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BACKGROUND: HER2-positive breast cancers, which accounts for 20% of breast cancers, is associated with aggressive clinical behavior and inferior survival. The approval of HER2 targeted therapy has changed the landscape of this disease and has reduced disease recurrence by 50% and has improved survival by 33%. (1) However, cardiotoxicity is a well-recognized adverse event associated with HER2-targeted therapies. Adjuvant trastuzumab emtansine (TDM1) is the current standard of care for patients with residual breast cancer after neoadjuvant HER2-targeted therapy. TDM1 is associated with a risk of cardiotoxicity defined as a decline in left ventricular ejection fraction (LVEF). In a pooled analysis of data from seven metastatic breast cancer trials with TDM1, the incidence of cardiac events such as congestive heart failure (CHF), cardiac ischemia, cardiac arrhythmia or grade 1/2 LVEF drop was 3.37%. Adjuvant breast radiation (RT) is routinely offered for patients at high risk for recurrence. Breast RT is also associated with long-term increased risk of cardiac disease more than 10 years after RT. The HERA trial which studied use of adjuvant trastuzumab showed that rates of cardiotoxicity were higher in patients receiving concurrent RT with trastuzumab (left sided > right sided breast cancer) compared to those who did not receive adjuvant RT, although not statistically significant. In the multivariate analysis, no treatment or baseline cardiovascular risk factors were strongly correlated with LVEF, but radiation therapy showed a borderline correlation (adjusted HR, 1.258; 95% CI, 1.00-1.58; P = .049). The risk of cardiotoxicity with concurrent TDM1 and RT has not been well studied. With increasing use of TDM1 in the adjuvant setting, it is important to understand the cardiotoxic effects of combination therapy in early-stage breast cancer. METHODS: We undertook a review of our clinical database to identify patients who received adjuvant TDM1 with concurrent RT for Stage I-III breast cancer from 1/2020 to 01/2022. Clinical parameters including age, date of diagnosis, history of cardiac disorders, echocardiogram findings, radiation dose, final pathologic stage and molecular subtypes of cancer were extracted. All patients had ejection fraction to monitor cardiac fraction. Global longitudinal strain (GLS), which is a more sensitive and reproducible indicator of cardiac dysfunction than LVEF, was also collected, if available. RESULTS: Of 32 patients identified in our retrospective analysis, two patients (6%) developed a drop in ejection fraction post radiation. Median age of patients was 57y. Majority of the patients were Caucasian (44%) followed by Hispanic (28%). 19 (60%) patients had right sided breast cancers and 13(40%) patients had left sided cancers. The mean pre-radiation ejection fraction was 60% and post radiation was 61%. Using paired t-testing, there was no statically significant difference in ejection fraction after radiation (p=0.343). Comparative GLS measurements were available for 16 patients and there was no statical difference with concurrent radiation (p=0.18). All patients tolerated radiation with mostly grade 2 skin dermatitis except four patients who had grade 3 skin dermatitis. One patient had to discontinue radiation early given grade 3 skin dermatitis. CONCLUSION: This institutional review of 32 patients suggests that adjuvant TDM1 with concurrent RT did not result in a significant change in ejection fraction or GLS. Most patients tolerated radiation without significant skin toxicities. One of the limitations of the study is the small sample size. A larger study should look at more broader conclusions; however this data has strong clinical implications. Cardiac Parameters pre and post RT Citation Format: Faheem Farooq, Dillon Cason, Nisha Ohri, Shicha Kumar, Allison Grann, Anna Litvak, Shridar Ganesan, Bruce G. Haffty, Deborah Toppmeyer, Coral Omene, Mridula A. George. Evaluating the risk of cardiotoxicity associated with concurrent trastuzumab emtansine (TDM1) and radiation therapy in patients with early-stage HER2 positive 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 P5-07-08.

Because the incidence of breast cancer increases decades after ionizing radiation exposure, aging has been implicated in the evolution of the tumor microenvironment and tumor progression. Here, we investigated radiation-induced carcinogenesis using a model in which the mammary glands of 10-month-old BALB/c mice were transplanted with Trp53-null mammary tissue 3 days after exposure to low doses of sparsely ionizing γ-radiation or densely ionizing particle radiation. Mammary transplants in aged, irradiated hosts gave rise to significantly more tumors that grew more rapidly than those in sham-irradiated mice, with the most pronounced effects seen in mice irradiated with densely ionizing particle radiation. Tumor transcriptomes identified a characteristic immune signature of these aggressive cancers. Consistent with this, fast-growing tumors exhibited an immunosuppressive tumor microenvironment with few infiltrating lymphocytes, abundant immunosuppressive myeloid cells, and high COX-2 and TGFβ. Only irradiated hosts gave rise to tumors lacking cytotoxic CD8+ lymphocytes (defined here as immune desert), which also occurred in younger irradiated hosts. These data suggest that host irradiation may promote immunosuppression. To test this, young chimera mice were fed chow containing a honeybee-derived compound with anti-inflammatory and immunomodulatory properties, caffeic acid phenethyl ester (CAPE). CAPE prevented the detrimental effects of host irradiation on tumor growth rate, immune signature, and immunosuppression. These data indicated that low-dose radiation, particularly densely ionizing exposure of aged mice, promoted more aggressive cancers by suppressing antitumor immunity. Dietary intervention with a nontoxic immunomodulatory agent could prevent systemic effects of radiation that fuel carcinogenesis, supporting the potential of this strategy for cancer prevention.

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Background: The addition of neoadjuvant platinums to standard chemotherapy regimens for triple negative breast cancer (TNBC) improves rates of pathologic complete response (pCR). Our prior trial combining carboplatin (CAR) with liposomal doxorubicin (DOX) for metastatic TNBC showed good response rates with minimal side effects and allows for higher platinum dosing. We hypothesized that the doublet of DOX+CAR is effective and tolerable in the neoadjuvant setting for TNBC and tumor genomics may identify biomarkers of complete response and actionable targets. Methods: A phase II single arm trial was conducted for patients (pts) diagnosed with stage II-III TNBC. Patients received 4 cycles of neoadjuvant carboplatin (AUC 5) and liposomal doxorubicin (30mg/m2) administered every 28 days, then underwent definitive breast surgery followed by 12 weeks of adjuvant paclitaxel 80 mg/m2 administered weekly. Primary and secondary clinical endpoints were rate of pCR and two year recurrence free survival (RFS) and overall survival (OS), respectively. Cardiac safety of the combination was assessed. Fresh residual tumor samples were obtained at time of surgery for generation of patient derived xenografts (PDX). Whole genome sequencing (WGS) and RNA sequencing will be performed to evaluate specific patterns of small-variant mutations and patterns of structural variants to identify which patients are likely to have pCR. Results: From 2/2015 to 6/2021, 62 pts were enrolled, 8 pts withdrew consent either prior to treatment or completion of neoadjuvant therapy and/or surgery. Median age of the cohort was 55.4 years. There was high participation by under-represented groups: 17.0% African American, 20.8% Asian, 13.2% Hispanic. Most histologies were invasive ductal carcinoma, but included apocrine, pleomorphic lobular, and metaplastic subtypes. 53 pts completed all 4 cycles of neoadjuvant DOX+CAR, followed by definitive surgery; 1 pt progressed after 1 cycle and proceeded to surgery thereafter. 30.2% (16) pts achieved pCR, and residual cancer burden (RCB) will be reported in patients with residual disease. Of the 53 pts who completed 4 cycles of treatment, 5 pts progressed (recurrence or death) within two years from time of surgery (4 distant and 1 local recurrence), yielding the 2-year recurrence-free-survival rate of 90.3% (95%CI 81.0%, 99.6%). The most common toxicities during DOX+CAR were grade 1 fatigue in 50 pts (92.6%), grade 1 anemia in 44 pts (81.5%), and grade 3/4 neutropenia in 16 pts (29.6%); these pts received GCSF support with subsequent cycles; febrile neutropenia occurred in 1 pt (1.9%) in this group. Only 10 pts (18.5%) had grade 1 alopecia, 5 pts (9.3%) had grade 2 alopecia, 2 pts (3.7%) had grade 3 thrombocytopenia. There were no delays in treatment due to cardiotoxicity or complications from surgical healing. Of the 37 pts who had residual disease, PDX was attempted in 21 pts, and 16 (76%) PDX were established, including those for 4 patients experiencing recurrence. WGS and RNA sequencing data from pre-treatment and residual disease samples will be analyzed and reported.Conclusion: 4 cycles of neoadjuvant DOX+CAR achieved rate of pCR similar to standard regimens and has good tolerability. Post chemotherapy PDX is feasible and may help identify targeted approaches for patients with resistant disease. These results warrant further evaluation of this combination for early stage TNBC. Citation Format: Nancy Chan, Shou-en Lu, Yue Wang, Gregory M Riedlinger, Coral Omene, Mridula George, Jyoti Malhotra, Maria Kowzun, Firas G Eladoumikdachi, Lindsay B Potdevin, Shicha Kumar, Kant Matsuda, Shruti Desai, Nayana Patel, Deborah L Toppmeyer, Shridar Ganesan, Kim Hirshfield. Neoadjuvant liposomal doxorubicin and carboplatin is effective and tolerable for the treatment of early stage triple negative breast cancer [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 P2-12-15.

