Plasticity of the ERBB receptor network has been suggested to cause acquired resistance to anti-human epidermal growth factor receptor 2 (HER2) therapies. Thus, we studied whether a novel approach ...using an ERBB1-3-neutralizing antibody mixture can block these compensatory mechanisms of resistance.
HER2+ cell lines and xenografts (n ≥ 6 mice per group) were treated with the ERBB1-3 antibody mixture Pan-HER, trastuzumab/lapatinib (TL), trastuzumab/pertuzumab (TP), or T-DM1. Downregulation of ERBB receptors was assessed by immunoblot analysis and immunohistochemistry. Paired pre- and post-T-DM1 tumor biopsies from patients (n = 11) with HER2-amplified breast cancer were evaluated for HER2 and P-HER3 expression by immunohistochemistry and/or fluorescence in situ hybridization. ERBB ligands were measured by quantitative reverse transcription polymerase chain reaction. Drug-resistant cells were generated by chronic treatment with T-DM1. All statistical tests were two-sided.
Treatment with Pan-HER inhibited growth and promoted degradation of ERBB1-3 receptors in a panel of HER2+ breast cancer cells. Compared with TL, TP, and T-DM1, Pan-HER induced a similar antitumor effect against established BT474 and HCC1954 tumors, but was superior to TL against MDA-361 xenografts (TL mean = 2026 mm 3 , SD = 924 mm 3 , vs Pan-HER mean = 565 mm 3 , SD = 499 mm 3 , P = .04). Pan-HER-treated BT474 xenografts did not recur after treatment discontinuation, whereas tumors treated with TL, TP, and T-DM1 did. Post-TP and post-T-DM1 recurrent tumors expressed higher levels of neuregulin-1 (NRG1), HER3 and P-HER3 (all P < .05). Higher levels of P-HER3 protein and NRG1 mRNA were also observed in HER2+ breast cancers progressing after T-DM1 and trastuzumab (NRG1 transcript fold change ± SD; pretreatment = 2, SD = 1.9, vs post-treatment = 11.4, SD = 10.3, P = .04). The HER3-neutralizing antibody LJM716 resensitized the drug-resistant cells to T-DM1, suggesting a causal association between the NRG1-HER3 axis and drug resistance. Finally, Pan-HER treatment inhibited growth of HR6 trastuzumab- and T-DM1-resistant xenografts.
These data suggest that upregulation of a NRG1-HER3 axis can mediate escape from anti-HER2 therapies. Further, multitargeted antibody mixtures, such as Pan-HER, can simultaneously remove and/or block targeted ERBB receptor and ligands, thus representing an effective approach against drug-sensitive and -resistant HER2+ cancers.
Clinical trials have demonstrated the efficacy of combining phosphoinositide 3-kinase (PI3K) inhibitors with endocrine therapies in hormone therapy-refractory breast cancer. However, biomarkers of ...PI3K pathway dependence in ER+ breast cancer have not been fully established. Hotspot mutations in the alpha isoform of PI3K (
) are frequent in ER+ disease and may identify tumors that respond to PI3K inhibitors. It is unclear whether PIK3CA mutations are the only biomarker to suggest pathway dependence and response to therapy. We performed correlative molecular characterization of primary and metastatic tissue from patients enrolled in a phase Ib study combining buparlisib (NVP-BKM-120), a pan-PI3K inhibitor, with letrozole in ER+, human epidermal growth factor-2 (HER2)-negative, metastatic breast cancer. Activating mutations in
and inactivating
mutations marked tumors from patients with clinical benefit (≥6 months of stable disease). Patients harboring mutations in both genes exhibited the greatest likelihood of clinical benefit. In ER+ breast cancer cell lines, siRNA-mediated knockdown of MAP3K1 did not affect the response to buparlisib. In a subset of patients treated with buparlisib or the PI3Kα inhibitor alpelisib each with letrozole where PAM50 analysis was performed, nearly all tumors from patients with clinical benefit had a luminal A subtype. Mutations in MAP3K1 in ER+ breast cancer may be associated with clinical benefit from combined inhibition of PI3K and ER, but we could not ascribe direct biological function therein, suggesting they may be a surrogate for luminal A status. We posit that luminal A tumors may be a target population for this therapeutic combination.
