related to clinical prognosis, but it shows limited activity in breast cancer (BC). Therefore, this study aimed to explore the infiltration pattern of immune cells in BC, and to find potential prognostic markers and new therapeutic targets.Patients and methods We downloaded the immune genome data of BC from the Cancer Genome Atlas (TCGA), and analyzed the tumor- infiltrating immune cells (TIICs) in BC for the first time using the CIBERSORT algorithm. The aim of this study was to assess the proportions of 22 immune cell subsets in BC and examine the correlation between each TIIC and overall survival (OS) as well as clinical characteristics.Results The results indicated that: (1) there was a significant difference between the immune infiltration spectrum of cancerous and adjacent tissues, with M2 macrophages, M0 macrophages, and CD4 + T cells being highly expressed in BC; (2) CD8 + T cells were positively correlated with activated CD4 + memory T cells and negatively correlated with M0 macrophages, and M2 macrophages was inversely correlated with M1 macrophages, T cells regulatory, T cells CD8; (3) T cells, macrophages and BC TNM stage, age, clinical stage were correlated (P < 0.05); and (4) high expression of M2 macrophage markers could be an independent biomarker of poor prognosis and a potential therapeutic target for BC.Conclusion This study provides a new research method for the systematic study of immune cells in the BC tumor microenvironment, and provides theoretical guidance for further experiments to verify M2 macrophages and T cell subsets as a potential target for immunotherapy and prognosis.

scholarly journals VWCE as a potential biomarker associated with immune infiltrates in breast cancer

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Qin Huo ◽  
Zhenwei Li ◽  
Siqi Chen ◽  
Juan Wang ◽  
Jiaying Li ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Immune Cell ◽  
Her2 Status ◽  
Cancer Tissue ◽  
M2 Macrophage ◽  
Breast Cancer Patients ◽  
Immune Infiltration ◽  
Potential Biomarker ◽  
Significant Difference ◽  
Factor C

Abstract Purpose Von Willebrand Factor C and EGF Domains (VWCE) is an important gene that regulates cell adhesion, migration, and interaction. However, the correlation between VWCE expression and immune infiltrating in breast cancer remain unclear. In this study, we investigated the correlation between VWCE expression and immune infiltration levels in breast cancer. Methods The expression of VWCE was analyzed by the tumor immune estimation resource (TIMER) and DriverDB databases. Furthermore, genes co-expressed with VWCE and gene ontology (GO) enrichment analysis were investigated by the STRING and Enrichr web servers. Also, we performed the single nucleotide variation (SNV), copy number variation (CNV), and pathway activity analysis through GSCALite. Subsequently, the relationship between VWCE expression and tumor immunity was analyzed by TIMER and TISIDB databases, and further verified the results using Quantitative Real-Time PCR (RT-PCR), Western blotting, and immunohistochemistry. Results The results showed that the expression of VWCE mRNA in breast cancer tissue was significantly lower than that in normal tissues. We found that the expression level of VWCE was associated with subtypes, estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2) status of breast cancer patients, but there was no significant difference in the expression of VWCE was found in age and nodal status. Further analyses indicated that VWCE was correlated with the activation or inhibition of multiple oncogenic pathways. Additionally, VWCE expression was negatively correlated with the expression of STAT1 (Th1 marker, r = − 0.12, p = 6e−05), but positively correlated with the expression of MS4A4A (r = 0.28, p = 0). These results suggested that the expression of VWCE was correlated with immune infiltration levels of Th1 and M2 macrophage in breast cancer. Conclusions In our study, VWCE expression was associated with a better prognosis and was immune infiltration in breast cancer. These findings demonstrate that VWCE is a potential prognostic biomarker and correlated with tumor immune cell infiltration, and maybe a promising therapeutic target in breast cancer.

scholarly journals Global immune characterization of HBV/HCV-related hepatocellular carcinoma identifies macrophage and T-cell subsets associated with disease progression

