Background:Hormone receptor (HR)-low human epidermal growth factor receptor 2 (HER2)-negative breast cancers (BC) have similar outcomes to triple-negative BC; however, there is a lack of consensus on treatment recommendations for this subset. We present results from a US National Cancer Database (NCDB) analysis of patients with stage I-III HER2-negative BC categorized into groups by estrogen and progesterone receptor (PR) expression: HR-Neg, HR-Low, HR-Intermediate (HR-Int), and HR-High. Objectives:The primary objective was to assess the effect of HR expression on neoadjuvant chemotherapy (NAC) pathologic complete response (pCR) rates. Secondary objectives included assessment of clinico-pathologic characteristics and practice patterns. Design and methods:Patients with stage I-III HER2-negative BC diagnosed in 2018 were identified in the NCDB, a nationwide oncology outcomes database in the United States. Quantitative HR expression was unavailable prior to 2018. Data were categorized into four groups by estrogen receptor (ER) and PR expression: ER <1% and PR <1% (HR-Neg); ER 1%-10% and/or PR 1%-10% (HR-Low); ER >11%-30% and/or PR >11%-30% (HR-Int); and ER >30% and/or PR >30% (HR-High). Those with undocumented HR status (3%) or without curative intent surgery (5%) were excluded. Results:Significant differences were found between HR groups with higher grade, clinical stage, and Ki-67 in HR-Low versus HR-Int or HR-High groups. pCR rates in those receiving NAC were significantly different by HR status, with higher pCR rates in HR-Low versus HR-High groups (p < 0.001). NAC utilization significantly differed between groups, with a higher proportion of patients with HR-Low BC receiving NAC than other HR-positive groups (p < 0.001). Less than half of patients with HR-Low BC received endocrine therapy compared to higher rates in the HR-Int and HR-High groups (p < 0.001). Conclusion:This large real-world analysis shows variability in NAC utilization and endocrine therapy for HR-Low and HR-Int BC, with further work needed to enhance representation of these in trials.

ObjectiveSeventy percent of newly diagnosed breast cancers are estrogen receptor-α positive and HER2/neu negative. First-line treatments incorporate endocrine therapy and cyclin-dependent kinase 4/6 inhibitors. However, therapy resistance occurs in most patients. Hence, there is an urgent need for effective second-line treatments. We previously showed that the potent estrogen receptor-β agonists, OSU-ERβ-12 and LY500307, synergized with the selective estrogen receptor modulator, tamoxifen, in vitro. Furthermore, we showed that these compounds inhibited endocrine-resistant and cyclin-dependent kinase 4/6-inhibitor-resistant estrogen receptor α-positive cell lines in vitro. Here, we used fulvestrant- and abemaciclib-resistant T47D-derived cell line xenografts to determine the efficacy of the combination of OSU-ERβ-12 and LY500307 with tamoxifen in vivo.ResultsDespite efficacy in vitro, treatments failed to reduce xenograft tumor volumes. Hence, we conclude that this treatment strategy lacks direct cancer cell-intrinsic cytotoxic efficacy in vivo.

BackgroundBreast cancer, one of the most common forms of cancer, is associated with the highest cancer-related mortality among women worldwide. In comparison to other types of breast cancer, patients diagnosed with the triple-negative breast cancer (TNBC) subtype have the worst outcome because current therapies do not produce long-lasting responses. Hence, innovative therapies that produce persisting responses are a critical need. We previously discovered that hyperactivating purinergic receptors (P2RXs) by increasing extracellular adenosine triphosphate (eATP) concentrations enhances TNBC cell lines' response to chemotherapy. Heparan sulfate inhibits multiple extracellular ATPases, so it is a molecule of interest in this regard. In turn, heparanase degrades polysulfated polysaccharide heparan sulfate. Importantly, previous work suggests that breast cancer and other cancers express heparanase at high levels. Hence, as heparan sulfate can inhibit extracellular ATPases to facilitate eATP accumulation, it may intensify responses to chemotherapy. We postulated that heparanase inhibitors would exacerbate chemotherapy-induced decreases in TNBC cell viability by increasing heparan sulfate in the cellular microenvironment and hence, augmenting eATP.MethodsWe treated TNBC cell lines MDA-MB 231, Hs 578t, and MDA-MB 468 and non-tumorigenic immortal mammary epithelial MCF-10A cells with paclitaxel (cytotoxic chemotherapeutic) with or without the heparanase inhibitor OGT 2115 and/or supplemental heparan sulfate. We evaluated cell viability and the release of eATP. Also, we compared the expression of heparanase protein in cell lines and tissues by immunoblot and immunohistochemistry, respectively. In addition, we examined breast-cancer-initiating cell populations using tumorsphere formation efficiency assays on treated cells.ResultsWe found that combining heparanase inhibitor OGT 2115 with chemotherapy decreased TNBC cell viability and tumorsphere formation through increases in eATP and activation of purinergic receptors as compared to TNBC cells treated with single-agent paclitaxel.ConclusionOur data shows that by preventing heparan sulfate breakdown, heparanase inhibitors make TNBC cells more susceptible to chemotherapy by enhancing eATP concentrations.