Background:ER positive, HER2-negative breast cancer is the most common subtype in both early and late stages. Previous studies have demonstrated a subset of patients may derive benefit from immune check point inhibitors in combination with endocrine therapy. We hypothesize that Pembrolizumab in combination with fulvestrant will demonstrate anti-tumor activity measured by frequency of response. Trial Design: This is a non-randomized, multi-site, open-label Phase II trial for subjects with metastatic, hormone receptor positive, HER2 negative breast cancer. The study will enroll 47 patients to evaluate the anti-tumor activity of pembrolizumab with fulvestrant as measured by RECIST 1.1 and irRECIST tumor response and by progression free survival. (ClinicalTrials.gov Identifier: NCT03393845) Eligibility Criteria: Patients with advanced non-resectable/metastatic breast cancer that is estrogen receptor positive (ER+) and/or (PR+), HER2 negative (HER2-), with no more than two lines of prior hormonal therapy or no more than two lines of prior chemotherapy. Specific aims: To evaluate the overall response rate (ORR) of pembrolizumab plus fulvestrant in patients with hormone receptor positive, HER2 negative advanced/metastatic breast cancer. Secondary aims include safety and tolerability of the combination. Tumor genomic profiling on solid and/or liquid specimens using next generation sequencing assays to help identify biomarkers of response to immunotherapy. Statistical Methods: This is a phase II study that will enroll a total of 47 patients to evaluate the anti-tumor activity of pembrolizumab with fulvestrant as measured by RECIST 1.1 tumor response and by progression free survival in patients with breast cancer. For maximal 47 patients, 21 will be accrued during stage I and 26 during stage II. If 6 or fewer responses are observed during stage I, then the trial is stopped early for futility. If there are 7 or more responses during stage I, the trial will go on to stage II, in which an additional 26 patients will be accrued. If a total of 21 or more responses are observed by the end of stage II, then the trial will be considered successful, and the combination therapy considered worthy of further investigation. Present accrual and target accrual: The study is currently enrolling patients at Rutgers Cancer Institute of New Jersey, Michigan State University, and University of Nebraska. 20 of 47 patients have been enrolled on study to date. Citation Format: Nancy Chan, Dirk Moore, Pavankumar Tandra, Jatin Rana, Coral Omene, Mridula George, Jairam Krishnamurthy, Shruti Desai, Nayana Patel, Yue Wang, Marc Bicomong, Xiao-wei Tan, Kim Hirshfield, Shridar Ganesan, Deborah Toppmeyer. A phase II study of pembrolizumab plus fulvestrant in hormone receptor positive, HER-2 negative advanced/metastatic 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 OT1-17-02.

Background: Triple negative breast cancer (TNBC) is an aggressive subtype accounting for 15% of all breast cancer subtypes. It is characterized by larger tumor size, higher grade, early peak of recurrence, and a worse 5-year overall survival rate compared to other breast cancer subtypes. Treatment with immunotherapy has been shown to be beneficial in PD-L1 positive patients in the metastatic setting. Priming the tumor environment can upregulate PD-L1 expression. Pelareorep, a proprietary isolate of the unmodified replication competent reovirus type 3 Dearing (T3D) strain, a non-enveloped reovirus has been shown to upregulate PD-L1 expression in tumor and inflammatory cells and promote a favorable CD8:regulatory T cell ratio indicating a less immunosuppressive tumor microenvironment. Retifanlimab (INCMGA00012) is PD-1 inhibitor currently in development. A phase 2 study is currently underway studying the combination with the rationale that the administration of pelareorep will prime the tumor microenvironment for enhanced tumor response to PD-1 inhibitor retifanlimab. Trial design: This is a phase II multi-site single-arm clinical trial to study the combination of PD-1 inhibitor retifanlimab and the oncolytic virus pelareorep in patients with metastatic triple negative breast cancer. Eligible patients will receive pelareorep 4.5x1010 TCID50/day, on Days 1, 2, 15 and 16 and retifanlimab 500 mg on day 3 of every 28-day cycle. Patients will be monitored clinically and radiologically for response to treatment. Tumor tissue, stool and blood samples will be collected during treatment to evaluate changes in PD-L1 expression, gut microbiome and inflammatory cells induced by the study drugs. (ClinicalTrials.gov Identifier: NCT04445844) Eligibility criteria: Premenopausal/postmenopausal women with metastatic TNBC who have previously received 1-2 prior lines of chemotherapy in the metastatic setting are eligible for the study. They must have adequate performance status (ECOG PFS 0-2). Specific aims: Primary endpoint will be objective response rate (ORR) and safety, determined by the number, frequency, duration, and severity of AEs using CTCAE v5.0. The Secondary end-points will be progression free survival (PFS), overall survival (OS), duration of response (DOR) and quality of life measures using EORTC QLQ-C30. Statistical methods: Simon’s optimal 2-stage design will be used to calculate sample size. In the first stage, 14 patients will be accrued. If there are 1 or fewer responses in these 14 patients, the study will be stopped. Otherwise, 11 additional patients will be accrued for a total of 25. The null hypothesis will be rejected if 4 or more responses are observed in 25 patients. The first 6 patients will be enrolled in a staggering interval for the safety run-in phase of the study. Accrual: The study is currently enrolling patients at Rutgers Cancer Institute of New Jersey and Ohio State University Comprehensive Cancer Center. There are no safety signals with the combination in patients who have received both drugs to date. Citation Format: Mridula George, Nicole Williams, Coral Omene, Nancy Chan, Shou-En Lu, Danielle Tang, Grey Wilkinson, Shridar Ganesan, Deborah Toppmeyer. Irene study: Phase 2 study of Incmga00012 and the oncolytic virus pelareorep in metastatic triple negative breast cancer [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 OT2-25-01.

Background: Black women have higher incidence rates of TNBC and may experience worse clinical outcomes compared with White women with TNBC, due to disparities, comorbidities, or differences in TNBC biology (Dietze EG, et al. Nat Rev Cancer. 2015;15:248-254). As a result, Black pts with TNBC may benefit from novel therapies to improve outcomes. SG is an antibody-drug conjugate composed of an anti-Trop-2 antibody coupled to the cytotoxic SN-38 payload via a proprietary, hydrolyzable linker. SG received FDA approval for pts with mTNBC who received ≥2 prior chemotherapies (at least 1 in the metastatic setting). The confirmatory phase 3 ASCENT study (NCT02574455) in pts treated in second line or greater (2L+) mTNBC setting demonstrated a significant survival benefit of SG over single-agent chemotherapy treatment of physician’s choice (TPC; median progression-free survival, [PFS]: 4.8 vs 1.7 mo, HR 0.43, P Citation Format: Lisa A. Carey, Amelia Zelnak, Hope S. Rugo, Florence Dalenc, Rita Nanda, Michael Danso, Mahasti Saghatchian, Kevin Kalinsky, Nelly Firmin, Manuel Ruiz-Borrego, Anne Favret, Jun Sun, Lee Schwartzberg, Christie Hilton, Coral Omene, Robyn Young, Sara A. Hurvitz, Eliza Harting, See Phan, Aditya Bardia. Assessment of sacituzumab govitecan (SG) in Black patients (pts) from the phase 3 ASCENT study in metastatic triple-negative breast cancer (mTNBC) [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 P5-16-07.

Adjuvant therapy has become a crucial part in reducing risk of recurrence after surgical intervention. Bisphosphonates such as zoledronic acid have been used as part of adjuvant therapy. Among its many side-effects, zoledronic acid also carries a risk of uveitis. Uveitis remains one of many lesser-known side-effects; prompt recognition is essential to ensure patients receive appropriate and timely care to help prevent permanent vision loss. We report a case of anterior uveitis in a post-menopausal woman who presented with visual symptoms after receiving the first dose of zoledronic acid. This case report serves to educate and increase awareness of the risk of uveitis in patients who are given zoledronic acid. This is the first and only reported case of zoledronic acid, when used in the adjuvant setting for the treatment of breast cancer.