2020 ◽  
Vol 6 (1) ◽  
Author(s):  
Guohe Song ◽  
Yang Shi ◽  
Meiying Zhang ◽  
Shyamal Goswami ◽  
Saifullah Afridi ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Immune Cells ◽  
Immune Cell ◽  
T Cell Subsets ◽  
M2 Macrophage ◽  
Advanced Hcc ◽  
Immune Cell Subsets

AbstractDiverse immune cells in the tumor microenvironment form a complex ecosystem, but our knowledge of their heterogeneity and dynamics within hepatocellular carcinoma (HCC) still remains limited. To assess the plasticity and phenotypes of immune cells within HBV/HCV-related HCC microenvironment at single-cell level, we performed single-cell RNA sequencing on 41,698 immune cells from seven pairs of HBV/HCV-related HCC tumors and non-tumor liver tissues. We combined bio-informatic analyses, flow cytometry, and multiplex immunohistochemistry to assess the heterogeneity of different immune cell subsets in functional characteristics, transcriptional regulation, phenotypic switching, and interactions. We identified 29 immune cell subsets of myeloid cells, NK cells, and lymphocytes with unique transcriptomic profiles in HCC. A highly complex immunological network was shaped by diverse immune cell subsets that can transit among different states and mutually interact. Notably, we identified a subset of M2 macrophage with high expression of CCL18 and transcription factor CREM that was enriched in advanced HCC patients, and potentially participated in tumor progression. We also detected a new subset of activated CD8+ T cells highly expressing XCL1 that correlated with better patient survival rates. Meanwhile, distinct transcriptomic signatures, cytotoxic phenotypes, and evolution trajectory of effector CD8+ T cells from early-stage to advanced HCC were also identified. Our study provides insight into the immune microenvironment in HBV/HCV-related HCC and highlights novel macrophage and T-cell subsets that could be further exploited in future immunotherapy.

scholarly journals Prognostic Value of MUC1 and its Correlation with Tumor-Infiltrating Immune Cells in Breast Cancer

2021 ◽  
Author(s):  
Xiaomin Xi ◽  
Yilin You ◽  
Weidong Huang ◽  
jicheng zhan
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
Cancer Progression ◽  
Macrophage Polarization ◽  
Muc1 Expression ◽  
Breast Cancer Patients ◽  
Gene Markers ◽  
Clinical Prognosis ◽  
Luminal B ◽  
Immune Infiltration

Abstract Background: MUC1 is a transmembrane glycoprotein, aberrantly glycosylated and overexpressed in a variety of epithelial cancers, and plays a crucial role in cancer progression, especially in breast cancer. It is also an essential regulator for immune functionality, but the mechanisms whereby it effects immune infiltration in breast cancer remain uncertain. Methods: In this research, MUC1 expression was analyzed by the Oncomine and TIMER database. The association between MUC1 and prognosis was evaluated by Kaplan-Meier plotter database. The correlations of MUC1 with immune infiltration and immunological markers were assessed by TIMER database. Results: We found that MUC1 expression was significantly correlated with outcomes in multiple cancers, with the effect being particularly pronounced in breast cancer. Pathologically, elevated MUC1 expression was related with worse prognosis depending on intrinsic subtypes, ER status, patient stage, lymph node and TP53 mutation status in breast cancer. Specifically, low level of MUC1 seemed to be more favorable to luminal B patients with systemic treatment. MUC1 expression had significant negative correlations with infiltrating levels of CD8+ T cells, CD4+ T cells, B cells, macrophages, neutrophils, monocytes and dendritic cells (DCs) in breast cancer. Besides, MUC1 displayed strong regulations on macrophage polarization and diverse immunological gene markers. The patients with lower MUC1 and deeper immune infiltration predicted better prognosis. Conclusions: MUC1 is significantly associated with clinical prognosis and potentially plays an essential role in modulating cytotoxic T lymphocytes (CTLs), tumor associated macrophages (TAMs), natural killer cells (NK cells) and DCs in breast tumors. Collectively, MUC1 could be served as a valuable biomarker of predicting prognosis and immune infiltration for breast cancer patients.