Abstract Among cancers in women worldwide, breast cancer has the highest incidence and the highest mortality. Patients with triple negative breast cancer (TNBC) have a markedly worse outcome in comparison to patients diagnosed with hormone-dependent and human epidermal growth factor receptor-positive breast cancers due to the aggressive and rapidly progressive nature of the disease and the deficit in specifically targeted therapies available. Therefore, there is a need for new therapeutic approaches. Extracellular ATP has been shown to cause cell death in an autocrine and paracrine manner and is also a known danger signal that causes immune activation. Our published data reveals that at high micromolar concentrations extracellular ATP (eATP) can induce cell death through the eATP-gated ion channels, purinergic P2RX7 and P2RX4 receptors. We also showed that in the context of chemotherapy treatment, eATP modulates TNBC cell death at submicromolar concentrations. A feature of the tumor microenvironment is a pronounced increase in the concentrations of eATP (micromolar) to levels that are closer to the threshold for cytotoxicity than in normal tissues (nanomolar). In addition, we discovered that chemotherapy induces the release of eATP from TNBC cells, further augmenting its concentration. Despite this, eATP is rapidly degraded to adenosine by extracellular ATPases (eATPases). It should be noted that extracellular ATP release and activation of its receptor, P2XR7, are prerequisites for the activation of a complex of proteins known as the NLRP3 inflammasome that is critical for IL-1β and IL-18 secretion and the induction of a highly inflammatory form of programmed cell death known as pyroptosis. The objective of this study was to evaluate if augmenting eATP levels using extracellular ATPase inhibitors will increase the efficacy of chemotherapy against THP-1 and differentiated THP-1 cells to undergo an inflammatory form of cell death known as pyroptosis. We treated THP-1 and differentiated THP-1 cells with increasing concentrations of the chemotherapeutic agent paclitaxel in the presence of eATPase inhibitors POM-1 and PSB 069 with eATP content and cell viability being assessed. Additionally, we examined NLRP3 inflammasome activation by immunoblot analysis. We analyzed the oligomerization of NLRP3 and ASC, the cleavage of caspase 1 and gasdermin and the secretion of IL-1β and IL-18, all components of pyroptosis. These results reveal that eATP modulates the chemotherapeutic response in the monocytic THP-1 and differentiated THP-1 cells, which could be exploited to augment the anticancer effects of macrophages on TNBC cells. These preclinical experiments could be applied to develop effective therapies that increase the depth and length of responses to chemotherapy and immunotherapy in TNBCs by augmenting pyroptosis of tumor associate macrophages through enhanced eATP. Citation Format: Jasmine M. Manouchehri, Lynn M. Marcho, Mathew A. Cherian. The effects of extracellular ATP signaling on the anticancer activities of macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3276.

Inflammatory breast cancer (IBC) poses an ongoing challenge as rates of disease recurrence and mortality remain high compared to stage-matched controls. However, frontline therapy has evolved through the years, including the widespread use of neoadjuvant chemotherapy (NAC) given the prognostic importance of pathologic complete response (pCR). Due to these sweeping changes, we need new data to assess current recurrence and survival outcomes for locally advanced IBC in the context of matched non-inflammatory controls. We conducted a retrospective analysis of institutional IBC data from 2010 to 2016 with the primary objective of comparing overall survival (OS), relapse-free survival (RFS), and distant relapse-free survival (DRFS). We matched IBC patients to non-inflammatory controls based on age, receptor status, tumor grade, clinical stage, and receipt of prior NAC. Secondary objectives included assessing pCR rates and identifying prognostic factors. Among NAC recipients, we observed similar pCR rates (47.6 % vs. 49.4 %, p = 0.88) between IBC (n = 84) and matched non-IBC (n = 81) cohorts. However, we noted a significant worsening of OS (p = 0.0001), RFS (p = 0.0001), and DRFS (p = 0.001) in the IBC group. Specifically, 5-year OS in the IBC cohort was 58.9 % vs. 86.7 % for matched controls (p = 0.0003). Older age was a weak negative predictor for OS (HR 1.03, p = 0.001) and RFS (HR 1.02, p = 0.01). For DRFS, older age was also a weak negative predictor (HR 1.02, p = 0.02), whereas the use of NAC was a positive predictor (HR 0.47, p = 0.02). Despite no clear difference in pCR, survival outcomes remain poor for IBC compared to matched non-inflammatory controls.

BACKGROUND:Inflammation can have social consequences, which may be relevant to inflammation's link with depression. The current study tests whether a typhoid vaccine increases feelings of social disconnection and avoidance behavior. METHOD:In two full-day visits at least three weeks apart, 172 postmenopausal breast cancer survivors (Stage I-IIIA) each received a typhoid capsular polysaccharide vaccination and a saline placebo injection in a random sequence. Blood was drawn prior to the injection, as well as every 90 min thereafter for 8 h to assess the inflammatory response (interleukin-6, IL-6; interleukin-1 receptor antagonist, IL-1Ra). At both visits, women completed the Social Connection Scale at 0 and 8.5 h post-vaccination as well as implicit and explicit social avoidance tasks at 7 h post-vaccination. RESULTS:The typhoid vaccine triggered rises in both inflammatory markers (ps < 0.01), but it did not impact feelings of social connection (p = .32), or performance on the implicit (p = .34) or explicit tasks (p = .37). Inflammatory rises did not predict feelings of social connection (ps > 0.64) or performance on explicit (ps > 0.73) or implicit (ps > 0.88) social avoidance tasks. CONCLUSION:Milder inflammatory stimuli may not affect social processes. Higher levels of inflammation or, relatedly, more sickness symptoms may be necessary to recapitulate prior findings of social avoidance.