PURPOSE: Limited epidemiologic data are available on the expression of adipokines leptin (LEP) and adiponectin (ADIPOQ) and adipokine receptors (LEPR, ADIPOR1, ADIPOR2) in the breast tumor microenvironment (TME). The associations of gene expression of these biomarkers with tumor clinicopathology is not well understood. METHODS: NanoString multiplexed assays were used to assess the gene expression levels of LEP, LEPR, ADIPOQ, ADIPOR1, and ADIPOR2 within tumor tissues among 162 Black and 55 White women with newly diagnosed breast cancer. Multivariate mixed effects models were used to estimate associations of gene expression with breast tumor clinicopathology (overall and separately among Blacks). RESULTS: Black race was associated with lower gene expression of LEPR (P=0.002) and ADIPOR1 (P=0.01). Lower LEP, LEPR, and ADIPOQ gene expression were associated with higher tumor grade (P=0.0007, P

Background The molecular mechanisms underlying the association of overall and central body fatness with poorer breast cancer outcomes remain unclear; altered gene and/or protein expression of the adipokines and their respective receptors in the breast tumor microenvironment may play a role. Methods In a sample of Black and White women with primary invasive breast cancer, we investigated associations of body mass index (BMI), waist circumference, hip circumference, waist-to-hip ratio (WHR), fat mass index, and percent body fat with protein expression (log-transformed, n = 722) and gene expression (log2-transformed, n = 148) of leptin receptor (LEPR) and adiponectin receptors 1 and 2 (ADIPOR1, ADIPOR2). Multivariable linear models, adjusting for race, menopausal status, and estrogen receptor status, were used to assess these associations, with Bonferroni correction for multiple comparisons. Results In multivariable models, we found that increasing BMI (β = 0.0028, 95% CI: 0.0011, 0.0045), waist circumference (β = 0.0013, 95% CI: 0.0005, 0.0022), hip circumference (β = 0.0015, 95% CI: 0.0007, 0.0024), and fat mass index (β = 0.0041, 95% CI: 0.0015, 0.0067) were associated with higher LEPR protein expression. These findings reflect a 16.8%, 17.6%, 17.7%, 17.2% increase in LEPR protein expression for each standard deviation increase in BMI, waist circumference, hip circumference, and fat mass index, respectively. These associations were stronger among White and postmenopausal women and ER + cases; formal tests of interaction yielded evidence of effect modification by race. We found no associations of any measure of body fatness with LEPR gene expression or with gene or protein expression of ADIPOR1 and ADIPOR2. Conclusions These findings support an association of increased body fatness – beyond overall body size measured using BMI – with higher LEPR protein expression in breast tumor tissues. Clarifying the impact of adiposity-related adipokine receptor expression in breast tumors on long-term breast cancer outcomes is a critical next step.

Triple negative breast cancer (TNBC) is a high-risk and aggressive malignancy characterized by the absence of estrogen receptors (ER) and progesterone receptors (PR) on the surface of malignant cells, and by the lack of overexpression of human epidermal growth factor 2 (HER2). It has limited therapeutic options compared to other subtypes of breast cancer. There is now a growing body of evidence on the role of immunotherapy in TNBC, however much of the data from clinical trials is conflicting and thus, challenging for clinicians to integrate the data into clinical practice. Landmark phase III trials using immunotherapy in the early-stage neoadjuvant setting concluded that the addition of immunotherapy to chemotherapy improved the pathologic complete response (pCR) rate compared to chemotherapy with placebo while others found no significant improvement in pCR. Phase III trials have investigated the utility of immunotherapy in previously untreated metastatic TNBC, and these studies have similarly arrived at inconsistent conclusions. Some studies showed no benefit while others demonstrated a clinically significant improvement in overall survival in the PD-L1 positive population. It is not yet clear which biomarkers are most useful, and assays for these biomarkers have not been standardized. Given the often serious and severe side effects of immunotherapy, it is important and necessary to identify predictive biomarkers of response and resistance in order to enhance patient selection. In this review, we will discuss both the challenges of traditional biomarkers and the opportunities of emerging biomarkers for patient selection.

Targeted therapies such as Cyclin Dependent Kinase 4 and 6 (CDK 4/6) inhibitors have improved the prognosis of metastatic hormone receptor (HR) positive breast cancer by combating the resistance seen with traditional endocrine therapy. The three approved agents currently in the market are palbociclib, ribociclib and abemaciclib. Besides the overall similarities associated with CDK4/6 inhibition, there are differences between the three approved agents that may explain the differences noted in unique clinical scenarios- monotherapy, patients with brain metastases or use in the adjuvant setting. This review article will explore the preclinical and pharmacological differences between the three agents and help understand the benefits seen with these agents in certain subgroups of patients with metastatic HR positive breast cancer.

Importance Obesity disproportionately affects Black women, who also have a higher risk of death after a breast cancer diagnosis compared with women of other racial/ethnic groups. However, few studies have evaluated the association of measures of adiposity with mortality among Black breast cancer survivors. Objective To assess the association of measures of adiposity with survival after a breast cancer diagnosis among Black women. Design, Setting, and Participants This prospective population-based cohort study comprised 1891 women with stage 0 to IV breast cancer who self-identified as African American or Black and were ages 20 to 75 years. The New Jersey State Cancer Registry was used to identify women living in 10 counties in New Jersey who were recruited from March 1, 2006, to February 29, 2020, and followed up until September 2, 2020. Exposures Measures of adiposity, including body mass index, body fat distribution (waist circumference and waist-to-hip ratio), and body composition (percent body fat and fat mass index), were collected during in-person interviews at approximately 10 months after breast cancer diagnosis. Main Outcomes and Measures All-cause and breast cancer–specific mortality. Results Among 1891 women, the mean (SD) age at breast cancer diagnosis was 54.5 (10.8) years. During a median follow-up of 5.9 years (range, 0.5-14.8 years), 286 deaths were identified; of those, 175 deaths (61.2%) were associated with breast cancer. A total of 1060 women (56.1%) had obesity, and 1291 women (68.3%) had central obesity. Higher adiposity, particularly higher waist-to-hip ratio, was associated with worse survival. Women in the highest quartile of waist-to-hip ratio had a 61% increased risk of dying from any cause (hazard ratio [HR], 1.61; 95% CI, 1.12-2.33) and a 68% increased risk of breast cancer death (HR, 1.68; 95% CI, 1.04-2.71) compared with women in the lowest quartile. The risks of all-cause and breast cancer–specific death were similarly high among women in the highest quartile for waist circumference (HR, 1.74 [95% CI, 1.26-2.41] and 1.64 [95% CI, 1.08-2.48], respectively), percent body fat (HR, 1.53 [95% CI, 1.09-2.15] and 1.81 [95% CI, 1.17-2.80]), and fat mass index (HR, 1.57 [95% CI, 1.11-2.22] and 1.74 [95% CI, 1.10-2.75]); however, the risk was less substantial for body mass index (HR, 1.26 [95% CI, 0.89-1.79] and 1.33 [95% CI, 0.84-2.10]). In analyses stratified by estrogen receptor status, menopausal status, and age, a higher waist-to-hip ratio was associated with a higher risk of all-cause death among women who had estrogen receptor–negative tumors (HR, 2.24; 95% CI, 1.14-4.41), women who were postmenopausal (HR, 2.15; 95% CI, 1.28-3.61), and women who were 60 years or older at diagnosis (HR per 0.10-U increase, 1.76; 95% CI, 1.37-2.26). Conclusions and Relevance In this population-based cohort study, central obesity and higher adiposity were associated with higher all-cause and breast cancer–specific mortality among Black breast cancer survivors. Simple measures of body fat distribution and body composition were found to be useful tools for identifying Black women with a higher risk of death after a breast cancer diagnosis.