Abstract
The breast cancer microenvironment comprises a complex stroma including tumor-infiltrating lymphocytes (TILs), which can either stimulate tumor progression or promote anti-tumor immunity in ...response to tumor-derived cues. In general, of all clinical subtypes, triple-negative breast cancer (TNBC) is characterized by the most extensive infiltration of TILs within tumor stroma, which is consistent with the observation that TNBC seems to clinically respond to immunotherapies at the highest rates. Immune checkpoint blockade (ICB), an immunotherapy that promotes prolonged activation of cytotoxic immune cells to mount robust anti-tumorigenic responses, has yielded limited success in treating breast cancer. IMpassion130 was the first clinical trial to indicate that combining anti-PD-L1 with standard-of-care chemotherapy (nab-paclitaxel) to treat TNBC increases progression-free survival in patients exclusively those with PD-L1 positive tumors. Furthermore, the KEYSTONE-522 trial showed that administering anti-PD-1 in addition to various neoadjuvant chemotherapies increased the pathologic complete response in early stage TNBC patients. Despite promising evidence for immunotherapy success, both clinical trials lacked an experimental ICB-only group, and thus cannot address the therapeutic benefit of ICB alone, or which chemotherapy combination would maximize this benefit. Finally, mechanisms of resistance to ICB in breast cancer remain unexplored. We sought to model ICB response in vivo to elucidate the mechanisms responsible for immunotherapy efficacy in breast cancer, explore the synergistic effects of ICB with chemotherapies, and model ICB resistance.In this study, we investigated the efficacy of anti-PD-L1 as single-agent or in combination with paclitaxel or doxorubicin in the EMT6 (Balb/c) orthotopic mammary tumor model. In this model, single-agent immunotherapy was efficacious in reducing primary tumor growth compared to combination treatment, with a small proportion of complete responses, whereas modest benefit was observed with either chemotherapy alone. Following two rounds of treatment, we analyzed the tumor-immune microenvironment by flow cytometry and gene expression analysis. Anti-PD-L1 alone or in combination with either chemotherapy enhanced infiltration of cytotoxic and effector T cell as well as natural killer cells into the tumor microenvironment. Using gene expression analysis, we observed elevated expression of myeloid recruitment and activation markers in combination-treated tumors, supporting a known role of chemotherapy-induced cell death in myeloid recruitment; however as chemotherapy did not add benefit to tumor response or survival, it is unclear if this effect is detrimental or supportive. Interestingly, completely responsive anti-PD-L1 treated tumors that eventually recurred retained resistance to ICB upon re-implantation in naïve recipient mice, suggesting that tumor-intrinsic factors may contribute to resistance.Herein, we explore an in vivo model that corroborates clinical response to combinatorial immunotherapy approaches in breast cancer patients. We report the immunogenic efficacy of single-agent ICB that upregulates tumoricidal immune cell infiltration into the primary tumor, thereby controlling tumor growth, albeit without achieving complete response in all mice. Additionally, post-therapy recurrent tumors retain resistance upon transplantation, indicating tumor-specific adaptive resistance. This study has potentially significant clinical implications for re-evaluating the contributions of chemotherapy in combination with ICB in TNBC patients.
Citation Format: Ann Hanna, Paula I. Gonzalez-Ericsson, Violeta Sanchez, Melinda E. Sanders, Justin M. Balko. Evaluating the efficacy of immunotherapy in triple negative breast cancer abstract. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-14.
