Emerging mechanisms of triple-negative breast cancer radioresistance: Interplay between cancer cell mechanisms and the tumor immune microenvironment.

Background. The AXL receptor tyrosine kinase is associated with poor overall survival in breast cancer. AXL signaling is an important regulator of tumor plasticity related to epithelial-to-mesenchymal transition (EMT) and stem cell traits that drive metastasis and drug resistance. Upregulation of AXL has been associated with reduced response to anti-PD-1 therapy. Signaling via AXL is also a key suppressor of the anti-tumor innate immune response, and AXL is expressed on several cells associated with the tumor immune microenvironment. Hence AXL signaling contributes uniquely to both tumor cell intrinsic and microenvironmental anti-tumor immune suppression mechanisms. We show that AXL is required for tumor immune evasion in the 4T1/Balb/C mammary adenocarcinoma model and that blocking AXL signaling with BGB324, a selective clinical-stage small molecule AXL kinase inhibitor, enhanced the effect of immune checkpoint blockade. BGB324 + anti-CTLA-4/anti-PD-1 treated tumors displayed enhanced infiltration of cytotoxic T lymphocytes and Natural Killer cells. Importantly, responding animals rejected orthotopic 4T1 tumor cell re-challenge, demonstrating sustained tumor immunity. These data provided a translational rationale for combining AXL targeted therapy with immune checkpoint inhibitors to enhance anti-cancer immune response. Study Design. BGBC007 (NCT03184558) is an open-label, single arm, multi-center phase II study designed to assess the anti-tumor activity of BGB324 in combination with pembrolizumab in patients with previously treated, locally advanced and unresectable, or metastatic TNBC or TN-IBC. Secondary objectives include safety and pharmacokinetic profile of BGB324 and pembrolizumab in combination. A single arm, extension of Simon's 2-stage design is employed with an interim and final analysis. Up to 56 evaluable patients will be enrolled. Recruitment will be halted once 28 evaluable patients have been entered to determine the Objective Response Rate (ORR, complete response and partial response). If 5 or fewer responses are observed in up to 28 patients, the trial will be terminated in favor of the null for futility. If 11 or more responses are observed, then the trial will be stopped in favor of the alternative for demonstration of activity. If 6 to 10 patients have an observed response then a further 28 patients may be evaluated. This design provides an overall power of 80.6% to test the stated null and alternative hypothesis. BGB324 will be administered orally, once daily, in a fasted state. Days 1, 2 and 3 of BGB324 administration consists of a 'loading' dose of 400 mg followed by a dose of 200 mg daily. A fixed dose of 200 mg pembrolizumab will be given by intravenous infusion over 30 minutes every 3 weeks. BGB324 and pembrolizumab will be given until disease progression, unacceptable dose toxicity, or until 106 weeks (35 cycles). Efficacy endpoints including ORR, Duration of Response, Progression Free Survival are based on tumor imaging evaluation by RECIST 1.1. Tumor specimens will be taken to assess AXL and PD-L1 expression. Citation Format: Yule M, Wnuk-Lipinska K, Davidsen K, Blø M, Hodneland L, Engelsen A, Kang J, Lie M, Bougnaud S, Aguilera K, Ahmed L, Rybicka A, Milde Nævdal E, Deyna P, Boniecka A, Straume O, Thiery J-P, Chouaib S, Brekken RA, Gausdal G, Lorens JB. A phase II multi-center study of BGB324 in combination with pembrolizumab in patients with previously treated, locally advanced and unresectable or metastatic triple negative breast cancer (TNBC) or triple negative inflammatory breast cancer (TN-IBC) [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr OT1-01-03.