scholarly journals EVA1B to Evaluate the Tumor Immune Microenvironment and Clinical Prognosis in Glioma

2021 ◽  
Vol 12 ◽  
Author(s):  
Shanqiang Qu ◽  
Jin Liu ◽  
Huafu Wang
Keyword(s):  
T Cells ◽  
B Cells ◽  
Immune Cells ◽  
Cox Regression ◽  
Prognostic Biomarker ◽  
Area Under The Curve ◽  
Biological Databases ◽  
Clinical Prognosis ◽  
Immune Infiltration ◽  
Potential Biomarker

BackgroundPrevious research indicated that the tumor cells and microenvironment interactions are critical for the immunotherapeutic response. However, predicting the clinical response to immunotherapy remains a dilemma for clinicians. Hence, this study aimed to investigate the associations between EVA1B expression and prognosis and tumor-infiltrating immune cells in glioma.MethodsFirstly, we detected the EVA1B expression in glioma tissues through biological databases. The chi-squared test, Kaplan-Meier, and univariate and multivariate Cox regression analyses were used to analyze the clinical significance of EVA1B expression. The correlation between EVA1B expression and levels of tumor-infiltrating immune cells in glioma tissues was investigated. Receiver operating characteristic (ROC) analysis was performed to compare the predictive power between EVA1B and other commonly immune-related markers.ResultsIn the CGGA cohort of 325 glioma patients, we found that EVA1B was upregulated in glioma, and increased with tumor grade. High EVA1B expression was prominently associated with unfavorable clinicopathological features, and poorer survival of patients, which were further confirmed by TCGA (n=609) and GEO (n=74) cohorts. Furthermore, multivariate analysis indicated that EVA1B is an independent prognostic biomarker for glioma. Importantly, EVA1B overexpression was associated with a higher infiltration level of CD4+ T cells, CD8+ T cells, B cells, macrophages, and neutrophils in glioma. ROC curves showed that, compared with PD-L1, CTLA-4, and Siglec15, EVA1B presented a higher area under the curve (AUC) value (AUC=0.824) for predicting high immune infiltration levels in glioma.ConclusionsWe found that EVA1B was upregulated and could act as a poor prognostic biomarker in glioma. Importantly, EVA1B overexpression was associated with the immune infiltration levels of immune cells including B cells, CD4+ T cells, CD8+ T cells, macrophages, and neutrophils, and strongly with the overall immune infiltration levels of glioma. These findings suggested that EVA1B might be a potential biomarker for evaluating prognosis and immune infiltration in glioma.

scholarly journals PCOLCE Is Potent Prognostic Biomarker and Associates With Immune Infiltration in Gastric Cancer

2020 ◽  
Vol 7 ◽  
Author(s):  
Aizhai Xiang ◽  
Xia Lin ◽  
Lvping Xu ◽  
Honggang Chen ◽  
Jufeng Guo ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
T Cells ◽  
Cancer Patients ◽  
Prognostic Biomarker ◽  
M2 Macrophage ◽  
Th1 Cell ◽  
Clinical Prognosis ◽  
Immune Infiltration ◽  
Kaplan Meier ◽  
Gastric Cancer Patients

