Quantitative assessment of the immune microenvironment in African American Triple Negative Breast Cancer: a case–control study

Abstract Purpose Triple negative breast cancer (TNBC) is more common in African American (AA) than Non-AA (NAA) population. We hypothesize that tumor microenvironment (TME) contributes to this disparity. Here, we use multiplex quantitative immunofluorescence to characterize the expression of immunologic biomarkers in the TME in both populations. Patients and methods TNBC tumor resection specimen tissues from a 100-patient case: control cohort including 49 AA and 51 NAA were collected. TME markers including CD45, CD14, CD68, CD206, CD4, CD8, CD20, CD3, Ki67, GzB, Thy1, FAP, aSMA, CD34, Col4, VWF and PD-L1 we quantitatively assessed in every field of view. Mean expression levels were compared between cases and controls. Results Although no significant differences were detected in individual lymphoid and myeloid markers, we found that infiltration with CD45+ immune cells (p = 0.0102) was higher in TNBC in AA population. AA TNBC tumors also had significantly higher level of lymphocytic infiltration defined as CD45+ CD14− cells (p = 0.0081). CD3+ T-cells in AA tumors expressed significantly higher levels of Ki67 (0.0066) compared to NAAs, indicating that a higher percentage of AA tumors contained activated T-cells. All other biomarkers showed no significant differences between the AA and NAA group. Conclusions While the TME in TNBC is rich in immune cells in both racial groups, there is a numerical increase in lymphoid infiltration in AA compared to NAA TNBC. Significantly, higher activated T cells seen in AA patients raises the possibility that there may be a subset of AA patients with improved response to immunotherapy.

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Quantitative Assessment of the Immune Microenvironment in African American Triple Negative Breast Cancer: A Case Control Study

10.21203/rs.3.rs-633060/v1 ◽

2021 ◽

Author(s):

Vesal Yaghoobi ◽

Myrto Moutafi ◽

Thazin Nwe Aung ◽

Vasiliki Pelekanou ◽

Sanam Yaghoubi ◽

...

Keyword(s):

Breast Cancer ◽

T Cells ◽

African American ◽

Triple Negative Breast Cancer ◽

Immune Cells ◽

Triple Negative ◽

Tumor Resection ◽

Lymphocytic Infiltration ◽

Case Control ◽

Activated T Cells

Abstract PURPOSETriple Negative Breast Cancer (TNBC) is more common in African American (AA) than Non-AA (NAA) population. We hypothesize that Tumor Microenvironment (TME) contributes to this disparity. Here we use multiplex quantitative immunofluorescence (QIF) to characterize the expression of immunologic biomarkers in the TME in both populations.PATIENTS AND METHODSTNBC tumor resection specimen tissues from a 100-patient case: control cohort including 49 AA and 51 NAA were collected. TME markers including CD45, CD14, CD68, CD206, CD4, CD8, CD20, CD3, Ki67, GzB, Thy1, FAP, aSMA, CD34, Col4, VWF and PD-L1 we quantitatively assessed in every field of view. Mean expression levels were compared between cases and controlsRESULTSAlthough no significant differences were detected in individual lymphoid and myeloid markers, we found that infiltration with CD45+ immune cells (p=0.0102) was higher in TNBC in AA population. AA TNBC tumors also had significantly higher level of lymphocytic infiltration defined as CD45+CD14- cells (p=0.0081). CD3+T-cells in AA tumors expressed significantly higher levels of Ki67 (0.0066) compared to NAAs, indicating that a higher percentage of AA tumors contained activated T-cells. All other biomarkers showed no significant differences between the AA and NAA group.CONCLUSIONSWhile the TME in TNBC is rich in immune cells in both racial groups, there is a numerical increase in lymphoid infiltration in AA compared to NAA TNBC. Significantly higher activated T cells seen in AA patients raises the possibility that there may be a subset of AA patients with improved response to immunotherapy.