Rationale and purpose: Triple negative breast cancers (TNBCs), characterized by lack of hormone receptors' expression in absence of HER2 amplification, partially overlap with the basal-like subtype, with frequent occurrence in BRCA1 mutation carriers (BRCA1+). TNBCs are often associated with earlier onset, interval cancer diagnosis, larger size and aggressive clinical course with peak risk of recurrence at 1-3 years and increased mortality rate in the first 5 years. Thus, when screening women at high risk of breast cancer, special attention should be paid to patient outcome for TNBCs in comparison with non-TNBCs. Our aim was to compare TNBCs and non-TNBCs diagnosed during a prospective, non-randomized, multimodality screening study – including clinical breast examination (CBE), mammography, ultrasound (US) and magnetic resonance imaging (MRI) – on women at familial/genetic high risk of breast cancer conducted in 18 centres from June 2000 to March 2008 (ISS-HIBCRIT-1; Sardanelli F et al, Invest Radiol 2011). Methods: Comparisons were performed using Mann-Whitney U, Fisher exact and χ2 tests. Results: Among the 44 patients diagnosed with invasive cancers, 14 (31%) were TNBCs and 30 (69%) non-TNBCs, the former being 13 invasive ductal (IDC) and 1 atypical medullary carcinoma, the latter also including 15/30 lobular subtypes and/or DCIS component (p=0.005). Of the 14 TNBCs, 10 (71%) were found in BRCA1+, 2 (14%) in BRCA2+ and 2 (14%) in BRCA-untested women with strong family history of breast/ovarian cancer; the same data for 30 non-TNBCs were 9 (30%), 6 (20%) and 15 (50%) respectively (p=0.028). We had only three interval cancers, all TNBCs. The median age at diagnosis was 49 years (range 36-62) for TNBCs and 53 years (range 35-72) for non-TNBCs (p=n.s). TNBCs presented a higher rate (11/14, 79%) of pathological grade 3 IDCs compared with non-TNBCs (8/30, 27%) (p=0.002). The mean tumor size was 1.6 cm for TNBCs and 1.2 cm for non-TNBCs (p=n.s). Nodal status was negative in 12/14 (86%) TNBCs and in only 16/30 (53%) non-TNBCs (p=0.038). MRI similarly outperformed CBE, mammography and US in both TNBCs and non-TNBCs. Clinical course and survival could be monitored for 40/44 patients (91%), 13 TNBCs and 27 non-TNBCs, with a follow-up of 5.8 and 6.3 years (p=n.s) respectively. The rate of disease-free patients for over 5 years was 8/13 (62%; mean disease-free interval 7.0 years, range 5.0-8.0) for TNBCs and 17/27 (63%; mean 7.2 years, range 5.2-9.9) for non-TNBCs. Death due to BC occurred for 2/13 TNBC (15%, at 3.5 and 4.2 years) and 3/27 non-TNBC patients (11%; at 2.0, 4.9 and 5.7 years). The rate of locoregional relapse was 1/13 (8%, at 4.4 years) and 5/27 (19%; mean time of 5.0 years, range 2.4-7.0) respectively. Distant recurrence was reported for only 2 non-TNBC patients. Conclusion: TNBCs showed stronger association with BRCA1+ status, lower rate of lobular subtypes or DCIS component, and less frequent nodal involvement. Despite a more frequent pathological grade 3 and the tendency to be diagnosed as interval cancers, under the current treatment protocols TNBCs showed relapse and BC-related death rates and over-5-year disease-free intervals similar to those of non-TNBCs. These data provide outcome evidence supporting the value of entering women at high risk of TNBC (in particular BRCA1+) in intensive screening programs including MRI. Citation Format: Franca Podo, Filippo Santoro, Siranoush Manoukian, Clelia de Giacomi, Laura Cortesi, Lorenzo Preda, Stefano Corcione, Francesco Sardanelli. High-risk patients found affected with breast cancer during a multimodality screening program: Triple negative versus non-triple negative breast cancers. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B13.

BackgroundEstrogen receptor (ER) positive human epidermal growth factor receptor 2 (HER2) negative breast cancer (ER+/HER2−BC) and triple-negative breast cancer (TNBC) are two distinct breast cancer molecular subtypes, especially in tumor immune microenvironment (TIME). The TIME of TNBC is considered to be more inflammatory than that of ER+/HER2−BC. Natural killer (NK) cells are innate lymphocytes that play an important role of tumor eradication in TME. However, studies focusing on the different cell states of NK cells in breast cancer subtypes are still inadequate.MethodsIn this study, single-cell mRNA sequencing (scRNA-seq) and bulk mRNA sequencing data from ER+/HER2−BC and TNBC were analyzed. Key regulator of NK cell suppression in ER+/HER2−BC, S100A9, was quantified by qPCR and ELISA in MCF-7, T47D, MDA-MB-468 and MDA-MB-231 cell lines. The prognosis predictability of S100A9 and NK activation markers was evaluated by Kaplan–Meier analyses using TCGA-BRAC data. The phenotype changes of NK cells in ER+/HER2−BC after overexpressing S100A9 in cancer cells were evaluated by the production levels of IFN-gamma, perforin and granzyme B and cytotoxicity assay.ResultsBy analyzing scRNA-seq data, we found that multiple genes involved in cellular stress response were upregulated in ER+/HER2−BC compared with TNBC. Moreover, TLR regulation pathway was significantly enriched using differentially expressed genes (DEGs) from comparing the transcriptome data of ER+/HER2−BC and TNBC cancer cells, and NK cell infiltration high/low groups. Among the DEGs, S100A9 was identified as a key regulator. Patients with higher expression levels of S100A9 and NK cell activation markers had better overall survival. Furthermore, we proved that overexpression of S100A9 in ER+/HER2-cells could improve cocultured NK cell function.ConclusionIn conclusion, the study we presented demonstrated that NK cells in ER+/HER2−BC were hypofunctional, and S100A9 was an important regulator of NK cell function in ER+BC. Our work contributes to elucidate the regulatory networks between cancer cells and NK cells and may provide theoretical basis for novel drug development.