Background: The use of neoadjuvant platinum with taxane for triple negative breast cancer (TNBC) has gained increased attention for improving rates of pathologic complete response (pCR). Our prior trial combining carboplatin (CAR) with liposomal doxorubicin (DOX) for metastatic TNBC showed good response rates with minimal side effects while allowing for greater platinum dosing compared to a taxane combination. We hypothesized that the doublet of DOX+CAR is effective and tolerable in the neoadjuvant setting for TNBC and that tumor genomics may aid in determining those patients most likely to benefit. Methods: A phase II single arm trial was conducted for patients (pts) diagnosed with stage II-III TNBC. Patients received 4 cycles of neoadjuvant carboplatin (AUC 5) and liposomal doxorubicin (30mg/m2) administered every 28 days, then underwent definitive breast surgery followed by 12 weeks of adjuvant paclitaxel 80 mg/m2 administered weekly. Primary and secondary clinical endpoints were rate of pCR and two year recurrence free survival (RFS) and overall survival (OS), respectively. Cardiac safety of the combination was assessed. Fresh residual tumor samples were obtained at time of surgery for generation of patient derived xenografts (PDX). Tumor genomic profiling was done to determine the mutational spectrum, association of this spectrum in primary tumors with achieving pCR, and identifying alternative treatment strategies for PDX evaluation for patients with resistant disease. Results: From 2/2015 to 5/2018, 36 pts were enrolled and 32 completed treatment; 4 pts await definitive surgery; 12 (33%) are two years from diagnosis. Median age of the cohort was 53 years. There was high participation by under-represented groups: 23% African American, 20% Asian, 14% Hispanic. Most histologies were invasive ductal but included apocrine, pleomorphic lobular, and metaplastic subtypes. Of the 32 pts who completed surgery, 34% (11) achieved pCR and 64% (23) had clinical response on serial physical exam. At 2 years, there were 2 distant and 1 local recurrence. The most common toxicities during DOX+CAR were grade 1 nausea in 19 pts (53%), grade 3/4 neutropenia occurred in 10 pts (28%); these pts received GCSF support with subsequent cycles; febrile neutropenia occurred in 1 pt (3%) in this group. Grade 3 thrombocytopenia (2 pts), pruritis (1 pt), and mucositis (1 pt) were observed. Only 6 pts (17%) had grade 1 alopecia. There were no delays in treatment due to cardiotoxicity or complications from surgical healing. TP53 (93%), PI3K/PTEN (26.6%), and NOTCH (20%) were the most commonly altered pathways. Structural variants, such as amplifications, rearrangements, and frameshifts were the most frequent alterations detected. Of the 25 pts who had residual disease, PDX was attempted from 14 pts, and 10 (71%) PDX were established, including those for all 3 patients experiencing recurrence. Conclusion: Neoadjuvant DOX+CAR demonstrated good efficacy and tolerability. Post-chemotherapy PDX is feasible and may help identify targeted approaches for patients with resistant disease. These results warrant further evaluation of this combination for early stage TNBC. Citation Format: Chan N, Riedlinger GM, Lu S-e, Pham KT, Kirstein LJ, Eladoumikdachi FG, George MA, Potdevin LB, Kowzun MJ, Desai SA, Tang DM, Omene CO, Wong ST, Rodriguez-Rust L, Kumar S, Kearney TJ, Liu C, Ganesan S, Toppmeyer DL, Hirshfield KM. Neoadjuvant liposomal doxorubicin and carboplatin is effective and tolerable for the treatment of triple negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-15-14.

Because the incidence of breast cancer increases decades after ionizing radiation exposure, aging has been implicated in the evolution of the tumor microenvironment and tumor progression. Here, we investigated radiation-induced carcinogenesis using a model in which the mammary glands of 10-month-old BALB/c mice were transplanted with Trp53-null mammary tissue 3 days after exposure to low doses of sparsely ionizing γ-radiation or densely ionizing particle radiation. Mammary transplants in aged, irradiated hosts gave rise to significantly more tumors that grew more rapidly than those in sham-irradiated mice, with the most pronounced effects seen in mice irradiated with densely ionizing particle radiation. Tumor transcriptomes identified a characteristic immune signature of these aggressive cancers. Consistent with this, fast-growing tumors exhibited an immunosuppressive tumor microenvironment with few infiltrating lymphocytes, abundant immunosuppressive myeloid cells, and high COX-2 and TGFβ. Only irradiated hosts gave rise to tumors lacking cytotoxic CD8+ lymphocytes (defined here as immune desert), which also occurred in younger irradiated hosts. These data suggest that host irradiation may promote immunosuppression. To test this, young chimera mice were fed chow containing a honeybee-derived compound with anti-inflammatory and immunomodulatory properties, caffeic acid phenethyl ester (CAPE). CAPE prevented the detrimental effects of host irradiation on tumor growth rate, immune signature, and immunosuppression. These data indicated that low-dose radiation, particularly densely ionizing exposure of aged mice, promoted more aggressive cancers by suppressing antitumor immunity. Dietary intervention with a nontoxic immunomodulatory agent could prevent systemic effects of radiation that fuel carcinogenesis, supporting the potential of this strategy for cancer prevention.

Background The molecular mechanisms underlying the association between increased adiposity and aggressive breast cancer phenotypes remain unclear, but likely involve the adipokines, leptin (LEP) and adiponectin (ADIPOQ), and their receptors (LEPR, ADIPOR1, ADIPOR2). Methods We used immunohistochemistry (IHC) to assess LEP, LEPR, ADIPOQ, ADIPOR1, and ADIPOR2 expression in breast tumor tissue microarrays among a sample of 720 women recently diagnosed with breast cancer (540 of whom self-identified as Black). We scored IHC expression quantitatively, using digital pathology analysis. We abstracted data on tumor grade, tumor size, tumor stage, lymph node status, Ki67, estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) from pathology records, and used ER, PR, and HER2 expression data to classify breast cancer subtype. We used multivariable mixed effects models to estimate associations of IHC expression with tumor clinicopathology, in the overall sample and separately among Blacks. Results Larger proportions of Black than White women were overweight or obese and had more aggressive tumor features. Older age, Black race, postmenopausal status, and higher body mass index were associated with higher LEPR IHC expression. In multivariable models, lower LEPR IHC expression was associated with ER-negative status and triple-negative subtype (P Conclusions Lower LEPR IHC expression within the breast tumor microenvironment might contribute mechanistically to inter-individual variation in aggressive breast cancer clinicopathology, particularly ER-negative status and triple-negative subtype.

Purpose: Pathologic complete response (pCR) to neoadjuvant chemotherapy is associated with improved event-free and distant recurrence free survival in women with stage II or III breast cancer (BC). While there is strong evidence that obesity is associated with reduced BC survival, its role on pCR after neoadjuvant chemotherapy among BC patients is unclear. Moreover, data regarding the association of pre-treatment BMI with response to neoadjuvant chemotherapy in Black women – who have the highest obesity prevalence than any other racial/ethnic groups and 40% worse survival outcomes compared to White women – are lacking. The purpose of this study was to examine the association of pretreatment BMI and subsequent pCR after neoadjuvant chemotherapy in Black BC patients. Methods: Data were abstracted from medical records of 131 Black BC patients who received neoadjuvant chemotherapy in the Women's Circle of Health Follow-up Study, a population-based cohort study of Black BC survivors in New Jersey. pCR was defined as absence of detectable invasive cancer in the breast and lymph nodes (ypT0/Tisand ypN0) at the time of surgery (noninvasive residual BC, i.e. DCIS was allowed). Tumor subtypes were defined by hormone receptor (HR, i.e., ER, PR) and HER2 status. Multivariable logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CIs) of the association between pretreatment obesity (BMI ≥30 kg/m2)vs. non-obese ( Citation Format: Coral Omene, Saber Amin, Nur Zeinomar, Bo Qin, Dhanya Chanumolu, Adana A.M. Llanos, Chi-Chen Hong, Kitaw Demissie, Elisa V. Bandera. Association of body mass index with pathological complete response after neoadjuvant chemotherapy in a population-based cohort of Black breast cancer patients [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-119.

CAPE is the major active component of propolis, a widely available, safe, honeybee natural product with anti-inflammatory, antioxidant, and antitumor properties. We have previously shown diverse effects of CAPE in breast cancer. We postulated that CAPE may be useful in chemoprevention for women at high risk for triple-negative breast cancers (TNBC) and evaluated this in a radiation chimera mouse model in which the tumor spectrum is shifted to TNBC. The radiation chimera model consists of surgically clearing the mammary epithelium from the inguinal glands of 3-week old BALB/c mice, the mice were irradiated with 1 Gy or sham irradiated at 10–12 weeks of age, and bilaterally transplanted 3 days later with syngeneic Trp53 null mammary fragments. Mice were placed on a CAPE diet or a control diet at 1 month post transplantation that was maintained for the course of the experiment. Mammary tumor development was monitored by palpation for up to 18 months. The first tumor was resected at 1cm3 and mice monitored for tumor recurrence or second tumor formation. Tumors were immunostained for estrogen receptor (ER) status and ER negative tumors were selected from all the groups for RNA sequencing. No difference in body weight or tumor incidence was observed between mice on the CAPE versus control diet. Host irradiation significantly accelerated tumor growth rate compared to the control sham irradiated mice. CAPE treatment blocked this effect, but did not affect the control tumor growth rate. Resected tumors in CAPE treated mice recurred at significantly longer intervals (average of 40 days in the control group versus 90 days with CAPE treatment) and much less frequently than tumors from mice on a control diet. Mean expression analysis showed that CAPE induces a distinct gene expression pattern in ER negative tumors from irradiated mice. As previously described, the transcriptional profile of ER negative tumors was enriched in immune response genes in tumors from nonirradiated mice on a control diet, while irradiation suppresses these genes. Interestingly, this difference was abrogated in mice on the CAPE diet where we found that CAPE reverses the suppression of immune response genes in the ER negative tumors from the irradiated mice. These findings support the potential use of CAPE to modify the aggressive behavior of TNBC. This may be due to effects on the immune system in which CAPE acts to re-establish anti tumor immunity. Thus, CAPE may be useful in chemoprevention both for women at high risk for TNBC and to prevent or delay TNBC breast cancer recurrence. Citation Format: Omene C, Patel M, Illa Bochaca I, Barcellos-Hoff MH. Modification and reversal of the aggressive behavior of triple negative breast cancer by caffeic acid phenethyl ester (CAPE) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-06-04.