BACKGROUND:Prior evidence has linked inflammation with impulsivity, but most of this evidence is cross-sectional. In this study, we provoked an acute inflammatory cytokine response to see whether it lowered prepotent response inhibition on three cognitive tasks. METHOD:This study features secondary analyses from a randomized crossover trial in which 171 postmenopausal breast cancer survivors (Stage I-IIIA) each received a typhoid capsular polysaccharide vaccination and a saline placebo injection in a random sequence at two separate visits at least one month apart. Participants completed the Stroop Color-Discrepant Task, the 2-back, and the Conners Continuous Performance Test (CPT) on the computer between 5 and 7 h after the injections. They had their blood drawn once before and repeatedly after the injection to measure interleukin-1 receptor antagonist and interleukin-6 responses. RESULTS:Women committed marginally fewer errors on the Stroop color-discrepant trials after the typhoid vaccine (M = 0.36, SE = 0.08), compared to placebo (M = 0.54, SE = 0.09, p = .076). Injection type did not predict 2-back accuracy (p = .80) or CPT commission errors (p = .47). Inflammatory cytokine responses were also unrelated to the outcomes of interest (ps>.16). CONCLUSION:We found no evidence that an acute inflammatory cytokine response provokes response disinhibition - an important facet of impulsivity. In fact, our only marginally non-significant result suggested that women were better able to inhibit their prepotent responses on the Stroop after receiving the typhoid vaccine, compared to placebo. Further experimental tests of the acute inflammatory cytokine response's effect on other aspects of impulsivity are warranted. LIMITATIONS:The sample was female, primarily White, highly educated cancer survivors, and recruitment was not premised on impulsive traits or diagnosis with an impulsive-related disorder. Also, there are many facets of impulsivity, and this study only measured response inhibition.

About one-in-three breast cancer survivors have lingering cognitive complaints and objective cognitive impairment. Chronic inflammation and intestinal permeability (i.e., leaky gut), two risk factors for cognitive decline, can also fuel depression—another vulnerability for cognitive decline. The current study tested whether depression accompanied by high levels of inflammation or intestinal permeability predicted lower subjective and objective cognitive function in breast cancer survivors. We combined data from four breast cancer survivor studies (n = 613); some had repeated measurements for a total of 1015 study visits. All participants had a blood draw to obtain baseline measures of lipopolysaccharide binding protein—a measure of intestinal permeability, as well as three inflammatory markers that were incorporated into an inflammatory index: C-reactive protein, interleukin-6, and tumor necrosis factor-α. They reported depressive symptoms on the Center for Epidemiological Studies depression scale (CES-D), and a binary variable indicated clinically significant depressive symptoms (CES-D ≥ 16). The Kohli (749 observations) and the Breast Cancer Prevention Trial (591 observations) scales assessed subjective cognitive function. Objective cognitive function tests included the trail-making test, Hopkins verbal learning test, Conners continuous performance test, n-back test, FAS test, and animal-naming test (239–246 observations). Adjusting for education, age, BMI, cancer treatment type, time since treatment, study visit, and fatigue, women who had clinically elevated depressive symptoms accompanied by heightened inflammation or intestinal permeability reported poorer focus and marginally poorer memory. However, poorer performance across objective cognitive measures was not specific to inflammation-associated depression. Rather, there was some evidence of lower verbal fluency; poorer attention, verbal learning and memory, and working memory; and difficulties with visuospatial search among depressed survivors, regardless of inflammation. By themselves, inflammation and intestinal permeability less consistently predicted subjective or objective cognitive function. Breast cancer survivors with clinically significant depressive symptoms accompanied by either elevated inflammation or intestinal permeability may perceive greater cognitive difficulty, even though depression-related objective cognitive deficits may not be specific to inflammation- or leaky-gut-associated depression.

