Exploring the role of ESR1 mutations in metastatic hormone receptor-positive breast cancer T cell immune surveillance disruption

Abstract

Background: Breast Cancer (BC) is the most common type of cancer in women around the world and 70% of cases are hormone-receptor positive (HR+). In 40% of cases, a key mechanism of endocrine resistance to the standard first line is a mutation of the ligand-binding domain (LBD) of Estrogen Receptor 1 (ESR1) encoding estrogen receptor α (ER). Most common ESR1 mutations that occur at positions 537 and 538 have been associated with poor clinical outcomes. ESR1 mutations have the potential to provide neoantigens. This study aims to identify if ESR1 mutations generate specific T cell responses against ESR1 neoantigens in patients with HR⁺ HER2^- BC, and to investigate if ESR1 mutations might correlate with a gene expression profile related to immune surveillance disruption.

Methods: We identified candidate ESR1-derived peptides by predictive software (SYFPEITHI and NetMHCpan 3.0). Then the immunogenicity of ESR1-derived peptides was assessed in Peripheral-Blood-Mononuclear-Cells from 31 healthy donors (HD) and 25 patients with metastatic HR-positive BC by IFN-γ ELISpot assay. A vaccination assay on a humanized mouse model (HLA-A2/DR1) was used to validate the immunogenicity and the presentation of these peptides. Finally, we used Bulk RNA-Seq sequencing along with MCPcounter, a cellular deconvolution method, to investigate the immune contexture of ESR1-mutated BC.

Results: Preliminary results showed recognition of ESR1-derived peptides by women HD lymphocytes but not in men. Frequencies and intensities of such immune responses were increased in patients with BC. Our results showed that 40% of patients had specific immune responses. In addition, we demonstrated the HLA-A2 ESR1 peptide immunogenicity in humanized HLA-A2/DR1 mice. In a data set generated from BC patients refractory to conventional therapy we showed that ESR1 mutations are correlated in advanced diseases with downregulation of molecules involved in antigen presentation and with loss HLA Class I gene expression. ESR1-mutated BC had a decrease in immune cell infiltration.

Conclusion: These results support that common ESR1 mutations generate neoantigens in hormone-receptor positive metastatic breast cancers. If ESR1 peptides-restricted lymphocytes were detectable in BC patients, ESR1 mutations promote immune escape at advanced stages.

Trial registration: ClinicalTrials.gov, NCT02838381. Registered on June 2012.

Keywords: ESR1 mutation; Hormone-receptor positive breast cancer; Neoantigen; Specific immune responses; T cell.

Fig. 1

Specific T cell responses induced byESR1 -derived peptides in healthy donors and metastatic breast patients. (A) Peripheral blood mononuclear cells (PBMC) from healthy donors and breast cancer patients were stimulated using ESR1 derived peptides, and an IFN-γ ELISpot assay was performed. (B) Frequency of positive responses induced by ESR1-derived peptides in healthy donors and metastatic breast cancer patients. (C) Distribution of women healthy donors (n = 12) vs. patients (n = 21) according to their T cell responses against 0, 1, and 2 or more ESR1 derived peptides

Fig. 2

Validation of HLA-A2-mediated presentation of ESR1. A. Peptides bind to HLA-A*02:01. T2 cells were pulsed with the indicated peptide for 12 h and surface HLA-A*02:01 was stained. MFI of pulsed T2 cells were normalized to experimentally matched non-pulsed T2 cells, and data were expressed as fold change in MFI. The L10L peptide was used as a positive control and V8L murine peptide was used as a negative control. The dashed horizontal line indicates negative peptide (V8L) pulsed T2 HLA-A*02:01. B. Vaccination assay on humanized mice at days 0, 7, 14 and 21. C-D Magnitude and frequency of spontaneous T cell responses against ESR1 derived peptides in (C) splenocytes from humanized HLA-A2/DR1 mice and (D) popliteal lymph nodes

Fig. 3

ESR1^D538G specific T cells are polyfunctional producing cells. (A) Representative FACS histogram of ESR1^D538G (p1) tetramer staining of respectively irrelevant T cells and specific p1 clone (HDB.5). (B) Functional avidity of HDB.5 T cell clone was evaluated after stimulation with a range of the indicated increasing p1 peptide concentrations (p0 was used as an irrelevant peptide). C-E. Representative histograms of ESR1 specific CD8 T clone cytokine production upon p1 peptide unstimulated (medium), stimulated, and PMA/Iono stimulated; IFN-γ (C), TNF-α (D) and IL-2 (E). F. Representative histograms of HDB.5 T cell clone IFN-γ production unstimulated (medium), stimulated with PMA/Iono (positive control) stimulated with p1 or p0 peptides, stimulated with MCF7 mutant pulsed with p1, stimulated with MCF7 mutant E: T 10:1 or MCF7 mutant E: T 20:1. All data are representative of at least 3 independent experiments

Fig. 4

MHC Class I alterations in breast cancer ESR1 mutated patients. (A) Oncoprint of ESR1 Mutated tumors (33 h+/HER2- patients, 12 of which are ESR1 mutated). All mutations are missense. Overall, 20.4% of HR+/HER2- metastatic tumors harbor ESR1 mutation, uniquely missense (B) ViolinPlot of transcriptomic expression of MHC Class I (left) and MHC Class II (right). ESR1 mutated tumors have a significantly lower expression of every gene in those antigen-presentation mechanisms. C. Expression of ESR1 status in CD8 T cells and CD8 cytotoxic T cells. Blue: ESR1 Wild Type; Pink: ESR1 mutated. D. Number of unique TCR clones established that advanced ESR1-mutated BC (pink) had a lower number of TCR clonotypes detected compared to ESR1 wild type BC (blue). E. Antigen processing and presentation (KEGG) pathway regulation. Genes with lower expression in ESR1 mutated vs. ESR1 wild type IFNγ and TNFα; TAP1/2; the MHCI molecule and the class II molecule (HLA-DM) are depicted in blue