A Novel Immunological Role of Hydrogen Sulphide in Shaping Natural Killer Cells Cytoxicity in Breast Cancer Patients

Tumor-initiating cells with reprogramming plasticity or stem-progenitor cell properties (stemness) are thought to be essential for cancer development and metastatic regeneration in many cancers; however, elucidation of the underlying molecular network and pathways remains demanding. Combining machine learning and experimental investigation, here we report CD81, a tetraspanin transmembrane protein known to be enriched in extracellular vesicles (EVs), as a newly identified driver of breast cancer stemness and metastasis. Using protein structure modeling and interface prediction-guided mutagenesis, we demonstrate that membrane CD81 interacts with CD44 through their extracellular regions in promoting tumor cell cluster formation and lung metastasis of triple negative breast cancer (TNBC) in human and mouse models. In-depth global and phosphoproteomic analyses of tumor cells deficient with CD81 or CD44 unveils endocytosis-related pathway alterations, leading to further identification of a quality-keeping role of CD44 and CD81 in EV secretion as well as in EV-associated stemness-promoting function. CD81 is coexpressed along with CD44 in human circulating tumor cells (CTCs) and enriched in clustered CTCs that promote cancer stemness and metastasis, supporting the clinical significance of CD81 in association with patient outcomes. Our study highlights machine learning as a powerful tool in facilitating the molecular understanding of new molecular targets in regulating stemness and metastasis of TNBC.

SummaryM2-tumor-associated macrophages (M2-TAMs) in the tumor microenvironment represent a prognostic indicator for poor outcome in triple-negative breast cancer (TNBC).Here we show that Prune-1 overexpression in human TNBC patients has positive correlation to lung metastasis and infiltrating M2-TAMs. Thus, we demonstrate that Prune-1 promotes lung metastasis in a genetically engineered mouse model of metastatic TNBC augmenting M2-polarization of TAMs within the tumor microenvironment. Thus, this occurs through TGF-β enhancement, IL-17F secretion, and extracellular vesicle protein content modulation.We also find murine inactivating gene variants in human TNBC patient cohorts that are involved in activation of the innate immune response, cell adhesion, apoptotic pathways, and DNA repair. Altogether, we indicate that the overexpression of Prune-1, IL-10, COL4A1, ILR1, and PDGFB, together with inactivating mutations of PDE9A, CD244, Sirpb1b, SV140, Iqca1, and PIP5K1B genes, might represent a route of metastatic lung dissemination that need future prognostic validations.