Abstract
Background: Immune checkpoint inhibitors (ICI) have improved patient overall and progression-free survival in some cancer types but yielded limited success in breast cancer. Phase-III ...clinical trials in triple negative breast cancer (TNBC) patients, who harbor extensive tumor-infiltrating lymphocytes within tumor stroma, have demonstrated increased progression-free survival (IMpassion130) and pathologic complete response (KEYNOTE-522). Consequently, combinations of ICI and chemotherapy have been FDA-approved for metastatic TNBC patients, and potentially in the early breast cancer setting. Despite FDA-approval, the therapeutic benefit of ICI alone and the most efficacious chemotherapy combinations are poorly characterized. Objective: We sought to model ICI response in vivo to elucidate the mechanisms responsible for immunotherapy efficacy in breast cancer and ascertain the therapeutic benefits of different chemotherapeutic combinations with ICI. Methods: In this study, we used an immunocompetent EMT6 orthotopic mammary tumor model to investigate the efficacy of single-agent immunotherapy and in combination with standard-of-care chemotherapy (paclitaxel PAC or doxorubicin DOX). We used single-cell RNA sequencing to analyze the cellular landscape of the primary tumor in response to combinatorial therapeutic strategies. Additionally, we serially sampled and analyzed peripheral blood from mice with differential responses by bulk and T-cell receptor (TCR) sequencing to identify systemic genetic alterations and T-cell expansion. Results: Single-agent anti-PD-L1 robustly suppressed primary tumor growth (p =0.0046) and extended survival (p<0.0001) beyond the isotype control group. While either PAC or DOX demonstrated moderate therapeutic efficacy, neither agent potentiated single-agent anti-PD-L1 benefit. Interestingly, despite using a genetically identical tumor model and murine host, anti-PD-L1 induced heterogeneous responses, ranging from complete response to complete intrinsic resistance. The longitudinal analysis of peripheral blood from heterogeneously responding mice uncovered signatures of myeloid cell recruitment corresponding to transient responses ultimately converting to resistance. We also identified specific clonal T cell expansion present only in responders. Single-cell transcriptomic profiling of the tumor microenvironment revealed an increase of T cells and natural killer cells and reduction of regulatory T cells in the combination groups versus chemotherapy alone, although this did not translate into improved benefit. Finally, we performed gene-set enrichment analysis on infiltrating T cells and identified a robust signature of cytotoxic T cell activation characterized by a significant enrichment in inflammatory pathways in both single-agent anti-PD-L1 and in combination with chemotherapy. Conclusions: This study identifies a heterogeneously ICI-responsive in vivo model that emulates TNBC patient response to combinatorial ICI approaches. We describe the efficacy of single-agent ICI in upregulating cytotoxic immune cell infiltration and expansion within the primary tumor, thereby diminishing tumor growth and enhancing survival. Moreover, this study describes differential responses in a genetically similar host, which reflects heterogeneous patient response to ICI. Further characterization may identify systemic biomarkers and tumor antigen-specific T cell clones to accurately predict immunotherapy response in patients and uncover mechanisms for sensitizing tumors refractory to ICI. This study also has potentially significant clinical implications for re-evaluating the benefits of chemotherapy in combination with ICI in TNBC patients.
Citation Format: Ann Hanna, Xiaopeng Sun, Paula I. Gonzalez-Ericsson, Violeta M. Sanchez, Melinda E. Sanders, Justin M. Balko. Host myeloid response to tumor and immunotherapy is associated with heterogeneity in outcomes to anti-PDL1 abstract. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-04-03.