BackgroundThe exact biological role of PCOLCE was not yet clear and there were few reports study the correlation of PCOLCE gene expression level with the occurrence and development of gastric cancer.MethodsThe expression of PCOLCE was analyzed by performing the Oncomine and Ualcan database. We evaluated the function of PCOLCE on clinical prognosis with the use of Kaplan–Meier plotter database. The relationship between PCOLCE and cancer immune in filtrates was researched by Tumor Immune Estimation Resource (TIMER) site database.ResultsPCOLCE significantly upregulated in gastric cancer patients compared to normal gastric samples. And the increased expression of PCOLCE mRNA was closely linked to shorter overall survival (OS), progress-free survival (PFS) in all gastric cancers. Besides, PCOLCE expression displayed a tight correlation with infiltrating levels of macrophages and dendritic cells (DCs) in gastric cancer. Moreover, PCOLCE expression was positively correlated with diverse immune marker sets in gastric cancer.ConclusionAll the results above suggested that overexpression of PCOLCE indicated unfavorable prognosis in patients with gastric cancer. PCOLCE was correlated with immune infiltrating levels including those of B cells, CD8 + T cells, CD4 + T cells, macrophages, neutrophils, and DCs in gastric cancer patients. All the findings suggested that PCOLCE could be used as a prognostic biomarker for determining prognosis and immune infiltration in gastric cancer. Additionally, PCOLCE expression potentially contributed to the regulation of monocyte, M2 macrophage, Tfh, CD8 + T cell, TAM, Th1 cell Thus PCOLCE is a potential target for gastric cancer therapy and these preliminary findings require further study to determine whether PCOLCE-targeting reagents might be developed for clinical application in gastric cancer.

scholarly journals Analysis of Immune Landscape Reveals Prognostic Significance of Cytotoxic CD4+ T Cells in the Central Region of pMMR CRC

2021 ◽  
Vol 11 ◽  
Author(s):  
Jingwen Qi ◽  
Xiaoyan Liu ◽  
Peian Yan ◽  
Shangwen He ◽  
Yuhao Lin ◽  
...  
Keyword(s):  
T Cells ◽  
Neoadjuvant Chemotherapy ◽  
Central Region ◽  
Immune Cells ◽  
Prognostic Value ◽  
Protective Factor ◽  
Prognostic Significance ◽  
Disease Free Survival ◽  
T Cell Subsets ◽  
Clinical Prognosis

BackgroundMismatch repair proficient colorectal cancer (pMMR CRC) lacks effective treatments and has a poor prognosis, which can be attributed to the complexity of tumor microenvironment. The coordinated function of immune cells is vital to anti-tumor immunity. However, the spatial characteristics of immune cells in the pMMR CRC immune microenvironment and their relationship with clinical prognosis are not fully understood. Meanwhile, the immune modulatory effect of neoadjuvant chemotherapy (NCT), which is the first-line treatment of pMMR CRC, needs further investigation. Therefore, this study aims to explore the spatial dynamics of immune cells and its prognostic value in pMMR CRC.MethodsWe analyzed the various immune cells in formalin-fixed, paraffin-embedded tumor tissues which were collected from 77 patients with stage II/III of pMMR CRC, including 39 non-NCT treated and 38 NCT treated patients. We used the optimized multiplex immunohistochemistry (mIHC) to identify and quantify the density, type and location of immune cells in pMMR CRC. Multivariate survival analysis was performed to assess the relationship of immune profiles and clinical prognosis of pMMR CRC patients.ResultsThe densities of most T cell subsets, B cells and macrophages were higher in the central region of the pMMR CRC than in the invasion margin. Tumor infiltrating lymphocytes (TILs), especially the infiltration of CD4+ GzmB+ T cells in the central region of the tumor was identified to be positively correlated with the prognosis of the patients. Multivariate analysis confirmed that CD4+ GzmB+ T cells population was an independent predictor of disease-free survival (DFS) in non-NCT group. Meanwhile, NCT enhanced the infiltration of CD4+ GzmB+ T cells in the central region of the pMMR CRC, which was also identified as an independent protective factor of overall survival (OS) and DFS in NCT group.ConclusionWe demonstrated that the level of CD4+ GzmB+ T cells located in the center of tumor could provide great prognostic value for pMMR CRC patients. And the application of neoadjuvant chemotherapy further improves the infiltration of CD4+ GzmB+ T cells in the central compartment. Further studies into the application of CD4+ GzmB+ T cells in tumor immunotherapy are needed.