Background: Few studies have empirically tested the association of allostatic load (AL) with breast cancer clinicopathology. The aim of this study was to examine the association of AL, measured using relevant biomarkers recorded in medical records before breast cancer diagnosis, with unfavorable tumor clinicopathologic features among Black women. Methods: In a sample of 409 Black women with nonmetastatic breast cancer who are enrolled in the Women's Circle of Health Follow-Up Study, we estimated prediagnostic AL using two measures: AL measure 1 [lipid profile–based—assessed by systolic and diastolic blood pressure (SBP, DBP), high-density lipoprotein, low-density lipoprotein, total cholesterol, triglycerides, and glucose levels; waist circumference; and use of diabetes, hypertension, or hypercholesterolemia medication] and AL measure 2 (inflammatory index–based—assessed by SBP, DBP, glucose, and albumin levels; estimated glomerular filtration rate; body mass index; waist circumference; and use of medications previously described). We used Cohen's statistic to assess agreement between the two AL measures and multivariable logistic models to assess the associations of interest. Results: AL measures 1 and 2 moderately agreed (κ = 0.504). Higher prediagnostic AL predicted higher grade (poorly differentiated vs. well/moderately differentiated) using AL measure 1 [OR = 2.16; 95% confidence interval (CI), 1.18–3.94] and AL measure 2 (OR = 1.60; 95% CI, 1.02–2.51), and larger tumor size (≥2 cm vs. Conclusions: Elevated prediagnostic AL might contribute to more unfavorable breast cancer clinicopathology. Impact: Addressing elevated prediagnostic levels of AL has potentially important clinical implications.

Background: Triple negative breast cancer (TNBC) is an aggressive subtype accounting for 15% of all breast cancer cases. It is characterized by larger tumor size, higher grade, early peak of recurrence, and a worse 5-year overall survival rate compared to other breast cancer subtypes. Chemotherapy serves as the backbone for the treatment of metastatic TNBC. Treatment with immunotherapy in combination with Abraxane, a taxane-based chemotherapy, is of benefit only in PD-L1 positive tumors, which represents a minority of the patients. Pelareorep, a proprietary isolate of the unmodified, replication competent reovirus type 3 Dearing (T3D), has been shown to upregulate PD-L1 expression in tumor and inflammatory cells and downregulate intra-tumoral regulatory T-cells in the tumor microenvironment in pre-clinical and early clinical studies. Retifanlimab is a PD-1 inhibitor currently in development. The rationale for this clinical study is that the administration of pelareorep will prime the tumor microenvironment for enhanced tumor response to PD-1 inhibitor retifanlimab.Trial design: This is a phase II multi-site single-arm clinical trial to study the combination of PD-1 inhibitor retifanlimab and the oncolytic virus Pelareorep in metastatic triple negative breast cancer who have progressed on chemotherapy. Eligible patients will receive pelareorep 4.5x1010 TCID50 /day IV, on Days 1, 2, 15 and 16 and retifanlimab 500mg IV on day 3 of every 28-day cycle until disease progression or unacceptable toxicity. Patient will be monitored clinically and radiologically for response to treatment. Tumor tissue, stool and blood samples will be collected while on treatment to evaluate changes in PD-L1 expression, gut microbiome and inflammatory cells induced by the study drugs. (ClinicalTrials.gov Identifier: NCT04445844) Eligibility criteria: Eligible patients will include premenopausal/postmenopausal women with metastatic TNBC who have previously received 1-2 prior lines of chemotherapy in the metastatic setting. ECOG PFS 0-2. Specific aims: Primary endpoint will be objective response rate (ORR) and safety, determined by the number, frequency, duration, and severity of AEs using CTCAE v5.0. The secondary end-points will be progression free survival (PFS), overall survival (OS) and duration of response (DOR) and quality of life measures using EORTC QLQ-C30. Statistical methods: Simon’s optimal 2-stage design will be used to calculate sample size. In the first stage, 14 patients will be accrued. If there are 1 or fewer responses in these 14 patients, the study will be stopped. Otherwise, 11 additional patients will be accrued for a total of 25. The null hypothesis will be rejected if 4 or more responses are observed in 25 patients. The first 6 patients will be enrolled in a staggering interval for the safety run-in phase of the study. Accrual: The study will enroll up to 25 patients at Rutgers Cancer Institute of New Jersey and Ohio State University Comprehensive Cancer Center Contact information: Mridula George, MD Email: mridula@cinj.rutgers.edu Citation Format: Mridula George, Nicole Williams, Maryam Lustberg, Coral Omene, Nancy Chan, Nisha Ohri, Maria Kowzun, Lindsay Potdevin, Firas Eladoumikdachi, Shicha Kumar, Robert Wesolowski, Grey Wilkinson, Danielle Tang, Sinae Kim, Shridar Ganesan, Bruce Haffty, Deborah Toppmeyer. Irene study: Phase 2 study of incmga00012 (retifanlimab)and the oncolytic virus pelareorep in metastatic 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 OT-32-02.

CAPE is the major active component of propolis, a widely available, non-toxic, honeybee natural product with anti-inflammatory, antioxidant, and antitumor properties. We have previously shown that CAPE inhibits growth of breast cancer cells and the tumorigenic potential of breast cancer stem cells. We have identified inhibition of histone deacetylase (HDAC) as one mechanism of action, which suggests that it mediates its effects through epigenetic modifications. We postulated that CAPE may be useful in chemoprevention for women at high risk for triple-negative breast cancers since the cell-of-origin hypothesis states that these cancers likely arise from transformation of mammary stem or progenitor cells, whose self-renewal is maintained via epigenetic states. We tested the effect of CAPE on wildtype (WT) and Trp53 null mammary stem cell (MaSC) self-renewal from BALB/c mice cultured as mammospheres (MMS). Primary mammary epithelial cells were cultured as MMS for 7 days, dissociated into single cells, re-cultured in the presence of CAPE for 7 days and passaged in secondary and tertiary passages without CAPE. MMS frequency and differentiation potential was analyzed using immunofluorescence detection of luminal marker, cytokeratin 18, basal marker, cytokeratin 14, and progesterone receptor (PR). Chromatin states were identified using ATAC-seq and open chromatin areas unique to CAPE treated murine MMS were used for pathway analysis performed by Ingenuity Pathway Analysis, Gene Set Enrichment Analysis and confirmed by Integrative Genome Viewer. CAPE treatment resulted in a dose dependent decrease in both WT and p53 null mammosphere forming efficiency that persisted in secondary and tertiary passages, suggesting reduced self-renewal. CAPE treatment also shifted differentiation from predominantly basal K14 to luminal K18-positive in both WT and p53 null MMS and increased PR expression in WT MMS. ATAC-seq of CAPE treated WT MMS showed significant pathway enrichment for p53 signaling, SOX2 signaling, and enrichment of open chromatin for several genes including the SMARCA4 gene, which regulates transcription of genes involved in stem cell renewal. ATAC-seq of CAPE treated Trp53 null MMS showed that genes defining early and late response to estrogen were particularly important. Significant canonical pathways included Aryl hydrocarbon receptor signaling, whose upregulation results in inhibition of self renewal and has been targeted as a potential drug target for estrogen receptor negative breast cancer. The integrin signaling pathway was also highly enriched. These data suggest that CAPE both inhibits MaSC self-renewal and shifts the lineage commitment to a luminal, ER + lineage. ATAC-seq demonstrated genomic effects that are important in differentiation, SC renewal and adhesion. These data suggest that CAPE may have an effect on lineage commitment in support of our chemoprevention strategy to reduce triple-negative breast cancer. Citation Format: Omene C, Patel M, Kannan K, Heguy A, Barcellos-Hoff MH. Mammary stem cell modulation of wildtype and Trp53 null stem cells by CAPE (caffeic acid phenethyl ester), a potential therapeutic agent. [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 P1-06-06.

e19074 Background: Estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) are determinants of both treatment and mortality for patients with breast cancer (BC). With a mortality to incidence ratio of 0.51, mortality rates for breast cancer in East Africa are among the world’s highest. Building on a meta-analysis of BC receptor subtypes in sub-Saharan Africa (2014) and including all recently available PubMed literature, our meta-analysis aims to investigate the differences among receptor subtype distribution in five East African countries. Methods: Receptor distributions for female samples >30 in all BC articles for Ethiopia, Kenya, Rwanda, Tanzania and Uganda were obtained from PubMed, January 1, 1998 - June 30, 2019. Outcomes were proportions of ER+, PR+, and HER2+ and/or molecular subtypes. Data included study characteristics and mean/median patient age (weighted by observations). Using metaprop for STATA 16, we estimated pooled proportions (ES) with 95% confidence intervals (CI) and assessed I2 statistics for heterogeneity across studies. Results: Among 35 BC studies with receptor data, 21 met criteria. Overall, the weighted mean age was 47.5 years (SD 3.2) and median, 48. For ER+ (18 studies/n=2,875), the overall ES was 0.55 (95% CI 0.47, 0.62), I2 of 94.4%, p2 of 70.2%, p2 of 84.0%, p

Obesity increases the risk of breast cancer and decreases the survival of patients with breast cancer, particularly for postmenopausal, oestrogen receptor-positive breast cancer. New findings now add to the evidence for the detrimental effects of obesity in patients with breast cancer by showing that obesity can cause resistance to therapy.