Abstract
Although neoadjuvant chemotherapy (NAC) induces complete response in 30-40% of triple-negative breast cancers (TNBC), patients with residual disease at surgery have poor prognosis and ...limited treatment options until recurrence. Tumor-infiltrating lymphocytes (TILs) in the residual disease are a positive prognostic factor, but the composition of TILs has not been explored in granularity, and how specific immune composition of the tumor guides outcome is unclear, resulting in a lack of understanding of how to employ immunotherapies in the adjuvant setting. We assessed multiple immunologic biomarkers in a series of 100 residual TNBCs after NAC. Immune markers were assessed by multiple methods, including H&E TILs analysis, standard immunohistochemistry (IHC; HLA-A, CD4, CD8, LAG-3) and multiplexed immunofluorescence (mIF; HLA-DR, GZMB, CD4, CD8, PD-L1, pan-CK). TILs and IHC were scored by clinical research pathologists and IF was scored by AQUA® analysis. Where multiple markers were multiplexed together, subset analyses was performed. Association among parameters were assessed (n=83) including clinical outcome after surgery (n=79). As previously demonstrated, TILs in residual tumors after NAC predicted both RFS and OS. H&E-scored TILs were correlated to both CD4 and CD8 as scored by IHC or IF (Pearson’s r range 0.41-0.55; all p<0.001). CD4 and CD8 measured by IHC and IF were highly correlated (r=0.68 and 0.71, respectively; p<0.0001). CD4 and CD8 were also highly correlated to one another (r=0.67; p<0.0001). Likewise, infiltration by CD4 or CD8 were each associated with improved recurrence-free (RFS; coxPH p=0.004 and 0.007, respectively) and overall survival (OS; p=0.003 and p=0.005, respectively). Tumor and stromal PD-L1 (E1L3N) staining was associated with marginally poorer RFS (p=0.06) but not OS (p=0.94). Colocalizing GZMB and CD8 cells by mIF yielded a paradoxical finding that cytotoxic CD8 cells were associated with significantly worse RFS and OS (p=0.004 and 0.0004, respectively). In a multivariate model, total PD-L1, total CD4, and GZMB+CD8+ T cell infiltration each remained significant, suggesting independence, and provided a strongly predictive signature for RFS (p=4.6e-06). In the same analysis to predict OS, only GZMB+CD8+ T cells remained significant (p=0.0002). High GZMB+ CD8+ T cells were not associated with PD-L1 expression, but were associated with low LAG-3 expression (p=0.0009). Although the results of this study remain to be validated in larger cohorts, the paradoxical finding that GZMB+ CD8+ T cells are a negative prognostic factor for long term outcome in NAC-treated TNBC is an intriguing result. We hypothesize it may be these patients whose T cells are poised for cytotoxic activity but remain restricted through high PD-1 expression, and thus may benefit from adjuvant immunotherapy.
Citation Format: Caroline Nebhan, Paula I. Gonzalez-Ericsson, Roberto Salgado, Jennifer Bordeaux, Ju Young Kim, Christine Vaupel, Henry Gomez, Justin Micah Balko. Molecular characterization of residual triple-negative breast cancers after neoadjuvant chemotherapy identifies immune composition and features associated with clinical outcome abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4050.
Abstract
Purpose: Neoadjuvant chemotherapy (NAC), the standard of care for many breast cancer patients, is known to have systemic immunologic effects and is increasingly being used in clinical trials ...in combination with immunotherapeutics. Currently, there are few biomarkers to predict NAC or immunotherapy response. Biomarkers are needed to identify patients who will benefit from combination therapy compared to those who are likely to respond to NAC alone, and thus avoid the added risk of toxicity and financial burden. Peripheral blood is an attractive site of biomarker development due to the relative ease of longitudinal sampling. We have previously shown that high expression of a cytotoxicity gene signature in the blood following NAC is associated with presence of residual disease (RD) and future breast cancer recurrence, demonstrating the feasibility of using blood-based transcriptional biomarkers. Methods: We used RNA sequencing to profile the peripheral blood of 53 breast cancer patients prior to definitive surgery (n=23 RD; 9 pathologic complete response (pCR); 21 no NAC). DESeq was used to identify differentially expressed genes and gene set enrichment analysis to identify differentially expressed pathways using the Molecular Signatures Database hallmark gene sets. CIBERSORTx was used to deconvolute cell type abundance. To extend