Background: Specific biomarkers can be essential for developing effective treatments for aggressive breast cancers, especially triple negative subtypes, for which treatment options are limited. Folate receptor alpha (FRα), a critical membrane protein for DNA synthesis and cell metabolism, has been suggested to participate in the transformation of breast cancer into aggressive subtypes. It has been shown to be strongly associated with poor prognosis in triple negative breast cancers (TNBC) as well as estrogen receptor (ER) positive and progesterone receptor (PR) negative subtypes. SOX10 is a nuclear transcription factor that participates in neural crest development and in the differentiation of cells of melanocytic lineage. Data suggests that SOX10 may contribute in stem cell or progenitor cell maintenance. Recently, SOX10 expression has also been documented in benign breast myoepithelial cells and in aggressive breast cancers. The correlation of FRα and SOX10 in breast cancer is not fully known. This is the first study to compare FRα and SOX10 immunohistochemical profiles in breast cancers with emphasis in TNBC. Design: 166 cases of whole breast cancer tissues were classified according to their ER, PR, and HER2 immunohistochemical (IHC) status. These same cases were then IHC stained for mouse monoclonal SOX10 and FRα. Cut-off values of 1% and 5% for SOX10 and FRα, respectively, were used to determine positivity. Results: SOX10 achieved a sensitivity of 42.1% (8/19) in ER+/PR-/HER2- cases and was negative in all ER+/PR-/HER2+ cases (p<0.05). FRα was positive in 7.6% (7/92) of ER+/PR+/HER2- cases and was negative in all ER+/PR+/HER2+ cases. SOX10 identified more ER+/PR-/HER2- cases (42.1%, 8/19) than ER+/PR+/HER2+ cases (7.7%, 1/13) (p<0.05). Similarly, FRα stained 52.6% (10/19) of ER+/PR-/HER2- cases and was negative in all ER+/PR+/HER2+ cases (p<0.005). SOX10 and FRα were observed in 3.3% (1/30) and 20% (6/30) of HER2+ cases, respectively. In ER-/PR-/HER2- (triple negative) cases, both markers were highly expressed with 40.0% (10/25) and 52.0% (13/25) positive cases with SOX10 and FRα, respectively, with 24.0% (6/25) of cases positive with both markers. Approximately one half of TNBC cases expressed SOX10 and FRα; however, most SOX10 positive TNBC cases did not overlap with FRα positive TNBC cases. Table 1: SOX10 and FRα expression in breast cancer subtypesER/PR/HER2 ClassificationSOX10+ (%)FRα+ (%)Co-expression of SOX10 and FRα (%)ER+/PR+/HER2+ (n=13)1 (7.7%)0 (0.0%)0 (0.0%)ER+/PR+/HER2- (n=92)6 (6.5%)7 (7.6%)2 (2.2%)ER+/PR-/HER2+ (n=10)0 (0%)5 (50.0%)0 (0.0%)ER+/PR-/HER2- (n=19)8 (42.1%)10 (52.6%)5 (26.3%)HER2+ (n=30)1 (3.3%)6 (20.0%)0 (0.0%)ER-/PR-/HER2- (n=25)10 (40.0%)13 (52.0%)6 (24.0%) Conclusion: SOX10 and FRα were frequently expressed in triple negative breast cancers and in progesterone receptor negative breast cancers. Our data suggests that there may be different mechanisms by which SOX10 and FRα are implicated in aggressive breast cancers. These findings may help achieve a better understanding of the two different pathways involving stem cells (SOX10) and growth factors (FRα), their potential prognosis and their therapeutic management in the future. Citation Format: Laura L Hoang, Weimin Qi, Charlie Yu, David Tacha. SOX10 and folate receptor alpha are frequently expressed in triple negative and progesterone receptor negative breast cancers [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-01-13.

Purpose: Treatment of breast cancer has been increasingly successful in recent years with the advent of HER2-receptor targeted treatment and endocrine treatment. However, the triple negative subgroup of breast cancer (TNBC) (estrogen-, progesterone- and HER2-receptor negative) still lacks targeted treatment options. TNBC is a type of breast cancer that often affects younger women, and generally has a worse prognosis than other types of breast cancer. Recently, the complex role of the immune system in cancer growth, elimination and metastasis has been the object of increased attention. There is hope that a more detailed understanding of the intricate roles of the constituents of the immune system, will hold potential both as prognostic or predictive markers of cancer progression, but also as treatment targets for a wide range of tumors, including TNBC. The aim of this review is to provide an overview of the cellular immune microenvironment in TNBC, and to highlight areas in which TNBC may differ from other types of breast cancer.Material and methods: A search of PubMed was made using the terms ‘triple negative breast cancer’ and ‘tumor infiltrating lymphocytes’, ‘CD8’, ‘CD4’, ‘B cells’, ‘natural killer cells’, ‘macrophages’, myeloid derived suppressor cells’, ‘dendritic cells’, ‘immune check point inhibitor’, ‘CTLA-4’ and ‘PD-L1’.Results: We find that whilst factors such as TILs and certain subgroups of TILs (e.g., CD8 + and regulator T-cells) have been extensively researched, none of these markers are currently applicable to routine clinical practice. Also, TNBC differs from other types of breast cancer with regards to cellular composition of the immune infiltrate and PD-L1 expression, and the prognostic significance of these.Conclusions: Immune-related factors have the potential as both prognostic and predictive biomarkers for new treatments targeting the immune system in breast cancer. However, multivariate analyses, taking other well-known factors into account, are required to determine the true value of these biomarkers. Also, differences between TNBC and other types of breast cancer may have implications for treatment and use of immune-related factors as biomarkers.