BACKGROUND: RAD001 is an oral mTOR inhibitor that has exhibited activity in breast cancer. Triple negative breast cancer cells are unable to repair double stranded DNA breaks and hence have sensitivity to platinum agents that cause interstrand cross-links. Rapamycin acts synergistically with platinum agents to induce apoptosis and inhibit proliferation in at least two different breast cancer cell lines (including ER/PR negative cell lines). We propose that combination RAD001 and carboplatin may have activity in triple-negative breast cancer. MATERIALS AND METHODS: The primary objective of the study is to determine clinical benefit (complete remission (CR) + partial remission (PR) + stable disease (SD more than 6 months)) and the toxicity of this combination in triple negative metastatic breast cancer who have had 0–3 prior chemotherapy regimens for metastatic disease. Twenty-five subjects were to be entered in this Phase II study. This design has greater than 80% power to test the null hypothesis that the clinical benefit rate is less than or equal to 10% versus the alternative hypothesis that clinical benefit rate is greater than or equal to 30%. Prior carboplatin is allowed. Women with treated brain metastasis are eligible. Secondary objectives are to determine progression free survival and relationship between pretreatment sensitivity (biopsy at baseline) and clinical response (biopsy post 2 cycles) using IHC staining for abundance of key proteins in the Akt-mTOR pathway and their activity using surrogate phosphorylation site-specific antibodies. According to the original study plan, carboplatin AUC 6, was to be given intravenously every three weeks. Five mg of RAD001 was to be given daily with a 3 patient run-in and then 10 mg daily if there were no dose-limiting toxicities. Due to a surprising amount of thrombocytopenia with this combination the dose of carboplatin was first amended to AUC 5 and most recently to AUC 4 with 5 mg of RAD001 (and no plan to escalate to 10 mg). RESULTS: 23 patients of a planned 25 have been recruited thus far. Median age is 59. Of the 20 patients assessable for response at this time, there have been 1 CR, 5 PRs, 8 SDs and 6 PDs. One SD was achieved in a patient progressing on single agent Carboplatin at study entry. Median duration of CR+ SD +PR thus far is 13 weeks (range: 6–74 weeks). 5 of 22 patients assessable for toxicity had grade 3/4 thrombocytopenia and 4 patients had grade 3 neutropenia (no febrile neutropenia). 13 out of eighteen patients have had treatment held and/or dose reductions secondary to hematological toxicity, however, since amendment for starting dose of Carboplatin to AUC 4 the regimen has been very well tolerated with only 1 out of eleven patients with grade 3 neutropenia and grade 3 thrombocytopenia. 1 patient suffered from grade 3 dehydration. The estimated clinical benefit rate is 45% (95% confidence interval: 23%, 67%). Median time to progression or death is 85 days from start of treatment. CONCLUSIONS: Our study has met the primary end point of demonstrating clinical benefit in triple negative metastatic breast cancer. Dose limiting thrombocytopenia was an unexpected side effect requiring protocol amendment. We continue to accrue study subjects at the amended dosing. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-20-05.

Background: RAD001 is an oral mTOR inhibitor that has exhibited activity in breast cancer. Triple negative breast cancer cells are unable to repair double stranded DNA breaks and hence have sensitivity to platinum agents that cause interstrand cross-links. Rapamycin acts synergistically with platinum agents to induce apoptosis and inhibit proliferation in at least two different breast cancer cell lines (including ER/PR negative cell lines). We propose that combination RAD001 and carboplatin may have activity in triple-negative breast cancer. Methods: The primary objective of the study is to determine clinical benefit (complete remission (CR) + partial remission (PR) + stable disease (SD)) and the toxicity of this combination in women with triple negative metastatic breast cancer who have had 0–3 prior chemotherapy regimens for metastatic disease. Secondary objectives are to determine progression free survival as well as investigating the relationship between pretreatment sensitivity (biopsy at baseline) and clinical response (biopsy post 2 cycles) using IHC staining for abundance of key proteins in the Akt-mTOR pathway and their activity using surrogate phosphorylation site-specific antibodies (Akt and phospho-serine 473, phospho-threonine and phospho-threonine 308 Akt; mTOR and phospho-serine 2448 mTOR; ribosome protein S6 kinase (S6K) and phospho-threonine 378 S6K; 4E-BP1 and phospho-serine 65 4E-BP1). Prior carboplatin is allowed. Women with treated brain metastasis are eligible. According to the original study plan, carboplatin AUC 6, was to be given intravenously every three weeks. Five mg of RAD001 was to be given daily with a 3 patient run-in and then 10 mg daily if there were no dose-limiting toxicities. Due to a unexpected amount of thrombocytopenia with this combination the dose of carboplatin was first amended to AUC 5 and most recently to AUC 4 with 5 mg of RAD001 (and no plan to escalate to 10 mg). Results: Fourteen patients of a planned 25 have been recruited thus far. Median age is 58.5. Median number of prior regimens is 2 (0-3). Of the 7 patients assessable for response at this time, there have been 2 PR's and 5 patients with SD. One SD was achieved in a patient progressing on single agent carboplatin at study entry. Median duration of SD + PR is 28.5 weeks (5 patients have ongoing response ranging from 8–46.5 weeks). Five of 8 patients assessable for toxicity had grade 3 or 4 thrombocytopenia and 2 patients had grade 3 neutropenia. No cases of febrile neutropenia were observed. Four patients have required blood transfusion and one patient has required platelet transfusion. All patients have had treatment held and/or dose reductions secondary to hematological toxicity, however, since amended carboplatin dose the regimen has been very well tolerated with only one out of six patients) with grade 3 neutropenia and grade 3 thrombocytopenia. There have been no non-hematological grade 3 or 4 toxicities. Conclusions: Clinical benefit was observed in all 7 assessable patients. Dose limiting thrombocytopenia was an unexpected side effect requiring protocol amendment. We continue to accrue study subjects at the amended dosing. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-17-07.

Summary Cancer stem cells (CSC) are chemoresistant and implicated in tumor recurrence, metastasis and high patient mortality; thus substances impairing CSC activity, could be invaluable as novel cancer therapeutics. We previously showed that CAPE (caffeic acid phenethyl ester), a component of propolis, a honeybee product, inhibits growth of MDA-MB-231 (MDA-231) cells, mdr gene expression, NF-κB, EGFR, and VEGF. We hypothesized that CAPE also acts by interfering with CSC-mediated effects. We isolated breast CSC (bCSC) from MDA-231 cells, a model of human triple-negative breast cancer, and mouse xenografts. bCSC grow as mammospheres (MMS) and when dissociated into single cells, form MMS again, a sign of self-renewal. bCSC exhibited the characteristic CD44+/CD24-/low phenotype and generated progenitors in the presence of serum, a CSC trait responsible for regenerating tumor mass. CAPE caused dose-dependent bCSC self-renewal inhibition and progenitor formation. Clonal growth on soft agar was inhibited dose-dependently, but apoptosis was not induced as determined by Annexin-V/PI assay. Instead, bCSC were noted to significantly progress from a quiescent cell cycle state in G0/G1 (82%), S phase (12%) to a cycling state with an increase in S phase (41%) and subsequent decrease in G0/G1 (54%). Treatment of bCSC with CAPE (4.5-days) decreased CD44 levels by 95%, while another cell population containing 10-100-fold lower CD44 content concurrently increased. Results suggest that CAPE causes pronounced changes in bCSC characteristics manifested by inhibition of self renewal, progenitor formation, clonal growth in soft agar, and concurrent significant decrease in CD44 content, all signs of decreased malignancy potential.