Background: Approximately 10% of breast cancers (BC) are hormone receptor positive (HR+) and HER2 positive (HER2+). Despite treatment advances in the modern era of HER2 targeted therapies for early-stage disease, there remains a risk for late relapses. However, the role of adjuvant endocrine therapy (ET) to reduce recurrence in this BC subtype is unclear. Oncologists employ clinical judgment given lack of consensus, resulting in differences in treatment patterns. The ideal endocrine agent including the role of adding ovarian suppression (OS) in premenopausal women is unknown. These patients are largely underrepresented in clinical trials such as the phase III SOFT and TEXT studies. Additionally, these trials were initiated prior to the widespread use of trastuzumab with chemotherapy, which is now standard of care for HER2+ disease. We aimed to describe real world patterns surrounding choice of adjuvant ET and clinicopathologic features which predicted treatment with OS in premenopausal women with HR+/HER2+ BC. Methods: We performed a multi-institutional retrospective analysis of premenopausal women with non-metastatic HR+/HER2+ BC in the American Society of Clinical Oncology CancerlinQ® Discovery database from January 2010 to May 2020. Electronic health record data was obtained from 74 participating academic and community oncology sites. We collected clinical data on women less than 50 years who received chemotherapy, anti-HER2 therapy (trastuzumab with or without pertuzumab), and ET. Adjuvant OS was defined as receipt of at least 6 months of goserelin or leuprolide or surgical bilateral oophorectomy. Demographics, clinical characteristics, and treatment history was collected. Patients were categorized into 1 of 4 groups based on type of adjuvant ET prescribed at treatment initiation: aromatase inhibitor (AI) + OS, OS, tamoxifen + OS, or tamoxifen. Multivariable logistic regression was conducted to assess the association between clinicopathologic features and OS use. Results: Out of 360,540 patients with invasive breast cancer in the database, 937 met inclusion criteria. Mean age was 41.7 (SD 5.9) years; 83% had stage 1 or 2 BC and 78% had node positive disease. The majority (n=818, 87%) were prescribed tamoxifen whereas only 4 (0.4%), 50 (5.3%), and 65 (6.9%) received OS, tamoxifen + OS, and AI + OS, respectively. Table 1 includes demographic and clinical characteristics of the cohort. No clinicopathologic features predicted OS use apart from age; patients ≥35 years were less likely to receive OS compared with those < 35 (p< 0.001) (table 2). Conclusion: To our knowledge, this is the first real world study evaluating OS treatment in HR+/HER2+ BC. The use of OS was uncommon; this suggests a perception of its limited benefit when added to HER2-targeted therapy. Most patients received tamoxifen as the ET of choice. Age was the only factor to predict OS treatment; high risk features including node positivity and higher stage was not associated with its use. This highlights the wide variability in real world practice surrounding the clinical indications for OS. Further investigation is warranted to characterize the utility of ET including addition of OS to prevent recurrence in premenopausal HR+/HER2+ BC. This will better inform a personalized approach to tailor therapy for optimal outcomes in this distinct BC subtype. Table 1. Demographic and clinical characteristics of study participants by endocrine therapy treatment group. Table 2. Multivariable logistic regression model of clinicopathologic characteristics to predict use of ovarian suppression. Citation Format: Jasmine S. Sukumar, Sagar Sardesai, Andy Ni, Nicole Williams, Bhuvaneswari Ramaswamy, Robert Wesolowski, Mathew A. Cherian, Daniel Stover, Margaret Gatti-Mays, Ashley C. Pariser, Preeti K. Sudheendra, Mridula A. George, maryam lustberg. Real World Treatment Patterns of Adjuvant Endocrine Therapy and Ovarian Suppression in Premenopausal HR+/HER2+ 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 P1-02-03.

ABSTRACT Individuals respond differently to inflammation. Pain, sadness, and fatigue are common correlates of inflammation among breast cancer survivors. Stress may predict response intensity. This study tested whether breast cancer survivors with greater exposure to acute or chronic social or nonsocial stress had larger increases in pain, sadness, and fatigue during an acute inflammatory response. In total, 156 postmenopausal breast cancer survivors (ages 36-78 years, stage I-IIIA, 1-9 years posttreatment) were randomized to either a typhoid vaccine/saline placebo or the placebo/vaccine sequence, which they received at 2 separate visits at least 1 month apart. Survivors had their blood drawn every 90 minutes for the next 8 hours postinjection to assess levels of interleukin-6 and interleukin-1 receptor antagonist (IL-1Ra). Shortly after each blood draw, they rated their current levels of pain, sadness, and fatigue. Women also completed the Test of Negative Social Exchange to assess chronic social stress and the Trier Inventory of Chronic Stressors screen to index chronic general stress. At each visit, a trained experimenter administered the Daily Inventory of Stressful Events to assess social and nonsocial stress exposure within the past 24 hours. After statistical adjustment for relevant demographic and behavioral covariates, the most consistent results were that survivors who reported more chronic social stress reported more pain and sadness in response to IL-1Ra increases. Frequent and ongoing social stress may sensitize the nervous system to the effects of inflammation, with potential implications for chronic pain and depression risk among breast cancer survivors.

