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Background: Aromatase inhibitors (AIs) are highly effective at reducing recurrences and mortality in postmenopausal patients with estrogen receptor positive breast cancer (ER+ BC). Poor anti-proliferative (Ki67) response or ER+ BCs to AIs after 2 weeks is associated with worse long-term outcomes. Factors that relate to the degree of the response may identify markers and/or mechansims of resistance. Methods: The PeriOperative Endocrine Therapy for Individualizing Care (POETIC) trial randomized 4,480 with ER+ BC to 2 weeks’ AI before surgery or no presurgical treatment. All patients within the bottom 15% of Ki67 responders to AI (poor responders [PRs]; n=177 with RNA extracted) were selected from and matched to good responders (GRs) within the 50% showing the best response (n=190). Matching was based on baseline Ki67 levels as measured by immunohistochemistry (IHC). Response to AI was measured by the percentage change in Ki67 after 2 weeks’ treatment. PRs were further divided into groups expressing high ESR1 (PRs ESR1HIGH; n=119) and low ESR1 (PRs ESR1LOW; n=58) levels since there were very few GRs with low ESR1. RNAseq, targeted exome DNA sequencing of 87 BC/resistance related genes and measurement of plasma estradiol levels by mass spectrometry were performed to understand mechanisms of de novo resistance. Intrinsic subtypes were estimated from RNAseq data. Results: More than 90% of PRs ESR1LOW were non-luminal subtypes with low expression of estrogen-responsive genes. In contrast, 11% of PRs ESR1HIGH were non-luminal compared to 4% of GRs but only HER2-enriched subtypes were significantly higher in PR ESR1HIGH (p=0.05, Fisher exact). While AI treatment had limited impact on Ki67 IHC values in PRs ESR1HIGH, PGR expression was more than 2-fold lower after 2 weeks of AI. Gene-set enrichment analysis showed significantly lower expression of estrogen-response genes in PRs ESR1HIGH compared to GRs (FDR< 10-9) at baseline despite similar percentage of Luminal subtypes in PRs ESR1HIGH and GRs. Plasma estradiol levels were correlated with expression of estrogen-response genes (FDR=0.01) and levels were significantly lower in PRs ESR1HIGH compared to GRs (p=0.003, Mann Whitney). PRs ESR1HIGH had significantly more mutations in RB1, TP53, ARID1B and DNAH11 genes (p< 0.05, Fisher exact). TP53 mutations were significantly enriched in Luminal-A PRs ESR1HIGH compared to GRs (22% and 3% respectively; p=0.003, Fisher exact), but not in Luminal-B tumors (23% and 15% mutated respectively). Discussion and conclusions: In approximately 33% of PRs, de novo AI resistance was associated with and most likely due to low expression of ER/ESR1 and estrogen-responsive genes in non-luminal tumors. In the remaining tumors, AI treatment still impacted some estrogen responsive genes but had limited downstream impact on suppressing proliferation. This might be due to mutations including in TP53 that limit suppression of proliferation downstream of estrogen signaling. The proportion of Luminal tumors in GRs and PRs ESR1HIGH was similar, suggesting better outcome of Luminal-A tumors on AI is likely due to their better intrinsic prognosis rather than better response to endocrine therapy. Citation Format: Eugene F. Schuster, Elena López-Knowles, Anastasia Alataki, Lila Zabaglo, Elizabeth Folkerd, David Evans, Kally Sidhu, Holly Tovey, Perry Maxwell, Nicholas Turner, Stephen Johnston, Manuel Salto-Tellez, Maggie Chon U Cheang, John Robertson, Ian Smith, Judith Bliss, Mitch Dowsett. PD10-07 Low plasma estradiol, low expression of estrogen responsive genes and TP53 mutations are associated with poor anti-proliferative response to aromatase inhibitors [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD10-07.