Limitations in the therapies of triple-negative breast cancer, the strongest invasive subtype of breast cancer, have led to unsatisfactory clinical outcomes for patients. The efficacy of immunotherapy, an emerging treatment choice for triple-negative breast cancer, is highly related to the functional expression of each immune cell in the tumor immune microenvironment. Therefore, improving the immune response of immune cells in the tumor immune microenvironment is beneficial to enhance the effects of immunotherapy for triple-negative breast cancer clinically. There is now growing evidence that the active ingredients in traditional Chinese medicine, especially herbal medicine can influence the correlation of 2 major immune cell subpopulations in the tumor microenvironment of triple-negative breast cancer: tumor-infiltrating lymphocytes (T lymphocytes, B lymphocytes, Natural killer cells) and tumor-associated myeloid cells (macrophages, myeloid-derived suppressor cells, dendritic cells, neutrophils) expression of immune effect. This suggests that traditional Chinese medicine can effectively improve the immunosuppressive state of tumor microenvironment and enhance the effects of clinical immunotherapy for triple-negative breast cancer.

Introduction: Triple negative breast cancer (TNBC) represents 10-20% of all breast cancer entities [1][2] and has a known aggressive behavior and poor outcome. Patients treated in the setting of randomized clinical trials often do not represent actual treatment characteristics in real-life scenarios. To determine the stage-related survival and effect of surgical performance in TNBC with current multimodal treatment, we set out to analyze data of a large population-based registry of primary breast cancers which covering >50% of all breast cancer cases in Germany. Patients and methods: We analyzed data from a prospectively collected cancer registry of >200 certified breast units of the West-German Breast Center (WBC) in Germany from 2009-2011. From a cohort of 39570 primary breast cancer patients treated in this period, 12759 underwent adjuvant systemic therapy, out of which 2037 were TNBC cases with adjuvant chemotherapy. Inclusion criteria were triple negative breast cancers (Her2-new1+/2+ (Fish negative) and estrogen receptor (ER) and progesterone receptor (PR) <10%) and adjuvant chemotherapy, unilateral and non-metastasized breast cancer. Only those patients were included who have been followed-up within the first 3 years. Exclusion criteria were neoadjuvant chemotherapy, bilateral breast cancer and metastatic disease. The use of first, second and third generation chemotherapy was analyzed as well as the effect of clear/unclear resection margins and its impact on survival data. Results: 2037 patients were eligible for this study. Overall survival rates were as follows: T1 a and T1b 100 %, T1c 90,7 %, T2 90,9 %, T3 68,1 % and T4 64,3 %. No statistical differences were detected in between stages T1 and T2, and also not in between T3 and T4. Combining T1/T2 and T3/T4 and performing group-wise comparisons, differences for combined stages were highly statistically significant (3,9 x E-09). Inflammatory TNBC was prognostically worst with a survival-rate of 33,3 % at 24-months. (p<0,001) Unclear resection-margins versus clear margins in TNBC exerted a negative impact on DFS (87 vs. 73 %; p=0,00002) and DDFS (p=0,0004). Age was an independent risk factor for survival with a cut-off at 35 years.(p=0,044) Third-generation chemotherapies (anthracycline+taxanes) were associated with a significant improved overall-survival at 24-months compared to first generation chemotherapies (non-anthracycline, non-taxane) (95 % vs. 87 %; p=0,0029) Conclusion: Standard 3rd generation (anthracycline- and taxane-containing) chemotherapy and optimal surgical performance with clear margins is vital for patients with early, triple-negative breast cancer (TNBC). Within T1 and T2 stages, no stage-related deterioration of prognosis was detected, however these stages were markedly different from stages T3/T4, declining from 90-100% to 64-68 %. This analysis of a large database of a population-based study demonstrates that tumor size, margins and guideline-adapted chemotherapy matter in triple-negative, early breast cancer. [1] Schwentner et al. 2013 [2] Elsawaf et al. 2013. Citation Format: Peter Kern, Gunter von Minckwitz, Carolin Pütter, Annika Flach, Sofia Pavlidou, Rainer Kimmig, Mahdi Rezai. Stage-related risk categorization and influence of free margins on survival in triple negative early breast cancer - a population-based study of 2037 TNBC patients with adjuvant chemotherapy [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-13-01.