IntroductionThe immunomodulatory impact of corticosteroids and concurrent chemotherapy is poorly understood within triple-negative breast cancer (TNBC). On a biochemical level, steroids have been linked to the signaling of chemotherapy-resistant pathways. However, on a clinical level, steroids play an essential role in chemotherapy tolerance through the prevention of chemotherapy-induced nausea and vomiting (CINV) and hypersensitivity reactions. Given these conflicting roles, we wanted to evaluate this interplay more rigorously in the context of early-stage TNBC.MethodsWe performed a retrospective analysis of patients with operable TNBC who received neoadjuvant chemotherapy (NAC) between January 2012 and November 2018, with the primary goal of examining the dose-dependent relationship between pathological complete response (pCR) rates and corticosteroid use. Secondary endpoints included the impact of steroid dosing on overall survival (OS) and recurrence-free survival (RFS), along with a breakdown in pCR rates based on steroid doses provided during each chemotherapy phase. Further adjusted analyses were performed based on patient age, diabetic status, and anatomical stage. Finally, we explored the relationship between tumor-infiltrating lymphocytes (TILs) seen on tissue samples at baseline and dexamethasone doses in terms of pCR rates.ResultsIn total, of the 174 patients screened within this study period, 116 met full eligibility criteria. Of these eligible patients, all were female, with a median age of 51.5 years (27.0 to 74.0) and a mean body mass index (BMI) of 29.7 [standard deviation (SD) 7.04]. The majority were nondiabetic (80.2%). For cancer stage, 69.8% (n = 81) had stage 2 breast cancer. We found no statistically significant association between pCR rates and dexamethasone use, both in terms of the total dose (p = 0.55) and mean dose per NAC cycle (p = 0.74). Similarly, no difference was noted when adjusting for diabetic status, metformin use, or age at diagnosis, regardless of the total steroid dose provided (p = 0.72) or mean dose per cycle (p = 0.49). No meaningful changes to pCR rate were seen with higher mean or higher total steroid doses during the paclitaxel (T) phase (adjusted p = 0.16 and p = 0.76, respectively) or doxorubicin and cyclophosphamide (AC) phase (adjusted p = 0.83 and p = 0.77, respectively). Furthermore, we found no clinically significant association between dexamethasone dose and either RFS (p = 0.45) or OS (p = 0.89). Of the 56 patients who had available pre-treatment biopsy tissue samples, 27 achieved pCR, with higher TILs at baseline being associated with higher pCR rates, regardless of the mean dexamethasone dose used.ConclusionOur findings demonstrate that dexamethasone has no clinically significant impact on pCR, RFS, or OS when given concurrently with NAC in patients with curative TNBC, regardless of diabetic status.

Background: Hormone receptor (HR) low (1-10%) HER2-negative breast cancer (BC) is emerging as a distinct subtype with similarities in clinical outcomes to triple-negative BC. However, there is a lack of consensus on treatment recommendations for chemo-immunotherapy and endocrine therapy in this subset. Here, we present results from a US National Cancer Database (NCDB) analyses of patients with HER2-negative BC evaluating response to neoadjuvant chemotherapy (NAC) and patterns of care by HR expression. Methods: Patients with stage I-III HER2-negative BC diagnosed in 2018 were identified in NCDB, a nationwide oncology outcomes database in the US. Quantitative HR expression was unavailable prior to 2018. Data were categorized into four groups by estrogen receptor (ER) and progesterone receptor (PR) expression: ER< 1% & PR< 1% (HR-Neg), ER 1-10% and/or PR 1-10% (HR-Low), ER >11-30% and/or PR>11-30% (HR-Int), ER> 30% and/or PR > 30% (HR-High). Those with undocumented HR status (3%) or without curative intent surgery (5%) were excluded. The primary outcome was pathologic complete response (pCR) by HR expression in those undergoing neoadjuvant chemotherapy. Key secondary objectives included assessment of clinicopathologic characteristics and practice patterns. The categorical variables were compared between the four groups using a Chi-square test. Age was compared using a Kruskal-Wallis test. Results: Out of 104,205 incident cases, 2541 (2.4%) were HR-Low and 1241 (1.2%) were HR-Int. Significant differences were found between HR groups with higher grade, clinical stage, and Ki-67 in HR-Low vs. HR-Int or HR-High groups (Table 1). Patients with HR-Low and HR-Int BC were more likely to receive chemotherapy than HR-High (74%, 70% vs. 20%; p < 0.001) and the use of adjuvant endocrine therapy correlated with HR expression. Only half of patients in the HR-Low group received any endocrine therapy compared to higher rates in the HR-Int and HR-High groups (52% vs. 74%, 92%; p< 0.001). pCR rates in those receiving neoadjuvant chemotherapy were significantly different by HR status, with higher pCR rates in HR-Low vs. HR-High groups (p< 0.001) (Table 2). NAC utilization significantly differed between groups. A higher proportion of patients with HR-Low BC received NAC than other HR-positive groups (p < 0.001). Additionally, there was an increased use of NAC in patients with HR-Low BC treated at academic vs. community cancer centers (p< 0.001). Conclusions: This is one of the largest real-world analyses comparing key differences in biology and practice patterns of HR-Low, HER2-negative BC. Consistent with prior studies, we report HR-Low BC to be a rare and distinct subtype with higher pCR rates compared to HR-High BC. Practice patterns show wide variability in utilization of neoadjuvant chemotherapy and endocrine therapy for these patients. Future studies should address this disparity and enhance representation of patients with HR-Low BC in clinical trials to improve long-term outcomes. Table 1: Patient demographics Table 2: Neoadjuvant chemotherapy response amongst differing HR expression levels Citation Format: Dionisia Quiroga, Michael Grimm, Julie Stephens, Kai Johnson, Nicole Williams, Preeti K. Sudheendra, Mathew A. Cherian, Daniel Stover, Ashley C. Pariser, Margaret Gatti-Mays, Robert Wesolowski, Jose G. Bazan, Sasha Beyer, Ko Un Park, Bridget Oppong, Bhuvaneswari Ramaswamy, Julia White, Sachin R. Jhawar, Sagar Sardesai. Impact of low hormone receptor expression on neoadjuvant chemotherapy response and patterns of care in early-stage HER2-negative breast cancer: a US National Cancer Database analysis [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 P4-02-17.