the cell type abundance data to a larger cohort, we used a de-identified electronic medical record to collect clinically measured cell type abundance data on breast cancer patients treated with NAC (n=110; 35 pCR; 75 RD). Results: We identified 1,238 (FDR corrected q-value <0.1) differentially expressed genes between pCR and RD samples. Interferon (IFN)-γ Response (q-value <0.0001; normalized enrichment score (NES)=3.32), IFNα Response (q-value <0.0001; NES=3.14), and Complement (q-value<0.001; NES=2.29) pathways were significantly enriched in the blood of patients experiencing pCR vs. RD. We combined expression of the unique leading-edge genes from each pathway into a 60-gene IFN/Complement score. Interestingly, expression of the IFN/Complement score was independent of expression of our previously published cytotoxicity score. Using single cell RNA sequencing on peripheral blood mononuclear cells, we localized expression of the IFN/Complement genes to monocytes whereas the cytotoxicity score was confined to CD8+ T cells and natural killer cells. A combination peripheral immunologic response score (PIRS; IFN/Complement score - Cytotoxic score) had improved predictive power compared to either signature alone. PIRS was highest in patients with pCR and lowest in patients with RD who experienced a breast cancer recurrence with 3 years. Using CIBERSORTx, we further identified relative monocyte abundance as higher in samples with pCR compared to those with RD. Clinically measured post-NAC, but not pre-NAC, monocytes were significantly higher in patients with pCR compared to those with RD. Using the synthetic derivative medical record, we further identified 110 breast cancer patients treated with NAC. In this cohort, relative monocytes were also statistically significantly higher in patients with pCR compared to those with RD (p=0.0367). Conclusions: Peripheral blood gene expression scores and cell type abundance may be useful biomarkers of NAC response and outcome in breast cancer. We identified an immunologic gene signature that was highest in patients with the best outcomes (pCR) and lowest in those with the worst outcome (RD with recurrence). We further identified monocyte abundance, which is routinely measured clinically, as highest in patients with pCR. Taken together, these results suggest that peripheral blood biomarkers following NAC may be useful in predicting long-term outcome. Future work will explore the utility of peripheral blood biomarkers in predicting immunotherapy response.
Citation Format: Margaret L Axelrod, Yu Wang, Yaomin Xu, Cosmin A. Bejan, Xiaopeng Sun, Paula I Gonzalez-Ericsson, Riley E Bergman, Joshua Donaldson, Sara Nunnery, Melinda Sanders, Chiara Massa, Barbara Seliger, Ingrid A Mayer, Justin M Balko. Peripheral immunity predicts therapeutic outcomes in breast cancer patients abstract. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-08-34.
Abstract
Triple-negative breast cancer (TNBC) is a collection of biologically diverse cancers characterized by distinct transcriptional patterns, biology and immune composition. TNBCs display ...transcriptional diversity with at least four tumor-intrinsic subtypes that include two basal-like (BL1, BL2), a mesenchymal (M) and a luminal androgen receptor (LAR) subtype. Using integrative analyses of transcriptomic, epigenetic, proteomic and phospho-proteomic patterns we have identified subtype-specific vulnerabilities, which advanced our understanding of the cellular origins of TNBC subtypes. In mesenchymal subtype tumors we observed high mutation load and genomic instability, absence of immune cells, low PD-L1 expression, loss of global DNA methylation and transcriptional repression of antigen presentation genes through the polycomb repressor complex 2 (PRC2). Using cell line models, we demonstrate that MHC-I expression is epigenetically silenced by the PRC2 in mesenchymal TNBC cell lines and inhibition of EZH2 restores expression. Pharmacological inhibition of EZH2 enhances chemotherapy efficacy in syngeneic murine tumor models providing a rationale for using PRC2 inhibitors in PD-L1 negative mesenchymal tumors.
Citation Format: Brian D Lehmann, Antonio Colaprico, Tiago C Silva, Jianjiao Chen, Hanbing An, Yuguang Ban, Lily Wang, Jamaal L James, Justin Balko, Paula I Gonzalez-Ericsson, Melinda E Sanders, Bing Zhang, Jennifer A Pietenpol, Xi S Chen. Multi-omics characterization of triple-negative breast cancer identifies therapeutic vulnerabilities and epigenetic immune suppression in the mesenchymal subtype abstract. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD3-04.
