Clinical Relevance of the Immune Contexture and AXL Kinase in Triple-Negative Breast Cancer

Bottai, Giulia (2017). Clinical Relevance of the Immune Contexture and AXL Kinase in Triple-Negative Breast Cancer. PhD thesis The Open University.



Triple-negative breast cancers (TNBC) are usually associated with an aggressive phenotype, an increased risk of early relapse, and poor outcome. Burgeoning evidence demonstrates that TNBC encompasses distinct molecular entities that are differentially characterized by specific hallmarks of cancer, including chromosomal instability (CIN), epithelial-to-mesenchymal transition (EMT), and cancer-related immune responses. In particular, the tumour immune microenvironment and the interaction between immune and cancer cells are emerging as crucial factors in tumour progression, prognosis, and response to therapy in TNBC. In this study, we aimed to evaluate the relationship between these three major hallmarks of TNBC. In particular, we assessed the composition and functionality of immune infiltrates in TNBC samples with different levels of CIN and investigated their clinical relevance in patients treated with adjuvant chemotherapy. Additionally, we explored the interactions between tumour-associated macrophages (TAM) and TNBC cells, particularly those with mesenchymal traits.

For this purpose, we integrated in vivo analysis of human TNBC tissues, in vitro experiments, and bioinformatics data. To assess the tumour immune microenvironment heterogeneity, we first identified different gene signatures from mRNA expression data, representing distinct immune components, and then evaluated their expression in different molecular subtypes of TNBC and in tumour samples characterized by low and high levels of CIN. To further explore the composition and functionality of immune infiltration in TNBC in vivo, formalin-fixed, paraffin-embedded tissues were retrospectively collected from large cohorts of early-stage TNBC patients treated with anthracycline-based chemotherapy. Stromal tumour-infiltrating lymphocytes (TIL) were evaluated on haematoxylin and eosin-stained sections. The density of CD4+, CD8+, CD103+, and FOXP3+ lymphocytes, CD68+ and CD163+ macrophages, and the expression of the immune checkpoints PD-1 and LAG-3 were assessed by immunohistochemistry. Furthermore, to understand the biological and clinical relevance of the interaction between TNBC cells and innate immune cells, we investigated several kinases functioning in the EMT process and their association with TAM in patients treated with adjuvant chemotherapy and in several TNBC cell lines.

We demonstrated that immune expression signatures were differentially expressed in TNBC characterized by varying levels of CIN and that TNBC molecular subgroups with a mesenchymal phenotype were enriched for immune signatures related to pro-tumour M2 macrophages. Conjunctly, by analysing human TNBC tissues, we showed that the presence of elevated TIL positively correlated with the density of all T cell subtypes, especially cytotoxic CD8+ lymphocytes. Among immune subpopulations, CD8+ lymphocytes were the main effectors of anti-tumour immune responses. We also found that PD-1 and LAG-3 were concurrently expressed in nearly 15% of TNBC. The expression of both checkpoint receptors positively correlated with the presence of TIL, but was not significantly associated with patient outcome. Furthermore, we showed that intraepithelial CD8+ cells frequently expressed the integrin CD103, which mediates the localization of cytotoxic lymphocytes within epithelial tissues. Importantly, the massive intraepithelial infiltration of cytotoxic CD103+ TIL co-expressing PD-1, correlated with prolonged survival in TNBC. In addition to TIL, we have demonstrated that the activation of the innate immune cells within the TNBC tumour stroma had a crucial role in tumour progression and chemoresistance, especially through the modulation of EMT. Accordingly, the EMT-related kinase AXL was highly associated with the presence of CD163+ TAM. Tumours from relapsing patients presented a high expression of AXL and CD163, although only AXL retained independent prognostic significance in multivariate analysis. In vitro analysis demonstrated that AXL-expressing TNBC cells were able to polarize human macrophages toward an M2-like phenotype. A selective inhibition of AXL impaired the activity of M2-like macrophages, reducing cancer cell invasiveness and restoring the sensitivity of breast cancer cells to chemotherapeutic drugs, especially anthracyclines.

Overall, our data indicate that TNBC subgroups with different biological and genomic features are characterized by distinct compositions of the immune microenvironment. Our results confirm that the evaluation of stromal TIL is the most reliable immune prognostic marker in TNBC patients. Our data also support the pharmacological and clinical evaluation of anti-PD-1/PD-L1 and anti-LAG-3 in a specific subset of TNBC patients and the inhibition of AXL as a novel strategy to simultaneously target TNBC cells and tumour promoting TAM.

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