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Epigenetic Quantification of Circulating Immune Cells in Peripheral Blood of Triple-Negative Breast Cancer patients

10.21203/rs.3.rs-508197/v1 ◽

2021 ◽

Author(s):

Mehdi Manoochehri ◽

Thomas Hielscher ◽

Nasim Borhani ◽

Clarissa Gerhäuser ◽

Olivia Fletcher ◽

...

Keyword(s):

Breast Cancer ◽

T Cells ◽

Triple Negative Breast Cancer ◽

Immune Cells ◽

Triple Negative ◽

Immune Cell ◽

Nk Cell ◽

Cell Type ◽

Cell Ratio ◽

Immune Cell Type

Abstract Background: A shift in the proportions of blood immune cells is a hallmark of cancer development. Here, we investigated whether methylation-derived immune cell type ratios and methylation-derived neutrophil-to-lymphocyte ratios (mdNLRs) are associated with triple-negative breast cancer (TNBC). Methods: Leukocyte subtype-specific un/methylated CpG sites were selected and methylation levels at these sites used as proxies for immune cell type proportions and mdNLR estimation in 231 TNBC cases and 231 age-matched controls. Data were validated using the Houseman deconvolution method. Additionally, the natural killer (NK) cell ratio was measured in a prospective sample set of 146 TNBC cases and 146 age-matched controls. Results: The mdNLRs were higher in TNBC cases compared with controls and associated with TNBC (odds ratio (OR) range (2.66-4.29), all Padj.<1e-04). A higher neutrophil ratio and lower ratios of NK cells, CD4+ T cells, CD8+ T cells, monocytes, and B cells were associated with TNBC. The strongest association was observed with decreased NK cell ratio (OR range (1.28-1.42), all Padj.<1e-04). The NK cell ratio was also significantly lower in pre-diagnostic samples of TNBC cases compared with controls (P=0.019).Conclusion: This immunomethylomic study shows that a shift in the ratios/proportions of leukocyte subtypes is associated with TNBC, with decreased NK cell showing the strongest association. These findings improve our knowledge of the role of the immune system in TNBC and point to the possibility of using NK cell level as a non-invasive molecular marker for TNBC risk assessment, early detection, and prevention.

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Epigenetic quantification of circulating immune cells in peripheral blood of triple-negative breast cancer patients

Clinical Epigenetics ◽

10.1186/s13148-021-01196-1 ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Mehdi Manoochehri ◽

Thomas Hielscher ◽

Nasim Borhani ◽

Clarissa Gerhäuser ◽

Olivia Fletcher ◽

...

Keyword(s):

Breast Cancer ◽

T Cells ◽

Triple Negative Breast Cancer ◽

Immune Cells ◽

Triple Negative ◽

Immune Cell ◽

Nk Cell ◽

Cell Type ◽

Cell Ratio ◽

Immune Cell Type

Abstract Background A shift in the proportions of blood immune cells is a hallmark of cancer development. Here, we investigated whether methylation-derived immune cell type ratios and methylation-derived neutrophil-to-lymphocyte ratios (mdNLRs) are associated with triple-negative breast cancer (TNBC). Methods Leukocyte subtype-specific unmethylated/methylated CpG sites were selected, and methylation levels at these sites were used as proxies for immune cell type proportions and mdNLR estimation in 231 TNBC cases and 231 age-matched controls. Data were validated using the Houseman deconvolution method. Additionally, the natural killer (NK) cell ratio was measured in a prospective sample set of 146 TNBC cases and 146 age-matched controls. Results The mdNLRs were higher in TNBC cases compared with controls and associated with TNBC (odds ratio (OR) range (2.66–4.29), all Padj. < 1e−04). A higher neutrophil ratio and lower ratios of NK cells, CD4 + T cells, CD8 + T cells, monocytes, and B cells were associated with TNBC. The strongest association was observed with decreased NK cell ratio (OR range (1.28–1.42), all Padj. < 1e−04). The NK cell ratio was also significantly lower in pre-diagnostic samples of TNBC cases compared with controls (P = 0.019). Conclusion This immunomethylomic study shows that a shift in the ratios/proportions of leukocyte subtypes is associated with TNBC, with decreased NK cell showing the strongest association. These findings improve our knowledge of the role of the immune system in TNBC and point to the possibility of using NK cell level as a non-invasive molecular marker for TNBC risk assessment, early detection, and prevention.