Cancer incidence increases with age and is a leading cause of death. Caloric restriction (CR) confers benefits on health and survival and delays cancer. However, due to CR's stringency, dietary ...alternatives offering the same cancer protection have become increasingly attractive. Short cycles of a plant-based diet designed to mimic fasting (FMD) are protective against tumorigenesis without the chronic restriction of calories. Yet, it is unclear whether the fasting time, level of dietary restriction, or nutrient composition is the primary driver behind cancer protection. Using a breast cancer model in mice, we compare the potency of daily CR to that of periodic caloric cycling on FMD or an isocaloric standard laboratory chow against primary tumor growth and metastatic burden. Here, we report that daily CR provides greater protection against tumor growth and metastasis to the lung, which may be in part due to the unique immune signature observed with daily CR.
Abstract
Background: Although neoadjuvant chemotherapy (NAC) induces complete response in 30-40% of triple-negative breast cancers (TNBC), patients with residual disease at surgery have poor ...prognosis and limited treatment options until recurrence. Tumor-infiltrating lymphocytes (TILs) in the residual disease are a positive prognostic factor, but how specific immune composition of the tumor guides outcome is unclear, resulting in a lack of understanding of how to employ immunotherapies in the adjuvant setting.
Methods: We assessed multiple immunologic biomarkers in a series of 99 residual TNBCs after NAC. Immune markers were assessed by multiple methods, including H&E-based TILs analysis, standard immunohistochemistry (IHC; CD8, CD20, CD56, FOXP3, LAG-3, B7-H4, HLA-A) and multiplexed immunofluorescence (mIF; HLA-DR, GZMB, CD4, CD8, PD-L1, pan-CK, PD1, CD3). Association among parameters were assessed (up to n=98) including clinical outcome after surgery (n=94).
Results: As previously demonstrated, TILs in residual tumors after NAC predicted both RFS and OS (p=0.0093 and p=0.0376, respectively). H&E-scored TILs were highly correlated to CD3, CD4 and CD8 positive T cells (Spearman r range 0.34-0.4850; all p<0.001), while CD20 and CD56 positive cells only make up a small and non-significant proportion of the TILs composition. Likewise, infiltration by overall T cell populations were associated with significantly improved recurrence free survival (RFS; HR range 0.34-0.54) and overall survival (OS; HR range 0.38-0.43), whereas CD20+ B cells were not. Additionally, infiltration by cells expressing LAG3 (HR 0.49) and CD56 (HR 0.44), were each associated with significantly improved OS. Interestingly, colocalizing GZMB+ and CD8+ T cells by mIF yielded a paradoxical finding that cytotoxic CD8+ T cells (CD8+GZMB+ T cells) were associated with worse RFS and OS (HR 1.8 p=0.0338 and HR=2.8 p=0.0013, respectively). In a multivariate model, only CD8+GZMB+ T cell infiltration remained significant for OS (p=0.0224), suggesting independence. CD8+GZMB+ T cell infiltration inversely correlated with TILs (p=0.0293) and CD8/mm2 counts in the tumor core (p=0.0020) and showed a positive correlation with B7H4 tumor expression (p=0.0183). Additionally, high CD8+GZMB+ cases showed low HLA-A (p=0.0189) expression in tumor cells. In accordance, high CD8+GZMB+ tumors mainly correspond to stroma-restricted, margin-restricted and immune-desert tumor immune microenvironment landscapes.
Conclusions: We have found a paradoxical association of GZMB+ CD8+ T cells with a negative prognosis in NAC-treated TNBC. We hypothesize that while these T cells are poised for cytotoxic activity, they remain restricted through sub-localization outside the tumor core, downregulation of HLA-A on tumor cells preventing interaction with the T cell receptor, and upregulation of B7H4 expression, which has been shown to inhibit cytotoxic T cell activity. Patients with high CD8+GZMB+ tumors in the post-NAC setting may benefit from adjuvant immunotherapy, particularly in combination with therapies that enhance MHC-I (e.g. HLA-A) antigen presentation.
Citation Format: Paula I Gonzalez-Ericsson, Violeta Sanchez, Roberto Salgado, Jennifer Bordeaux, Ju Y Kim, Christine Vaupel, Henry Gomez, Melinda E Sanders, Justin M Balko. The immune landscape of residual triple-negative breast cancers after neoadjuvant chemotherapy abstract. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD5-07.
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