Background: Over 30% of cancer patients routinely use alternative formulations, commonly in combination with allopathic treatment, which may be linked with fewer adverse events. This is especially true for refractory breast cancer (BC), which remains a major clinical challenge for women in whom conventional therapy has lost therapeutic benefit. Propolis (PL), and its active component Caffeic Acid Phenethyl Ester (CAPE), is a widely available honeybee product with an extended safety history credited with anti-inflammatory, antioxidant, and antitumor properties. We have previously shown that CAPE is innocuous to normal human mammary epithelial cells, but inhibits growth of MCF-7 (ER+/PR+) and MDA-231 (TNBC) cells by cell cycle arrest and apoptosis; inhibits mdr-1 gene expression, NF-kB, and VEGF. In addition, CAPE inhibits the growth and tumorigenic potential of BC stem cells derived from MDA-231 cells. These findings are reminiscent of the pleiotropic effects of histone deacetylases inhibitors (HDACi) like vorinostat, aliphatic acids and depsipeptide in models of both epithelial and hematopoietic origin. We hypothesized that CAPE, which is structurally similar to the hydroxamic acid class of HDAC inhibitors, may mediates its effects on BC through epigenetic modifications and thus, may modulate determinants such as ER, PR, Her2 neu, and EGFR. Materials & Methods: 3x10 5 cells/well of MDA-231, MCF-7, and SKBR3 (Her2+) cells were grown in 10% DMEM in 6 well tissue culture plates using 3ml of media for 12h and then incubated with different concentrations of CAPE (0-40μM) or PL (0-.05%) for 24h. Protein analysis of lysates was done by western blotting using various antibodies. Results: CAPE exposure leads to the acetylation of histone proteins in all the BC cell lines with subsequent decrease of EGFR over-expression and induction of ER in MDA-231 cells; decrease in ER and PR expression in MCF-7 cells; and decrease in p-Her 2 in SKBR3 cells. When normalized for the concentration of CAPE in PL, the HDAC inhibitory effects ofthe natural product, PL, are superior to single agent CAPE, establishing that the epigenetic effects of CAPE can be recapitulated in the natural product. CAPE's aliphatic structure is within the expected structural class of chemicals known to possess this unusual activity. Further, PL yielded similar results for ER, PR, EGFR and p-Her2 expression seen with CAPE. Discussion: The mechanism of CAPE's effects appears to lie in its ability to inhibit HDACs, making CAPE and PL a naturally occurring epigenetic therapy. PL represents an unusual formulation of a natural product that possesses the properties of an epigenetic agent, which has the broader appeal of being viewed as a ‘natural product’ possessing unique therapeutic properties seen in many of the most promising novel therapeutics being developed by the pharmaceutical industry. Further, the enhanced effects of HDAC inhibition with PL compared to CAPE alone, make it a suitable candidate for a clinical trial, especially in heavily pretreated metastatic BC patients who may likely benefit from therapy with minimal added toxicity. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-14-07.

Alternative remedies for cancer treatment is a multi-billion dollar industry. In particular, breast cancer (BC) patients use alternative and natural remedies more frequently than patients with other malignancies. Propolis is an example of a honeybee-produced naturopathic formulation, contents of which differ by geographic location. It is readily available, affordable, and in use safely since ancient times globally. Caffeic acid phenethyl ester (CAPE) is a major active component in propolis and is thought to be responsible for its varied properties, including antibacterial, antiviral, antifungal, antioxidant, anti-inflammatory and anticancer. CAPE is effective in many models of human cancer, including BC as we have previously shown. CAPE affects genes associated with tumor cell growth and survival, angiogenesis and chemoresistance. We demonstrate that these are related in part to CAPE’s role as a histone deacetylase inhibitor, a class of drugs designated as epigenetic agents that modulate the activities of oncogenes and tumor suppressor genes. CAPE and propolis, cause an accumulation of acetylated histone proteins in MCF-7 (ER+) and MDA-MB-231 (ER-/PR-/Her2-) cells with associated decreases in ER and PR in MCF-7 cells, and upregulation of ER and decrease in EGFR in MDA-231 cells. In addition, these products reduced activated phosphorylated Her 2 protein in SKBR3 (Her 2 +) cells. Interestingly, propolis, when normalized for CAPE content, appears to be more potent than CAPE alone similarly to the greater effects of complete foods than isolated components. These data provide a potential mechanistic basis for one of the oldest naturopathic agents used in medicine and cancer treatment.

CAPE is the major active component of propolis, a widely available, safe, honeybee natural product with anti-inflammatory, antioxidant, and antitumor properties. We have previously shown diverse effects of CAPE in breast cancer. We postulated that CAPE may be useful in chemoprevention for women at high risk for triple-negative breast cancers (TNBC) and evaluated this in a radiation chimera mouse model in which the tumor spectrum is shifted to TNBC. The radiation chimera model consists of surgically clearing the mammary epithelium from the inguinal glands of 3-week old BALB/c mice, the mice were irradiated with 1 Gy or sham irradiated at 10-12 weeks of age, and bilaterally transplanted 3 days later with syngeneic Trp53 null mammary fragments. Mice were placed on a CAPE diet or a control diet at 1 month post transplantation that was maintained for the course of the experiment. Mammary tumor development was monitored by palpation for up to 18 months. The first tumor was resected at 1cm3 and mice monitored for tumor recurrence or second tumor formation. Tumors were immunostained for estrogen receptor (ER) status and ER negative tumors were selected from all the groups for RNA sequencing. No difference in body weight or tumor incidence was observed between mice on the CAPE versus control diet. Host irradiation significantly accelerated tumor growth rate compared to the control sham irradiated mice. CAPE treatment blocked this effect, but did not affect the control tumor growth rate. Resected tumors in CAPE treated mice recurred at significantly longer intervals (average of 40 days in the control group versus 90 days with CAPE treatment) and much less frequently than tumors from mice on a control diet. Mean expression analysis showed that CAPE induces a distinct gene expression pattern in ER negative tumors from irradiated mice. As previously described, the transcriptional profile of ER negative tumors was enriched in immune response genes in tumors from irradiated mice on a control diet. Interestingly, this difference was abrogated in mice on the CAPE diet. These findings support the potential use of CAPE to modify the aggressive behavior of TNBC. This may be due to effects on the immune system in which CAPE acts to re-establish anti tumor immunity. Thus, CAPE may be useful in chemoprevention both for women at high risk for TNBC and to prevent or delay TNBC breast cancer recurrence. Citation Format: Coral O. Omene, Manan Patel, Irineu IllaBochaca, Mary Helen Barcellos-Hoff. Caffeic acid phenethyl ester (CAPE) reverses aggressive breast cancer in the radiation chimera model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5247.

Breast cancer (BC) patients use alternative and natural remedies more than patients with other malignancies. Specifically, 63%–83% use at least one type of alternative medicine and 25%–63% use herbals and vitamins. Propolis is a naturopathic honeybee product, and CAPE (caffeic acid phenethyl ester), is a major medicinal component of propolis. CAPE, in a concentration dependent fashion, inhibits MCF-7 (hormone receptor positive, HR+) and MDA-231 (a model of triple-negative BC (TNBC) tumor growth, both in vitro and in vivo without much effect on normal mammary cells and strongly influences gene and protein expression. It induces cell cycle arrest, apoptosis and reduces expression of growth and transcription factors, including NF-κB. Notably, CAPE down-regulates mdr-1 gene, considered responsible for the resistance of cancer cells to chemotherapeutic agents. Further, CAPE dose-dependently suppresses VEGF formation by MDA-231 cells and formation of capillary-like tubes by endothelial cells, implicating inhibitory effects on angiogenesis. In conclusion, our results strongly suggest that CAPE inhibits MDA-231 and MCF-7 human breast cancer growth via its apoptotic effects, and modulation of NF-κB, the cell cycle, and angiogenesis.

Publisher Summary Breast cancer is a disease that develops in both men and women. While there are similarities in this disease between the two genders, there are also differences. It is the most commonly diagnosed malignancy in women, second only to skin cancer, with associated immense socioeconomic ramifications. However, in men, breast cancer is rare. Is this disease biologically different in men and women? Or is it similar between the sexes with the same etiologic, prognostic, and clinical features? The data to date suggest that breast cancer in men is fundamentally identical to breast cancer in women with few exceptions. This chapter explores the classic features of breast cancer in both sexes, highlighting the differences and the similarities between them and what is as yet unknown. Despite the clear disparity in the incidence of breast cancer between the sexes, once it occurs in either a man or a woman its clinical presentation, pathologic appearance, response to treatment, and overall prognosis are not that different. Given that breast cancer in women is a prevalent disease and the second leading cause of cancer-related death, there is a great socioeconomic burden. This has led to extensive research into this disease. The risk factors, prognostic factors, and treatment algorithm have all been thoroughly explored, and clinicians have resources to draw on when treating their female patients. This is the major difference between the sexes. Breast cancer is a rarity in males; therefore, it is much less studied. Although there have been some emerging data in male breast cancer, most knowledge and treatment approaches for this disease in males come from the extrapolation of information about female patients with breast cancer.

