The immunogenicity of breast cancer (BC) is quite heterogeneous among the clinical subtypes, with immune responses identified most frequently in triple negative (TNBC) and HER2-positive tumors. The extent, spatial localization, distribution patterns, organization and phenotype of the BC immune infiltrate are currently being widely investigated but require standardization before they can be used clinically. One highly relevant unmet clinical need is to understand how immune features are linked to prognosis and potential benefit from treatments, particularly immunotherapy. The present work investigated tumor-infiltrating lymphocytes (TIL), tertiary lymphoid structures (TLS), the expression of multiple targetable inhibitory immune checkpoint molecules (PD-1, PD-L1 and PD-L2, CTLA-4, LAG3 and TIM3) and their clinical relevance in primary BC. Different technical approaches were employed including: flow cytometry (FC) on fresh tissue homogenates; immunohistochemistry (IHC) and immunofluorescence (IF) on formalin-fixed paraffin embedded (FFPE) tissue blocks from untreated primary tumors; and gene expression on a large dataset of BC patients with available long-term survival data. Flow cytometric analysis of PD-1 expression and its principal ligands PD-L1 and PD-L2 together with CTLA-4, LAG3 and TIM3 on TIL in fresh untreated primary tumors revealed that PD-1 and CTLA-4 are most highly expressed on BC TIL and PD-L1 is the principal PD-1 ligand in BC. Immune checkpoint molecule expression parallels the extent of TIL infiltration and TLS presence and number, with the patterns detected similar to that observed in secondary lymphoid organs. Significantly improved disease-specific survival (DSS) has been associated with PD-1hi HER2-enriched and PD-L1hi, PD-L2hi and CTLA-4hi basal-like BC; however there is significant heterogeneity between individual tumors even within the same subtype. These observations suggest that determining expression levels of multiple targetable inhibitory immune checkpoint molecules in patients might help to successfully target them in BC patients most likely to respond.We examined the concordance between two experienced immuno-pathologists who read 800 IHC-stained slides from five independent series over a period of four years to determine the reproducibility of assessing multiple immune biomarkers. This included scoring TIL, TLS, PD-1 and PD-L1 together with detailed information on the spatial localization and cell types expressing these molecules in the tumor microenvironment (TME). The interobserver reproducibility for the assessment of TIL and TLS was consistently good to excellent overtime, while the concordance for PD-L1 evaluation ranged from fair to excellent when it was only expressed on tumor cells (TC); and the concordance for PD-1 evaluation was fair to excellent when it was expressed in TLS and evaluated in primary tumors. Neither PD-L1 expression by TC, nor PD-1 expression within a TLS was significantly associated with prognosis in our datasets.The extent of TIL, TLS and PD-1 and PD-L1 expression were studied in a cohort of TNBC patients who underwent genetic counseling for their personal/familial history of BC or ovarian cancer (OC). This study revealed a remarkable similarity in patterns of immune infiltration between the two cohorts. Interestingly, a higher prevalence of TIL intermediate cases (≥10% and <50% TIL) was detected in the BRCA-mutated cohort, suggesting that this group may be more immunogenic.We next investigated whether the extent and presence of these immune parameters were associated with prognosis in the most highly infiltrated, aggressive BC subtypes (TNBC and HER2-positive). We determined the ideal cut-off for each subtype (TNBC and HER2-positive) to use TIL as a categorical variable. This study found a consistent prognostic impact from TIL (in any tumor compartment including stromal, intratumoral and global areas) and a novel association between PD-L1 expression within TLS and better survival in TNBC. This last effect was driven by baseline stromal TIL, strengthening the importance of reliably quantifying the levels of TIL in BC. Overall, our analyses show that among the targetable inhibitory immune checkpoint molecules investigated in BC, PD-1 and CTLA-4 are most highly expressed by BC TIL and are associated with higher infiltration of TIL; PD-L1 is the principal ligand for PD-1; TIL and TLS are reproducibly scored on IHC-stained tissues; and TIL levels are associated with a better prognosis in TNBC independent of their location in the TME at optimal cut-offs. Our data also provide new insight on targetable inhibitory immune checkpoint molecule expression and location as well as showing a prognostic role for TIL assessed by IHC in primary BC, which identifies these biomarkers as ideal candidates for further investigation. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished
| Identifer | oai:union.ndltd.org:ulb.ac.be/oai:dipot.ulb.ac.be:2013/282008 |
| Date | 24 January 2019 |
| Creators | Solinas, Cinzia |
| Contributors | Sotiriou, Christos, Piccart-Gebhart, Martine, Braun, Michel, Corazza, Francis, Fontaine, Christel, Coulie, Pierre G, Noël, Jean Christophe, Smits, Guillaume |
| Publisher | Universite Libre de Bruxelles, Université libre de Bruxelles, Faculté de Médecine – Médecine, Bruxelles |
| Source Sets | Université libre de Bruxelles |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/doctoralThesis, info:ulb-repo/semantics/doctoralThesis, info:ulb-repo/semantics/openurl/vlink-dissertation |
| Format | 294 p., 3 full-text file(s): application/pdf | application/pdf | application/pdf |
| Rights | 3 full-text file(s): info:eu-repo/semantics/closedAccess | info:eu-repo/semantics/restrictedAccess | info:eu-repo/semantics/closedAccess |
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