High-throughput gene expression and mutation analysis have provided increasing insight into the complex molecular biology of breast cancer. For many decades, detailed knowledge of the basic molecular biology of breast cancer did not have significant impact on routine clinical management of the disease. However, with increasing insights into both breast cancer molecular heterogeneity and targetable signal transduction pathways, scientists as well as clinicians have to understand the increasing complexity of the disease to be able to offer an individualized treatment strategy to patients with breast cancer. Molecular biology becomes relevant for treatment selection, understanding efficacy, side effects, and resistance as well as for generating new hypotheses for combinations of therapeutic agents. While 10 years ago, Perou et al. [1] have suggested breast cancer to be divided into 5 distinct molecular subtypes, it has recently been suggested that each breast cancer subtype is far from being homogeneous. For instance, triple negative breast cancer (TNBC) is understood to consist of several subtypes, presumably each with different prognostic and predictive implications. In order to understand recent developments in breast cancer molecular biology, it is of importance to have basic knowledge of the underlying technologies. In this special issue of Breast Care, Thomas Karn gives insights into the recent development of high-throughput gene expression and mutation profiling [2]. He provides an overview of the technologies used and their applicability in basic, translational, and clinical breast cancer research. Using these modern technologies, breast cancer has been shown to be comprised of several subtypes. Using complex gene expression profiling methods, basal-like, HER2-like, luminal, and presumably normal-like breast cancer subclasses may be assigned. These subgroups show significant overlap with the clinically defined triple-negative, HER2-positive and hormone receptor-positive breast cancer subtypes, however, it is important to recognize that there may be significant discordance between both classification systems. Both classifications show significant differences with regard to prognosis, response to (chemo-) therapy and underlying molecular biology. In this special issue of Breast Care, 2 breast cancer subtypes will be portrayed in detail. First, Gluz et al. [3], summarize the current data regarding hormone receptor-positive breast cancer. The most important clinical question for patients with hormone receptor-positive breast cancer is to identify those patients that have an excellent prognosis after being treated with endocrine therapy alone (usually referred to as luminal-A disease). In contrast, there is a subgroup of patients with highly proliferative, partially endocrine resistant disease that would additionally need (chemo)therapy. The authors summarize the current profiling technologies including immunohistochemical tools and gene expression profiling methods. The authors underscore, however, that current guidelines in Germany suggest only limited use of these tools in selected patients and, if possible, as part of clinical trials. TNBC is defined by an absence of hormone receptor expression and lack of HER2 overexpression / amplification. There is significant overlap between TNBC and the basal-like breast cancer subtype which is usually defined through complex gene expression profiling or a panel of (immunohistochemical) surrogate markers. In contrast to luminal breast cancer where major efforts are being made to identify patients that do not need chemotherapy with the goal of reducing chemotherapeutic frequency, patients with TNBC suffer from an unfavorable prognosis which is largely due to a lack of therapeutic agents. Therefore, there is an increasing need to identify promising therapeutic targets for patients with TNBC. Liedtke et al. [4] describe the efforts being made in this context. The authors summarize the current knowledge regarding the molecular biology of TNBC including the association with basal-like breast cancer and hereditary breast cancer with a particular focus regarding mutational and gene expression profiling methods and the association between TNBC and breast cancer stem cells.