Dynamic relationship between cycling kinetics of triple-negative breast cancer and tumor infiltrating immune cells.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. 1100-1100
Author(s):  
Ishita Choudhary ◽  
Sergey Klimov ◽  
Padmashree C.G. Rida ◽  
Angela Ogden ◽  
Andrew R. Green ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Immune Response ◽  
T Cells ◽  
Triple Negative Breast Cancer ◽  
Immune Cells ◽  
Triple Negative ◽  
Antitumor Immune Response ◽  
M2 Macrophage ◽  
Slow Cycling ◽  
Kinetics Of

1100 Background: Recent studies show strong correlation between tumor infiltrating lymphocytes (TILs) and triple-negative breast cancer (TNBC) patient survival. CD8+ T cells serve as a favorable prognostic marker for TNBC. In addition, other cells such as CD4+ T cells, macrophages, B cells, and Tregs also infiltrate tumors. In this study, we delineate a strong relationship between the cycling kinetics of proliferating cells in TNBCs and antitumor immune response. Methods: A multi-institutional study performed by our group has previously shown that KAMS (Ki67-Adjusted Mitotic Score) provides a measure of the cycling kinetics of proliferating tumor cells and robustly stratifies TNBC patients into slow cycling (low KAMS) cyclophosphamide-methotrexate-fluorouracil (CMF)-responsive and fast cycling (high KAMS) CMF-resistant subgroups. In this study, we reviewed clinical data from 124 CMF-treated TNBC patients from Nottingham Hospital and sought correlations between cycling kinetics (High/Low KAMS) and tumor infiltrating immune cells. Results: We found that slow cycling TNBCs had higher mean expression of tumor infiltrating immune cells than fast cycling TNBCs. Intratumoral CD68 (p = 0.003), CD3 (p = 0.006), CD20 (p = 0.01), FOXP3 (p = 0.01), and total numbers of intratumoral and stromal CD68 (p = 0.01) and CD3 (p = 0.03) expressing cells were found to be significantly higher in low KAMS tumors than in high KAMS tumors. Of these biomarkers, CD68 was significantly associated with patients’ breast cancer-specific survival (BCSS): (a) low KAMS, high CD68 TNBCs had better BCSS than low KAMS, low CD68 (p = 0.01) TNBCs, and (b) high KAMS, low CD68 cases had better BCSS than high KAMS, high CD68 cases. Conclusions: Our observation that there are more TILs in slow cycling TNBCs suggests that there may be a dynamic cross-regulation between cycling kinetics and antitumor immune response. From our surprising observation that CD68 exerts polar roles in low/high KAMS subgroups, we propose that distinctions in M1 and M2 macrophage subsets in slow and fast cycling TNBCs may correlate with distinct outcomes. In addition, metabolic competition between tumor and immune cells may determine the level and function of TILs.

scholarly journals Hyperthermia by near infrared radiation induced immune cells activation and infiltration in breast tumor

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Wan Fatin Amira Wan Mohd Zawawi ◽  
M. H. Hibma ◽  
M. I. Salim ◽  
K. Jemon
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
Side Effects ◽  
Immune Cells ◽  
Infrared Radiation ◽  
Near Infrared ◽  
Tumor Regression ◽  
Immunohistochemical Analysis ◽  
Therapeutic Effects ◽  
Near Infrared Radiation

AbstractBreast cancer is the most common cancer that causes death in women. Conventional therapies, including surgery and chemotherapy, have different therapeutic effects and are commonly associated with risks and side effects. Near infrared radiation is a technique with few side effects that is used for local hyperthermia, typically as an adjuvant to other cancer therapies. The understanding of the use of near NIR as a monotherapy, and its effects on the immune cells activation and infiltration, are limited. In this study, we investigate the effects of HT treatment using NIR on tumor regression and on the immune cells and molecules in breast tumors. Results from this study demonstrated that local HT by NIR at 43 °C reduced tumor progression and significantly increased the median survival of tumor-bearing mice. Immunohistochemical analysis revealed a significant reduction in cells proliferation in treated tumor, which was accompanied by an abundance of heat shock protein 70 (Hsp70). Increased numbers of activated dendritic cells were observed in the draining lymph nodes of the mice, along with infiltration of T cells, NK cells and B cells into the tumor. In contrast, tumor-infiltrated regulatory T cells were largely diminished from the tumor. In addition, higher IFN-γ and IL-2 secretion was observed in tumor of treated mice. Overall, results from this present study extends the understanding of using local HT by NIR to stimulate a favourable immune response against breast cancer.