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Epigenetic Quantification of Circulating Immune Cells in Peripheral Blood of Triple-negative Breast Cancer Patients

10.21203/rs.3.rs-508197/v2 ◽

2021 ◽

Author(s):

Mehdi Manoochehri ◽

Thomas Hielscher ◽

Nasim Borhani ◽

Clarissa Gerhäuser ◽

Olivia Fletcher ◽

...

Keyword(s):

Breast Cancer ◽

T Cells ◽

Triple Negative Breast Cancer ◽

Immune Cells ◽

Triple Negative ◽

Immune Cell ◽

Nk Cell ◽

Cell Type ◽

Cell Ratio ◽

Immune Cell Type

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CCL5 expression and tumor infiltrating immune cells in triple negative breast cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.11553 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. 11553-11553

Author(s):

Jhajaira Araujo ◽

Andrea C. Gomez ◽

Roberto Salgado ◽

Justin M. Balko ◽

Alfredo Aguilar ◽

...

Keyword(s):

Breast Cancer ◽

T Cells ◽

Triple Negative Breast Cancer ◽

Immune Cells ◽

Triple Negative ◽

Memory T Cells ◽

Residual Disease ◽

Univariate Analysis ◽

Rank Test ◽

Cd8 Cells

11553 Background: CCL5 is a chemo-attractant of regulatory T cells, promoting tumor immune avoidance and related to a poor outcome in several malignancies; however, in triple negative breast cancer (TNBC), it is related to a better outcome. Our aim was to evaluate the correlation between CCL5 and tumor infiltrating immune cells and their prognosis value. Methods: We evaluated 72 TNBC patients with residual disease after neoadjuvant chemotherapy with matched data of tumor infiltrating lymphocytes (TILs) count and CCL5 expression (profiled with NanoString). CCL5 expression levels were log2 transformed and median centered. Correlation between TILs (log2 transformed) and CCL5 was evaluated with the Spearman’s rank test. Cox PH model was used to investigate the effect of CCL5 (median as cutoff) and TILs ( < 20% and ≥20%) in distant-recurrence free survival. We used the CIBERSORT platform to evaluate the immune cells composition according to the expression of CCL5 (higher vs. lower or equal than median) in 3 independent TNBC datasets (GSE25066, GSE58812 and GSE76124). Results: There was a significant correlation between TILs and residual tumor size (P = 0.017) and CCL5 (ρ = 0.347, P = 0.003). In univariate analysis, TILs (HR = 0.276, 95%IC: 0.128-0.593; P = 0.001) and CCL5 (HR = 0.401; 95%CI: 0.206-0.781; P = 0.007) were both associated with outcome. In a multivariate analysis with CCL5 expression and TILs count, TILs was the only significant marker with a P = 0.008 (HR = 0.336; 95%CI: 0.150-0.753), in contrast to CCL5 (HR = 0.573; 95%CI: 0.285-1.154; P = 0.124). CIBERSORT analysis suggested that high CCL5 expression is associated with recruitment of CD8 cells (13% v 6%, P < 0.001; 6% v 1%, P < 0.001 and 12% v 8%, P = 0.003), activated CD4 memory T cells (4% vs. 2%, P < 0.001; 5% vs. 0%, P < 0.001; 3% vs. 0%, P < 0.001) and Macrophages M1 (9% vs. 7%, P = 0.022; 13% vs. 8%, P = 0.005; 11% vs. 5%, P < 0.001) in GSE25066, GSE58812 and GSE76124 datasets, respectively. Conclusions: TILs was the stronger and more significant prognostic immunological marker, even than CCL5 expression. High CCL5 expression was associated with enrichment of CD8 cells, activated CD4 memory T cells and Macrophages M1. Role of these cells in TNBC should be explored more deeply.