Background: Aromatase inhibitors (AIs) are one of the main treatment strategies for the clinical management of estrogen receptor-positive (ER+) breast cancer (BC). Despite prolonged time to recurrence and initial clinical responses, >20% of patients eventually relapse, and previous studies have shown an association of poor anti-proliferative response to AIs and worse outcome. High immune activity in ER+ tumors may be associated with worse outcome, in contrast to ER-negative BC where immune infiltration is a feature associated with better outcome. Our work focused on understanding the correlations between immune cell infiltration and response to AI. Methods: All patients with ER+ HER2- tumors within the bottom 15% of Ki67 anti-proliferative responders to AIs (poor responders [PRs]; n=177) were selected from the PeriOperative Endocrine Therapy for Individualizing Care (POETIC) trial and matched on baseline Ki67 levels to good responders (GRs) within the 50% showing the best response (n=190). Response to AI was measured by the Ki67 percentage change after 2 weeks of treatment. PRs were further divided into groups expressing high ESR1 (PRs ESR1HIGH; n=119) and low ESR1 (PRs ESR1LOW; n=58) levels to represent PR subgroups that showed partial or no response to AIs. The percentage of stromal tumor-infiltrating lymphocytes (TILs) was assessed. Multiple immunofluorescence was performed for ER, CD3, CD20, CD68, FOXP3, and CD3/FOXP3 in 15 baseline samples from each of the GR, PR ESR1HIGH, and PR ESR1LOW populations and immune cell density in stromal or tumor compartments was estimated. Spearman correlations of TILs with Consensus tumor microenvironment (TME) deconvolution and Molecular Signatures Database hallmark gene sets were conducted. The relationship between the immune markers’ density and genes, hallmark gene sets and Consensus TME was assessed. Results: The percentage of TILs was significantly higher in the PR ESR1HIGH and PR ESR1LOW compared to the GRs (adjusted p< 0.05). As expected, TILs were highly correlated with T cells (particularly T-regulatory cells) and immune hallmark gene sets. There was a tendency for higher density of each of the immune markers in PRs compared to GRs, with significant differences being observed in stromal B-cell marker CD20 density (p< 0.05). Analysis showed a significant correlation between TILs and stromal FOXP3 marker density (FDR< 0.05), and stromal biomarker density was highly correlated to the gene expression of the encoding genes of the same tumors (CD3/CD3D, FOXP3/FOXP3, and CD20/MS4A1) (FDRs< 0.05). There was also a strong and significant correlation between the stromal expression of CD20, CD3, FOXP3, and CD3/FOXP3 with the immune hallmark gene sets (FDRs< 0.05). Finally, the immune phenotyping showed the expected correlations with TME deconvolution, with particularly strong correlations of CD20 and CD3 with B- and T-cell gene signatures, respectively (FDRs< 0.05). Conclusions: Different immune features indicated a broad involvement of several immune cell types in PRs to AIs, suggesting that the immune system might be associated with resistance of ER+ breast tumors to AI treatment. Spatial gene expression profiling is ongoing to characterize these tumors further and investigate potential mechanisms of AI resistance. Citation Format: Anastasia Alataki, Gene Schuster, Lila Zabaglo, Perry Maxwell, Elena López-Knowles, Elizabeth Folkerd, David Evans, Kally Sidhu, Holly Tovey, Nicholas Turner, Stephen Johnston, Maggie Chon U Cheang, John Robertson, Manuel Salto-Tellez, Ian Smith, Judith Bliss, Mitch Dowsett. PD10-08 Immune cell infiltration associated with poor anti-proliferative response to aromatase inhibitors in postmenopausal women with primary ER-positive HER2-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD10-08.

Endocrine therapies are the main treatment strategies for the clinical management of hormone-dependent breast cancer. Despite prolonged time to recurrence in the adjuvant setting and the initial clinical responses in the metastatic setting, many patients eventually encounter tumour relapse due to acquired resistance to these agents. Other patients experience a lack of tumour regression at the beginning of treatment indicating de novo resistance that significantly limits its efficacy in the clinic. There is compelling evidence that human epidermal growth factor receptor-2 (HER2) overexpression contributes to resistance to endocrine therapies in oestrogen receptor-positive (ER+) breast cancer. ER+/HER2+ tumours comprise about 10% of all breast cancer cases and about 60% of the whole set of HER2+ tumours. Most patients with primary ER+/HER2+ disease will receive antibody-based HER2-targeted therapy, but this is generally for no more than one year while endocrine treatment is usually for at least 5 years. A number of HER2-kinase inhibitors are also now in clinical use or in clinical trials, and the interaction of these with endocrine treatment may differ from that of antibody treatment. In this review article, we aim to summarise knowledge on molecular mechanisms of breast cancer resistance to endocrine therapies attributable to the impact of HER2 signalling on endocrine sensitivity, to discuss data from clinical trials addressing the role of HER2 in the development of endocrine resistance in the metastatic, neoadjuvant and adjuvant settings and to explore rational new therapeutic strategies.

Purpose Changes occur in the expression of oestrogen-regulated and proliferation-associated genes in oestrogen receptor (ER)-positive breast tumours during the menstrual cycle. We investigated if Oncotype® DX recurrence score (RS), Prosigna® (ROR) and EndoPredict® (EP/EPclin) prognostic tests, which include some of these genes, vary according to the time in the menstrual cycle when they are measured. Methods Pairs of test scores were derived from 30 ER-positive/human epidermal growth factor receptor-2-negative tumours sampled at two different points of the menstrual cycle. Menstrual cycle windows were prospectively defined as either W1 (days 1–6 and 27–35; low oestrogen and low progesterone) or W2 (days 7–26; high oestrogen and high or low progesterone). Results The invasion module score of RS was lower (− 10.9%; p = 0.098), whereas the ER (+ 16.6%; p = 0.046) and proliferation (+ 7.3%; p = 0.13) module scores were higher in W2. PGR expression was significantly increased in W2 (+ 81.4%; p = 0.0029). Despite this, mean scores were not significantly different between W1 and W2 for any of the tests and the two measurements showed high correlation (r = 0.72–0.93). However, variability between the two measurements led to tumours being assigned to different risk categories in the following proportion of cases: RS 22.7%, ROR 27.3%, EP 13.6% and EPclin 13.6%. Conclusion There are significant changes during the menstrual cycle in the expression of some of the genes and gene module scores comprising the RS, ROR and EP/EPclin scores. These did not affect any of the prognostic scores in a systematic fashion, but there was substantial variability in paired measurements.