Background: Given the heterogeneity of TNBC, identification of biologically and clinically distinct TNBC subtypes with unique therapeutic vulnerabilities is critically needed. Preclinically, DNMT3A protein expression is predictive of sensitivity to DNMT inhibitors (DNMTi). Additionally, treatment with DNMTi enhances antitumor immune responses. Ongoing (NCT05673200) and completed (NCT02957968) trials are evaluating the addition of DNMTi to chemoimmunotherapy for TNBC. However, it is unknown whether DNMT3A expression is associated with distinct TNBC biology or clinical outcomes. Methods: We measured DNMT3A protein using immunohistochemistry in early-stage TNBC tumors and evaluated its association with clinicopathologic characteristics, gene expression (RNASeq), spatial tumor immune microenvironment features (52 proteins using Nanostring GeoMx® Digital Spatial Profiler [DSP]), and recurrence-free survival (RFS). DNMT3A expression was categorized as negative (no staining) or positive (high: moderate/strong staining in >25% of nuclei; low/intermediate: weak staining in any % nuclei or moderate/strong staining in ≤25% of nuclei). Results: Among 345 patients, the median age was 54 years; most tumors were > 2 cm (53%) and N0 (62%). 281 (81%) were DNMT3A+ (high: 127 [37%], low/intermediate: 154 [45%]). DNMT3A+ TNBC were more often grade 3 (94% vs 86%, p = 0.037), with Ki-67 >15% (85% vs 67%, p = 0.002), and N+ (40% vs 26%, p= 0.043). There were no differences in age, menopausal status, tumor size, PD-L1 or stromal tumor-infiltrating lymphocytes (sTILs) between DNMT3A+ or DNMT3A- TNBC. To assess the impact of DNMT3A on the natural history of early TNBC, we focused our survival analyses on a subset of 103 (30%) patients who did not receive systemic therapy. DNMT3A expression was associated with worse RFS on a multivariable model adjusting for tumor size, nodal status, and sTILs (aHR 4.6, 95% CI 1.05-19.96, p=0.043; univariable results: HR 3.3; 95% CI: 0.79, 13.99; p = 0.102). The 5-year RFS (95% CI) of DNMT3A+ TNBC without chemotherapy was 66% (53-76), compared with 93% (59-99) for DNMT3A- TNBC. While TNBC with sTILs ≥ 30% had favorable 5-year RFS regardless of DNMT3A expression (86% DNMT3A+ vs 100% DNMT3A-), TNBC with sTILs <30% had particularly poor outcomes when also expressing DNMT3A (5y RFS 54% [38-67] for sTIL low and DNMT3A+ TNBC vs 90% [47-99] for sTIL low and DNMT3A- TNBC). RNA-Seq data (available for 154 [63%] of 345 tumors, regardless of receipt of chemotherapy) revealed differential expression of 234 genes between DNMT3A+ (n=126) and DNMT3A- (n=28) TNBC (e.g. MUC19, CEACAM5, and ZNF606, among others). GeoMx® DSP data (available for 289 [84%] tumors) showed that DNMT3A+ TNBC exhibited lower expression of CD3, CD4, CD8 (T cells) and granzyme B in the tumor compartment; lower expression of HLA-DR (antigen presentation), CD56 (NK cell), FAP alpha, SMA (fibroblasts) and CTLA4 (immune checkpoint) in the stromal compartment; and lower expression of CD127 (memory T cell), CD11c, CD14, CD68 (myeloid/macrophages), CD34 (stem cell), EpCAM and PanCK (epithelium), fibronectin (extracellular matrix) and TIM3 (immune checkpoint) in both the tumor and stromal compartments. Expression of Ki-67 was higher in both the tumor and stromal compartments of DNMT3A+ TNBC. Conclusions: DNMT3A expression was associated with worse clinical outcomes in TNBC (particularly in sTIL-low tumors), higher tumor proliferation, and diverging gene expression and tumor immune microenvironment profiles. While no differences in sTILs were noted by H&E quantification according to DNMT3A status, GeoMX DSP analyses revealed an immunosuppressed tumor and stromal microenvironment in DNMT3A+ tumors, characterized by decreased T, NK, myeloid, and antigen presentation markers. These data support targeting DNMT along with immunotherapy in DNMT3A+ TNBC as is currently being evaluated in NCT05673200. Citation Format: Roberto Leon-Ferre, David M Zahrieh, Sarah K Reed, Jodi M Carter, Saba Yasir, David Hillman, Judy C Boughey, Krishna Kalari, Peter C Lucas, Saranya Chumsri, Jennifer M Kachergus, Yi Liu, E. Aubrey Thompson, Harry D Bear, Fergus J Couch, James N Ingle, Liewei Wang, Matthew P Goetz. DNA methyltransferase 3A (DNMT3A) protein expression in triple-negative breast cancer (TNBC): Impact on clinical outcomes, gene expression, and tumor microenvironment [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr PS12-07.

Innate immune cells in the tumor microenvironment.

Background: Triple negative breast cancer (TNBC) is known for its aggressive behavior, poor prognosis and still remains as a difficult disease since treatment options are limited. Despite some success in PARP inhibition in BRCA gene mutation patients or platinating agents that may offer superior outcomes in a subset of TNBC patients (pts), currently, there are no targeted therapies for TNBC available. Specific biomarkers are urgently needed for developing effective treatments to predict which patients will respond to the given therapy. In this regard, circulating tumor cells (CTCs) are discussed to be an ideal surrogate marker for individualized treatment options. Since TNBC is closely related to epithelial-mesenchymal transition (EMT), a stem cell phenotype and, in addition, androgen receptor (AR) expression has been detected in up to a third of TNBC pts, we here established a multi-marker gene panel for the characterization of CTCs in TNBC pts and compared these findings with CTC characteristics in non-TNBC pts. Methods: 2x5 ml blood of 30 TNBC pts before and/or after neoadjuvant therapy and 30 non- TNBC pts (E+/PR+: n=23; ER+/PR-: n=4; HER2+: n=1; HER2+/ER+: n=1; HER2+/ER+/PR+: n=1) before therapy were analyzed for CTCs applying positive immunomagnetic selection targeting EpCAM, EGFR and HER2 using the AdnaTest EMT-2/Stem Cell Select (QIAGEN Hannover GmbH, Germany). Subsequently, cDNA was gene specifically pre-amplified using TaqMan PreAmp Master Mix according to in house designed assays. Establishment of a 19 gene qPCR panel was performed for the markers PI3K, AKT2, ERCC1, Aurka, HER2, HER3, EGFR, ALK, AR (androgene receptor), BRCA1, c-KIT, c-MET, KRT5, mTOR, NOTCH1, PARP1, SRC1, CD45 (leucocyte control) and GAPDH (housekeeping gene) as well as an internal reference. The cutoff was calculated, taken the false positive rate in healthy donors into account and defined as Ct(cutoff)-Ct(sample)-[Ct(CD45cutoff)-Ct(CD45sample)]. Results: In general, the distribution of the markers across all patients was highly variable. However, different expression patterns were found when CTCs of TNBC pts were compared with those of non-TNBC pts. In TNBC pts, SRC1 was the gene that was predominantly expressed, followed by c-Kit, HER3, BRCA1 and AURKA expression, before as well as after therapy. Interestingly, AKT2, EGFR, ERCC1 and PARP1 expression could not be detected at any time point studied. In addition, ALK, AR, c-Met, HER2 and KRT5 were only detected before but not after therapy. All other genes were expressed below 15%. In contrast, in non-TNBC pts, AKT2 was the gene that was predominantly expressed, followed by c-MET, HER3 and PI3K whereas c-KIT, ERCC1, mTOR and NOTCH1 were never found. All other genes were expressed below 10%. Conclusion: We successfully established a gene panel for the detection of the heterogeneous CTC population and demonstrated that CTCs in TNBC pts and non-TNBC pts show different genetic profiles. Although these data have to be confirmed in a bigger patient cohort, the knowledge about the individual target gene expression profile might efficiently help to predict a personalized targeted therapy for these pts in the future. Citation Format: Bittner A-K, Hoffmann O, Hauch S, Kimmig R, Kasimir-Bauer S. Circulating tumor cells in triple-negative and non-triple negative breast cancer patients show different genetic profiles. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-02-05.