Can Intratumoral neutrophil lymphocyte ratio (NLR) be a prognostic biomarker in breast cancer patients?

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e12573-e12573
Author(s):  
Yoshihisa Tokumaru ◽  
Masanori Oshi ◽  
Vijayashree Murthy ◽  
Eriko Katsuta ◽  
Nobuhisa Matsuhashi ◽  
...  
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
Cancer Patients ◽  
Immune Cells ◽  
Breast Cancer Patients ◽  
Immune Microenvironment ◽  
High Group ◽  
Anti Cancer ◽  
Tumor Immune Microenvironment ◽  
Er Positive

e12573 Background: In breast cancer patients, it is well known that the elevation of neutrophil lymphocyte ratio (NLR) in the blood are reported to associate with poor prognosis based on the notion that neutrophils represent pro-cancer, and lymphocytes represent anti-cancer immune cells. Tumor immune microenvironment has been demonstrated to play critical roles in the outcome of breast cancer patients. However, there is scarce evidence on the clinical relevance of intratumoral NLR in breast cancer patients. In the current study, we hypothesized that intratumoral NLR high tumors are associated with worse survival particularly in TNBC that is known to have high immune cell infiltration. Methods: A total of 1904 breast cancer patients’ data from METABRIC (Molecular Taxonomy of Breast Cancer International Consortium) and analyzed. NLR was calculated by the gene expressions of CD66b (CEACAM8) and CD8 (CD8A). NLR high and low were divided by the median. Overall Survival (OS) and Disease-Free Survival were calculated utilizing Kaplan Meier method between intratumoral NLR high and low groups. xCell algorithm was used to analyze the infiltrated immune cells within the tumor immune microenvironment as we have previously published. Results: Intratumoral NLR high group was associated with worse OS in whole, ER-positive/HER2-negative, and triple negative (TN) subtypes, in agreement with the previous studies. TN subtype alone demonstrated worse DFS of NLR high group. Surprisingly, gene set enrichment analysis (GSEA) demonstrated no gene set enrichment to NLR high group, which implicates that there is no distinctive mechanism that associate with worse survival. Whereas, immune response-related gene sets significantly enriched to NLR low group in TN subtype. This enrichment was consistent in ER-positive/HER2-negative. Compared with ER-positive/HER2-negative subtype, anti-cancer immune cells such as CD4+ T cells, CD8+ T cells, M1 macrophage, and helper T helper type 1 cells were significantly infiltrated in TN patients (p < 0.001 for all genes), where M2 macrophages and neutrophils were less and regulatory T cells and T helper type 2 cells were more infiltrated in TN subtype. Furthermore, intratumoral NLR was significantly lower in TN compared with ER-positive/HER2-negative subtype (p < 0.001). These results suggest that intratumoral NLR low group is associated with better survival due to favorable tumor immune microenvironment in TN subtype rather than NLR high group has worse survival. Conclusions: Intratumoral NLR low tumor demonstrated more favorable OS and more favorable DFS in TN patients. Intratumoral NLR low breast cancer was associated with enhanced immune response and higher infiltration of anti-cancer immune cells were observed in TN subtype compared to ER-positive/HER2-negative which may contribute to the favorable outcome of in TN breast cancer.

scholarly journals P02.03 Automated cell type specific PD-L1 quantification by artificial intelligence using high throughput bleach & stain 15-marker multiplex fluorescence immunohistochemistry in human cancers