The leading cause of cancer-related death among women worldwide is breast cancer. The triple-negative breast cancer (TNBC) subtype has limited therapeutic options that produce durable responses. As a result, it is associated with an especially poor prognosis compared to other breast cancer subtypes. Hence, the development of innovative therapeutic strategies that produce deeper and more durable responses is critically needed. We found that increasing extracellular ATP (eATP) can enhance the susceptibility of TNBC cell lines to chemotherapy through the activation of purinergic receptors (P2RXs) P2RX4 and P2RX7. However, eATP levels are limited by several different classes of eATPases, making the design of a single molecule that effectively inhibits all classes of eATPase complicated. Heparan sulfate, a carbohydrate moiety that is covalently linked to several cell surface and extracellular matrix proteins has previously been identified as an endogenous inhibitor of several classes of eATPases. Heparan sulfate is desulfated by sulfatases 1 and 2 at the 6-O-hydroxyl groups of glucosamine residues in heparan sulfate. Sulfatase 2 has been determined to be highly expressed in a variety of cancers including breast cancer, whereas sulfatase 1 is not. Several sulfatase inhibitors have been previously developed, such as OKN-007, which is a specific inhibitor of sulfatase 2. We hypothesized that OKN-007 would augment eATP levels in TNBC cell lines exposed to chemotherapy by increasing levels of heparan sulfate in the tumor microenvironment, leading to intensified cell death. TNBC cell lines MDA-MB 231, Hs 578t, and MDA-MB 468 and non-tumorigenic immortal mammary epithelial MCF-10A cells were treated with increasing concentrations of the chemotherapeutic agent paclitaxel in the presence of heparan sulfate and/or OKN-007, and eATP content and cell viability were evaluated. Inhibitors to the purinergic receptors P2RX4 and P2RX7 were used to further confirm that the mechanism of enhanced cell death induced by OKN-007 was through these receptors. In addition, basal protein and cell surface expression of sulfatases 1 and 2 were determined in all the examined cell lines via ELISA, Western blot, and flow cytometry analysis. To assess the effects on cancer-initiating cell properties, TNBC cell lines were treated with paclitaxel in the presence of heparan sulfate and/or OKN-007, and the tumorsphere formation efficiency assay used to assess for cancer-initiating cells. In parallel, we assessed for effects on cancer-initiating cells in treated cells using flow cytometry. Our results showed that inhibiting the 6-O desulfation of heparan sulfate increases eATP accumulation, thereby, enhancing TNBC cell lines’ response to chemotherapy. These findings may lead to therapies for TNBC based on sulfatase 2 inhibition that results in more effective responses with fewer side effects. Citation Format: Jasmine M. Manouchehri, Jharna Datta, Mathew A. Cherian. The inhibition of sulfation of heparan sulfate augments the chemotherapeutic response in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 398.

Background:Breast cancer is the leading cause of cancer-related death among women worldwide. Patients diagnosed with triple-negative breast cancer (TNBC) have limited therapeutic options that produce durable responses. Hence, a diagnosis of TNBC is associated with a poor prognosis compared to other types of breast cancer. As a result, there is a critical need for novel therapies that can deepen and prolong responses.We previously found that chemotherapy causes the release of extracellular adenosine triphosphate (eATP). Augmenting eATP release can boost the response of TNBC cells to chemotherapy and cause increased cell death. However, eATP concentrations are limited by several families of extracellular ATPases, which complicates the design of compounds that attenuate eATP degradation.In this study, we hypothesized that heparan sulfate (HS) would inhibit extracellular ATPases and accentuate chemotherapy-induced cytotoxicity in TNBC by augmenting eATP. HS can be desulfated by sulfatase 1 and 2; sulfatase 2 is consistently highly expressed in a variety of cancers including breast cancer, whereas sulfatase 1 is not. We hypothesized that the sulfatase 2 inhibitor OKN-007 would exacerbate chemotherapy-induced eATP release and TNBC cell death. Methods:TNBC cell lines and nontumorigenic immortal mammary epithelial cells were treated with paclitaxel in the presence of heparan sodium sulfate and/or OKN-007; eATP content and cell viability were evaluated. In addition, protein and cell surface expression of sulfatases 1 and 2 were determined in all examined cell lines via ELISA, Western blot, and flow cytometry analyses. Results:Sulfatase 2 was highly expressed in TNBC cell lines and human breast cancer samples but not in immortal mammary epithelial cells and much less so in normal human breast tissue and ductal carcinoma in situ samples. OKN-007 exacerbated chemotherapy-induced eATP release and chemotherapy-induced TNBC cell death. When combined with chemotherapy, OKN-007 attenuated cells with a cancer-initiating cell phenotype. Conclusions:These results suggest that sulfatase 2 inhibitors in combination with chemotherapy attenuate the viability of TNBC cells more than chemotherapy alone by exacerbating eATP release. These effects, as well as their capacity to attenuate the cancer-initiating cell fraction, may translate into combination therapies for TNBC that induce deeper and more durable responses.