CXCL16, as a chemotactic cytokine, has been observed in various human solid cancers. The effects of CXCL16 on tumor behavior is controversial and showed tissue-specific manners. In breast cancers, previous studies have showed that CXCL16 mainly originated from mesenchymal stromal cells or cancer-associated fibroblasts and played pro-tumorigenic roles. CXCL16 is highly expressed in triple-negative breast cancers (TNBC) in TCGA dataset for breast cancer. This study aimed to investigate the therapeutic effects of CXCL16 neutralizing antibody and its mechanisms in TNBC. To establish a xenograft mouse model of TNBC, MDA-MB-231 cells were injected subcutaneously to athymic nude mice, and mice were randomized into anti-CXCL16 and control groups. When measurable tumors were established, anti-CXCL16 neutralizing antibody (CLS-A101, cellus inc.) or control IgG were intravenously injected. Tumor growth was significantly delayed in the anti-CXCL16 group than the control group by 67% for 3 weeks. Flow cytometric analysis showed that macrophages (CD11b+F4/80+ cells) and myeloid derived suppressor cells (MDSC, CD11b+GR1+ cells) were decreased in the anti-CXCL16 group than the control group. Histologic examination showed that anti-CXCL16 antibody significantly reduced tumor vessel formation and decreased macrophage infiltration in both intra- and peri-tumoral area. Collectively, treatment of anti-CXCL16 antibody reduced tumor growth by inhibiting macrophage and MDSC infiltration following reduced tumor angiogenesis. To explore the mechanistic insight into the monotherapy of anti-CXCL16 antibody, we further analyzed the metabolic changes. In human tumor tissues, TNBC harboring high expression of CXCL16 showed significant shift of glutamine metabolism pathway-related genes (upregulation of GLS and downregulation of GLUL, GLUD1, and GLUD2) toward accumulating glutamate. For cellular experiments, CXCL16-enriched cancer/macrophage co-cultured conditioned medium (co-CM) or cancer single-cultured conditioned medium (s-CM) were treated to cancer cells, and glutamine and glutamate levels were measured. In MDA-MB-231 cells, co-CM treatment significantly enhanced glutamate/glutamine ratio and anti-CXCL16 antibody significantly decreased it. Consistently, glutamate measures in tumor lysates from TNBC xenograft were lower in the anti-CXCL16 group than the control group. In conclusion, anti-CXCL16 antibody (CLS-A101) inhibited immune suppressive myeloid cell infiltration followed tumor angiogenesis and glutamate productions in cancer cells. These dual actions on both tumor immune microenvironment and cancer cell metabolism enables anti-CXCL16 antibody to be a potential monotherapy for TNBC. Citation Format: Min Joo Kim, Hyun Jin Sun, Geun-Hyung Kang, Seong Keun Kim, Sun Wook Cho. Monotherapy of anti-CXCL16 antibody is a potential therapeutic for macrophage-enriched triple-negative breast cancer by modulating tumor immune microenvironment and glutamine metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5556.