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Comprehensive genomic and immunophenotypic analysis of CD4 T cell infiltrating human triple-negative breast cancer

Cancer Immunology Immunotherapy ◽

10.1007/s00262-020-02807-1 ◽

2020 ◽

Author(s):

He Zhang ◽

Guohui Qin ◽

Hui Yu ◽

Xu Han ◽

Sha Zhu

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

T Cells ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Immune Cell ◽

Differential Expression Analysis ◽

Effector Memory ◽

Activated T Cells ◽

Cell Functions

AbstractThe aim of this study is to investigate the gene expression module of tumor-infiltrating CD4+T cells and its potential roles in modulating immune cell functions in triple-negative breast cancer. Differentially expressed genes were identified by comparison of the expression profile in CD4+T cells isolated from tumor tissues and peripheral blood of TNBC patients respectively. The differential expression analysis was conducted using R, and then the functional and pathway enrichment of the DEGs were analyzed using GSEA, followed by integrated regulatory network construction and genetic analysis of tumor-infiltrating immune cells based on a scientific deconvolution algorithm. As a result, abundant Treg and exhausted lymphocytes were detected, accompanied by largely decreased of effector/memory and cytotoxic T cells. Immune-related gene correlation analysis showed that the extent of follicular helper T cells gene expression signatures were inversely associated with those of CD4+ naive T cells and CD4+ memory resting T cells, but positively correlated with that of CD4+ memory activated T cells. In addition, we found five core genes including IFNG, CTLA4, FAS, CXCR6, and JUN were significantly over expressed in CD4+ TILs which may contribute to exhaustion of lymphocytes and participate in biological processes associated with regulation of chemotaxis. Study provides a comprehensive understanding of the roles of DEGs associated with the chemotactic and exhausted immunophenotypes of CD4+ TILs that are a valuable resource from which future investigation may be carried out to better understand the mechanisms that promote TNBC progression.

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CIBERSORT analysis of TCGA and METABRIC identifies subgroups with better outcomes in triple negative breast cancer

Scientific Reports ◽

10.1038/s41598-021-83913-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Kelly E. Craven ◽

Yesim Gökmen-Polar ◽

Sunil S. Badve

Keyword(s):

Breast Cancer ◽

T Cells ◽

Triple Negative Breast Cancer ◽

Molecular Mechanisms ◽

Triple Negative ◽

Immune Cell ◽

Molecular Taxonomy ◽

The Cancer Genome Atlas ◽

Activated T Cells ◽

Cancer Immunoediting

AbstractStudies have shown that the presence of tumor infiltrating lymphocytes (TILs) in Triple Negative Breast Cancer (TNBC) is associated with better prognosis. However, the molecular mechanisms underlying these immune cell differences are not well delineated. In this study, analysis of hematoxylin and eosin images from The Cancer Genome Atlas (TCGA) breast cancer cohort failed to show a prognostic benefit of TILs in TNBC, whereas CIBERSORT analysis, which quantifies the proportion of each immune cell type, demonstrated improved overall survival in TCGA TNBC samples with increased CD8 T cells or CD8 plus CD4 memory activated T cells and in Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) TNBC samples with increased gamma delta T cells. Twenty-five genes showed mutational frequency differences between the TCGA high and low T cell groups, and many play important roles in inflammation or immune evasion (ATG2B, HIST1H2BC, PKD1, PIKFYVE, TLR3, NOTCH3, GOLGB1, CREBBP). Identification of these mutations suggests novel mechanisms by which the cancer cells attract immune cells and by which they evade or dampen the immune system during the cancer immunoediting process. This study suggests that integration of mutations with CIBERSORT analysis could provide better prediction of outcomes and novel therapeutic targets in TNBC cases.

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

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.1100 ◽

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.

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