Background: POETIC was a phase III clinical trial, with patients randomised 2:1 to 2-week perioperative aromatase inhibitor (POAI) vs control for postmenopausal women with oestrogen receptor positive (ER+) early breast cancer (BC) (Smith et al., Lancet Oncology 2020). Our previous study on POETIC trial patients with ER+ human epidermal growth factor receptor 2 positive (HER2+) BC suggested both HER2 enriched subtype (HER2-E) and immune enrichment pre-POAI (baseline, B) are main drivers of poor early response to POAI (Bergamino et al., 2022). However, some patients with HER2-E or immune enriched BC at B still showed good response to POAI. In this study, we aim to further investigate a sub-cohort of ER+ HER2+ BC from the POETIC trial, including a subset of aforementioned HER2-E tumours, to further explore the multi-modal molecular characteristics of the tumours resistant to POAI. Methods: Proliferation rate was assessed as percentage of cancer cells stained by Ki67. Patient POAI response was determined by Ki67 reduction at 2 weeks of treatment. A sub-cohort of 37 patients were selected based on response and classified as poor responders (PR, reduction < 30%, n=18), good responders (GR, reduction > 90%, n=11) and good responders with HER2-E BC at B (GR, reduction > 65%, n=8). Paired B and post-POAI (surgery, S) samples were taken from each patient of the sub-cohort. Multiplex immunofluorescence (mIF) was performed on these samples, measuring the immune cell densities in stroma and tumour compartments using five biomarkers: CD3 (all T cells), CD20 (B cells), CD68 (Macrophages), FOXP3 (regulatory T cells), and CD3 FOXP3 co-expression. The samples were also profiled using Breast Cancer 360TM (NanoString, BC360), covering the expressions of 758 genes and 46 biological signatures. Wilcoxon test, hierarchical clustering and spearman correlation test were performed to compare the tumour characteristics of GR and PR. Results: In this study, two B and four S samples were not achievable for mIF experiments due to low tumour content. At B (n = 35), among the five mIF biomarker measurements in stroma and tumour, only the stromal CD3 density was significantly different between GR (median = 0.0013) and PR (median = 0.0003, p = 0.041). In GR, HER2-E BC at B were separated into immune-high and immune-low groups with mIF biomarkers at B; the immune-high group was more likely to change into luminal subtypes post-POAI, while the immune-low group remained HER2-E. After POAI, the density changes in five mIF biomarkers in stroma and CD68 in tumour were all significantly higher in PR than GR (Table 1, n of paired samples = 62). The BC360 signatures of BC p53 (p < 0.001), BC proliferation (p < 0.001), LumB (p < 0.001) and HER2-E correlation coefficients (p < 0.001) were significantly downregulated in GR after POAI, while LumA correlation coefficients (p < 0.001) were notably increased. Conclusions: Our results suggest that for this sub-cohort, increased stromal immune response is associated with poor response to 2-week POAI in ER+ HER2+ early BC. HER2-E GR display visible immune heterogeneity at B. Lower-risk BC characteristics were exhibited in GR after the 2-week treatment. Further integrating mIF imaging data and additional digital spatial profiling are ongoing to reveal additional characteristics of ER+ HER2+ BC and tumour microenvironment predicting POAI resistance. Table 1: List of medians of log2 fold changes in mIF biomarker densities between GR and PR among the 62 paired samples, and Wilcoxon test p-values. Citation Format: Xixuan Zhu, Hui Xiao, Elena López-Knowles, Milana A. Bergamino Sirvén, Anastasia Alataki, Perry Maxwell, Holly Tovey, Lucy Kilburn, Chris Holcombe, Anthony Skene, Ian Smith, John Robertson, Katherine A Hoadley, Roberto Salgado, Judith Bliss, Nicholas Turner, Manuel Salto-Tellez, Gene Schuster, Mitch Dowsett, Maggie Chon U Cheang. Deconstructing the molecular characteristics of ER+ HER2+ early breast cancer in the POETIC trial using multiplex immunofluorescence and gene expression profiles [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-03-08.