Introduction Breast cancer affects two million patients worldwide every year and is the most common cause of cancer-related death among women. The triple-negative breast cancer (TNBC) sub-type is associated with an especially poor prognosis because currently available therapies fail to induce long-lasting responses. Therefore, there is an urgent need to develop novel therapies that result in durable responses. One universal characteristic of the tumor microenvironment is a markedly elevated concentration of extracellular adenosine triphosphate (eATP). Chemotherapy exposure results in further increases in eATP through its release into the extracellular space of cancer cells via P2RX channels. eATP is degraded by eATPases. Given that eATP is toxic to cancer cells, we hypothesized that augmenting the release of eATP through P2RX channels and inhibiting extracellular ATPases would sensitize TNBC cells to chemotherapy. Methods TNBC cell lines MDA-MB 231, Hs 578t and MDA-MB 468 and non-tumorigenic immortal mammary epithelial MCF-10A cells were treated with increasing concentrations the chemotherapeutic agent paclitaxel in the presence of eATPases or specific antagonists of P2RXs with cell viability and eATP content being measured. Additionally, the mRNA, protein and cell surface expressions of the purinergic receptors P2RX4 and P2RX7 were evaluated in all examined cell lines via qRT-PCR, western blot, and flow cytometry analyses, respectively. Results In the present study, we observed dose-dependent declines of cell viability and increases in eATP of paclitaxel-treated TNBC cell lines in the presence of inhibitors of eATPases, but not of the MCF-10A cell line. These effects were reversed by specific antagonists of P2RXs. Similar results, as those observed with eATPase inhibitors, were seen with P2RX activators. All examined cell lines expressed both P2RX4 and P2RX7 at the mRNA, protein and cell surface levels. Conclusion These results reveal that eATP modulates the chemotherapeutic response in TNBC cell lines, which could be exploited to enhance the efficacy of chemotherapy regimens for TNBC.

Purpose: To investigate breast cancer survivors' inflammatory responses to typhoid vaccine as a window into their innate immune response to novel pathogens. Methods: This double-blind crossover trial randomized 158 breast cancer survivors to either the vaccine/saline placebo or the placebo/vaccine sequence. The relative contributions of age, cardiorespiratory fitness (VO2peak), type of cancer treatment, central obesity, and depression to interleukin (IL)-6, IL-1 receptor antagonist (IL1Ra), and WBC vaccine responses were assessed pre-injection and 1.5, 3, 4.5, 6, and 7.5 h post-injection. Results: The vaccine produced larger IL-6, IL-1Ra, and WBC responses than placebo, ps < 0.0001. Prior chemotherapy, higher central obesity, and lower VO2peak were associated with smaller vaccine responses after controlling for baseline inflammation. Vaccine response was summarized by the percent increase in area under the curve (IL-6, WBC) or average post-injection mean (IL-1Ra) for vaccine relative to placebo. Women who received chemotherapy had smaller vaccine responses than women who did not for both IL-6 (44% vs 78%, p <.001) and WBC (26% vs 40%, p <.001); IL-1ra response was not significantly moderated by chemotherapy. Women whose central adiposity was one standard deviation above the mean had smaller vaccine responses than women with average adiposity for IL-6 (33% vs 54%, p <.001), WBC (20% vs 30%, p <.001), and IL-1Ra (2.0% vs 3.2%, p <.001). Women with an average level of VO2peak had smaller vaccine responses than women whose VO2peak was one standard deviation above the mean for IL-6 (54% vs 73%, p <.001), WBC (30% vs 40%, p <.001), and IL-1Ra (3.2% vs. 4.1%, p = 0.01). Age and depression did not significantly moderate vaccine responses. Conclusions: This study provided novel data on chemotherapy's longer-term adverse immune consequences. The data also have an important public health message: even relatively low levels of fitness can benefit the innate immune response to a vaccine.