2021 ◽  
Vol 9 (Suppl 1) ◽  
pp. A8.2-A9
Author(s):  
NC Blessin ◽  
E Bady ◽  
T Mandelkow ◽  
C Yang ◽  
J Raedler ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Tumor Cells ◽  
Immune Cells ◽  
T Cell Subsets ◽  
Immune Microenvironment ◽  
L1 Data ◽  
Tumor Immune Microenvironment ◽  
Immune Phenotypes ◽  
Fluorescence Immunohistochemistry ◽  
Tumor Entities

BackgroundThe quantification of PD-L1 (programmed cell death ligand 1) has been used to predict patient’s survival, to characterize the tumor immune microenvironment, and to predict response to immune checkpoint therapies. However, a framework to assess the PD-L1 status with a high interobserver reproducibility on tumor cells and different types of immune cells has yet to be established.Materials and MethodsTo study the impact of PD-L1 expression on the tumor immune microenvironment and patient outcome, a framework for fully automated PD-L1 quantification on tumor cells and immune cells was established and validated. Automated PD-L1 quantification was facilitated by incorporating three different deep learning steps for the analysis of more than 80 different neoplasms from more than 10’000 tumor specimens using a bleach & stain 15-marker multiplex fluorescence immunohistochemistry panel (i.e., PD-L1, PD-1, CTLA-4, panCK, CD68, CD163, CD11c, iNOS, CD3, CD8, CD4, FOXP3, CD20, Ki67, CD31). Clinicopathological parameter were available for more than 30 tumor entities and overall survival data were available for 1517 breast cancer specimens.ResultsComparing the automated deep-learning based PD-L1 quantification with conventional brightfield PD-L1 data revealed a high concordance in tumor cells (p<0.0001) as well as immune cells (p<0.0001) and an accuracy of the automated PD-L1 quantification ranging from 90% to 95.2%. Across all tumor entities, the PD-L1 expression level was significantly higher in distinct macrophage/dendritic cell (DC) subsets (identified by CD68, CD163, CD11c, iNOS; p<000.1) and in macrophages/DCs located in the Stroma (p<0.0001) as compared to intratumoral macrophages/DC subsets. Across all different tumor entities, the PD-L1 expression was highly variable and distinct PD-L1 driven immune phenotypes were identified based on the PD-L1 intensity on both tumor and immune cells, the distance between non-exhausted T-cell subsets (i.e. PD-1 and CTLA-4 expression on CD3+CD8+ cytotoxic T-cells, CD3+CD4+ T-helper cells, CD3+CD4+FOXP3+ regulatory T-cells) and tumor cells as well as macrophage/(DC) subtypes. In breast cancer, the PD-L1 fluorescence intensity on tumor cells showed a significantly higher predictive performance for overall survival with an area under receiver operating curves (AUC) of 0.72 (p<0.0001) than the percentage of PD-L1+ tumor cells (AUC: 0.54). In PD-L1 positive as well as negative breast cancers a close spatial relationship between T- cell subsets (CD3+CD4±CD8±FOXP3±PD-1±CTLA-4±) and Macrophage/DC subsets (CD68±CD163±CD11c±iNOS) was found prognostic relevant (p<0.0001).ConclusionsIn conclusion, multiplex immunofluorescence PD-L1 assessment provides cutoff-free/continuous PD-L1 data which are superior to the conventional percentage of PD-L1+ tumor cells and of high prognostic relevance. The combined analysis of spatial PD-L1/PD-1 data and more than 20 different immune cell subtypes of the immune tumor microenvironment revealed distinct PD-L1 immune phenotypes.Disclosure InformationN.C. Blessin: None. E. Bady: None. T. Mandelkow: None. C. Yang: None. J. Raedler: None. R. Simon: None. C. Fraune: None. M. Lennartz: None. S. Minner: None. E. Burandt: None. D. Höflmayer: None. G. Sauter: None. S.A. Weidemann: None.

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