Background Among women, breast cancer is the leading cause of cancer-related death worldwide. Estrogen receptor α-positive (ERα+) breast cancer accounts for 70% of all breast cancer subtypes. Although ERα+ breast cancer initially responds to estrogen deprivation or blockade, the emergence of resistance compels the use of more aggressive therapies. While ERα is a driver in ERα+ breast cancer, ERβ plays an inhibitory role in several different cancer types. To date, the lack of highly selective ERβ agonists without ERα activity has limited the exploration of ERβ activation as a strategy for ERα+ breast cancer. Methods We measured the expression levels of ESR1 and ESR2 genes in immortalized mammary epithelial cells and different breast cancer cell lines. The viability of ERα+ breast cancer cell lines upon treatments with specific ERβ agonists, including OSU-ERb-12 and LY500307, was assessed. The specificity of the ERβ agonists, OSU-ERb-12 and LY500307, was confirmed by reporter assays. The effects of ERβ agonists on cell proliferation, cell cycle, apoptosis, colony formation, cell migration, and expression of tumor suppressor proteins were analyzed. The expression of ESR2 and genes containing ERE-AP1 composite response elements was examined in ERα+ human breast cancer samples to determine the correlation between ESR2 expression and overall survival and that of putative ESR2 -regulated genes. Results In this study, we demonstrate the efficacy of highly selective ERβ agonists in ERα+ breast cancer cell lines and drug-resistant derivatives. ERβ agonists blocked cell proliferation, migration, and colony formation and induced apoptosis and S and/or G2/M cell-cycle arrest of ERα+ breast cancer cell lines. Also, increases in the expression of the key tumor suppressors FOXO1 and FOXO3a were noted. Importantly, the strong synergy between ERβ agonists and ERα antagonists suggested that the efficacy of ERβ agonists is maximized by combination with ERα blockade. Lastly, ESR2 (ERβ gene) expression was negatively correlated with ESR1 (ERα gene) and CCND1 RNA expression in human metastatic ERα+/HER2- breast cancer samples. Conclusion Our results demonstrate that highly selective ERβ agonists attenuate the viability of ERα+ breast cancer cell lines in vitro and suggest that this therapeutic strategy merits further evaluation for ERα+ breast cancer.

Patients with hormone receptor (HR)-positive tumors breast cancer usually experience a relatively low pathological complete response (pCR) to neoadjuvant chemotherapy (NAC). Here, we derived a 10-microRNA risk score (10-miRNA RS)-based model with better performance in the prediction of pCR and validated its relation with the disease-free survival (DFS) in 755 HR-positive breast cancer patients (273, 265, and 217 in the training, internal, and external validation sets, respectively). This model, presented as a nomogram, included four parameters: the 10-miRNA RS found in our previous study, progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) status, and volume transfer constant (Ktrans). Favorable calibration and discrimination of 10-miRNA RS-based model with areas under the curve (AUC) of 0.865, 0.811, and 0.804 were shown in the training, internal, and external validation sets, respectively. Patients who have higher nomogram score (>92.2) with NAC treatment would have longer DFS (hazard ratio=0.57; 95%CI: 0.39-0.83; P=0.004). In summary, our data showed the 10-miRNA RS-based model could precisely identify more patients who can attain pCR to NAC, which may help clinicians formulate the personalized initial treatment strategy and consequently achieves better clinical prognosis for patients with HR-positive breast cancer.

Background:Triple negative breast cancer (TNBC) represents about 10-15% of all breast cancers. Given the lack of targeted therapies, chemotherapy and immunotherapy are the only treatment options. The five-year survival rate of TNBC patients is approximately 15% less than other forms of breast cancer. Therefore, there is an urgent need to develop novel effective therapeutics for TNBC.Material and Methods:RNA isolated from human immortalized mammary epithelial MCF10A, and various TNBC cell lines was DNase treated and quantitative RT-PCR (qRT-PCR) performed using gene-specific primers spanning exon-exon junctions that include introns in the corresponding genomic sequence to avoid genomic DNA amplification. Gene expression was calculated by ΔΔCt method using GAPDH as an internal control. Immuno-blot analyses were performed on lysates prepared from cells in log phase of growth. Proteins were detected by Enhanced Chemi-Luminescence (ECL) method. Viability of cells treated with ERβ specific agonists was determined by using CellTiter-Glo® 2.0 assay. MB231 cells were treated with ERβ agonists following transfection with pcDNA3 (vector) or Flag-tagged ESR2 plasmids. Western blot analyses using specific antibodies and qRT-PCR were performed with the protein extracts and RNA isolated from the cells, respectively. Results:Our experiments demonstrate that ESR2 (ERβ) is differentially expressed and its level of expression is about 3 to 22-fold higher in TNBC cell lines tested than MCF10A. We have shown that viability of estrogen receptor (ER) positive breast cancer cell lines can be significantly inhibited by selective activation of ERβ. A highly selective ERβ agonist has been invented in the Drug Development Institute at the Ohio State University. Treatment of TNBC lines with these ERβ agonists exhibited a dramatic reduction of cell viability. While comparable IC50 values were observed with our in house and commercially available compounds, TNBC had 2-3-fold lesser IC50 values than ER-positive cell lines. Increased expression of FOXO3 and c-Myc1 and decreased expression of c-Myc2 isoform was noted in ERβ overexpressing MB231 cells upon treatment with ERβ agonists.Conclusion:Our results demonstrate that treatment with highly selective ERβ agonists could be an effective therapeutic strategy for TNBC patients. To improve and develop novel therapeutics for TNBC understanding the mechanism of action of these compounds will be crucial. Citation Format: Jharna Datta, Natalie Willingham, Jasmine Manouchehri, Joel David, Mirisha Sheth, Bhuvaneswari Ramaswamy, Ramesh Ganju, Mathew A Cherian. Estrogen receptor beta agonists for 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 PS18-39.