Propolis, and its active component, CAPE, is a widely available, safe, honeybee natural product with anti-inflammatory, antioxidant, and antitumor properties. We have previously shown that CAPE, inhibits 1) growth of ER+/PR+, ER-/PR- and Her2 + breast cancer cells, 2) the tumorigenic potential of breast cancer stem cells, and 3) mediates its effects through epigenetic modifications via HDAC inhibition, resulting among others, in increased ER in ER- cells. We postulated that CAPE may be useful in chemoprevention for women at high risk for triple-negative breast cancers (TNBC). The cell-of-origin hypothesis states that these cancers arise from transformation of mammary stem cells (SC). We tested the ability of CAPE to inhibit SC self-renewal in murine mammary epithelial cells (MEC) and a human breast epithelial cell line (MCF10A). MEC isolated from BALB/c mice were cultured as mammospheres (MMS) for 7 days in ULA 96 well plates using media containing 5% FBS and 5% Matrigel. MMS were dissociated into single cells, recultured in the presence of CAPE for 7 days and passaged in secondary and tertiary passages in media alone. Alternatively, MCF10A transfected with a Let7c-miRNA reporter were sorted for Let7c-negative cells, and similarly cultured with CAPE. Murine MMS were analyzed for luminal marker, K18, basal marker K14, or progesterone receptor (PR) using immunofluorescence. Open chromatin states were identified using ATAC-seq and peaks were independently called using PeakDeck. Open chromatin areas unique to CAPE treated murine MMS were used for pathway analysis performed by Ingenuity Pathway Analysis and confirmed by Integrative Genome Viewer. CAPE treatment resulted in a dose dependent decrease in mammosphere forming efficiency that persisted in secondary and tertiary passages, indicating reduced self-renewal. CAPE shifted the cells within murine MMS from predominantly K14 to K18-positive and increased PR expression. Interestingly, ATAC-seq showed enrichment of open chromatin of the SMARCA4 gene in the exonic region. SMARCA4 is a chromatin remodeling gene that is involved in SWI/SNF complex and alters the positioning of the nucleosome, it is thought to regulate transcription of certain genes by altering the chromatin structure around those genes. In addition, SMARCA4 can bind BRCA1, and regulate the expression of the tumorigenic breast cancer SC marker, CD44. These data indicate that CAPE both inhibited mammary SC self-renewal and shifted the lineage commitment to a luminal lineage. ATAC-seq demonstrated effects at the chromatin level that may regulate the SMARCA4 gene, which controls the transcription of many genes involved in stem cell renewal, pluripotency, and embryonic development and is differentially expressed in the luminal phenotype of human MEC. Thus, CAPE may have an effect on lineage commitment in agreement with our chemoprevention strategy to reduce TNBC breast cancer development. Citation Format: Coral O. Omene, Manan Patel, Kasthuri Kannan, Adriana Heguy, Mary Helen Barcellos-Hoff. CAPE (caffeic acid phenethyl ester) induces a mammary stem cell lineage restriction to a luminal phenotype via chromatin remodeling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4232. doi:10.1158/1538-7445.AM2015-4232

108 Background: Triple-negative breast cancer cells are unable to repair double stranded DNA breaks and have sensitivity to platinum agents. Rapamycin acts synergistically with platinum agents to induce apoptosis and inhibit proliferation in breast cancer cell lines. RAD001 (oral mTOR inhibitor) and Carboplatin combination may have activity in triple-negative breast cancer. Methods: The primary objective is to estimate clinical benefit (complete remission (CR) + partial remission (PR) + stable disease (SD) lasting >6 months) and toxicity of the combination in triple negative metastatic breast cancer patients who have had 0-3 prior chemotherapy regimens. This design has > 80% power to test the null hypothesis i.e. clinical benefit rate is ≤ 10% vs. alternative hypothesis that clinical benefit rate is ≥ 30%. Prior carboplatin is allowed. Women with treated brain metastasis are eligible. Originally, carboplatin AUC 6 was to be administered every 3 weeks along with daily 5mg of RAD001 with a 3 patient run-in and then 10 mg daily. Due to a surprising amount of thrombocytopenia, the dose of carboplatin was first amended to AUC 5 and most recently to AUC 4 with 5 mg of RAD001 (no escalation to 10 mg). Results: 23 out of 25 patients have been recruited. Median age is 59. Thus far, there have been 1 CR, 5 PR’s, 8 SD's and 6 PD’s. One SD was achieved in a patient progressing on single agent carboplatin at study entry. Median duration of response is 13 weeks (range: 6-74 weeks). 5 patients had grade 3/4 thrombocytopenia and 4 patients had grade 3 neutropenia (no febrile neutropenia). 13 patients had treatment held and/or dose reductions secondary to hematological toxicity. Since dosing amendment for carboplatin to AUC 4 the regimen has been well tolerated (only 1 patient with grade 3 neutropenia and thrombocytopenia). 1 patient had grade 3 dehydration. The estimated clinical benefit rate is 45% (95% C.I.: 23%, 67%). Median time to progression or death is 85 days from start of treatment. Conclusions: Our study has met the primary end point of demonstrating clinical benefit in triple-negative metastatic breast cancer. Dose-limiting thrombocytopenia was an unexpected side effect requiring protocol amendment. Patient accrual continues at the amended dosing.

e11529 Background: Triple negative breast cancer cells are unable to repair double stranded DNA breaks and hence have sensitivity to platinum agents. Rapamycin acts synergistically with platinum agents to induce apoptosis and inhibit proliferation in breast cancer cell lines. Combination of RAD001 (oral mTOR inhibitor) and Carboplatin may have activity in triple-negative breast cancer. Methods: The primary objective of the study was to estimate the clinical benefit (complete remission (CR) + partial remission (PR) + stable disease (SD) >6 months) and the toxicity of this combination in women with triple negative metastatic breast cancer who have had 0-3 prior chemotherapy regimens for metastatic disease. 25 subjects were to be entered into a single stage open label Phase II study. Prior Carboplatin is allowed. Treated brain metastasis are eligible. The null hypothesis that the clinical benefit rate is ≤10% could be rejected if number of CR/PR/SD >6 months was ≥6. Originally, intravenous Carboplatin AUC 6 was to be administered every 3 weeks along with daily 5mg of RAD001 with a 3 patient run-in and then 10 mg daily. Due to a surprising amount of thrombocytopenia with this combination, the dose of Carboplatin was first amended to AUC 5 and most recently to AUC 4 with 5 mg of RAD001. Results: 18 patients have been recruited thus far. Median age is 59. There have been 1 CR, 4 PR’s and 2 SD's lasting > 6 months. One SD was achieved in a patient progressing on single agent Carboplatin at study entry. Median duration of CR+ SD +PR thus far is 13 weeks (range: 6-60 weeks). 5 patients had grade 3/4 thrombocytopenia and 4 patients had grade 3 neutropenia (no febrile neutropenia). However, since amendment of Carboplatin dose to AUC 4 the regimen has been well tolerated. 1 patient suffered from grade 3 dehydration. The estimated clinical benefit rate is 50% (95% C.I.: 24%, 76%). Median time to progression or death is 87.5 days from start of treatment; there is only 1 death to date on this study. Conclusions: The study has achieved it’s primary objective of demonstrating clinical benefit of RAD 001-Carboplatin combination in triple negative metastatic breast cancer. Dose limiting thrombocytopenia was an unexpected side effect requiring protocol amendment.

Introduction: Cancer stem cells (CSC) are implicated in tumor metastasis, recurrence, and high patient mortality, thus, substances impairing their activity, e.g., self-renewal, could be invaluable as novel cancer therapeutics. We propose that CAPE (caffeic acid phenethyl ester), a component of propolis, a honeybee product, could be such an agent. CSC are known for their chemoresistance. We previously showed that CAPE inhibits growth of MDA-231 (231) cells, a model of human triple-negative breast cancer, multidrug resistance gene expression, NF-κB, EGFR, and VEGF. We hypothesized that CAPE could also affect CSC-mediated effects. Methods: We isolated CSC from 231 cells and mouse xenografts, and propagated them in low adherence tissue culture plates without serum. CSC characteristics, ie Hoechst 33342 dye exclusion and CD44+/CD24-/low/EpCAMlow phenotype, were assessed by flow cytometry, while clonal growth was assayed in soft agar. CSC were treated with CAPE (0-40 μM) for 4 or 7 days. Apoptosis was assessed by Annexin V/propidium iodide staining. Cell cycle analysis was done according to standard protocol and analysis by ModFit software. Results: 231-derived CSC grow as mammospheres (MMS), and when dissociated into single cells, form MMS again. This process was repeated for many generations and is considered a sign of self-renewal. These cells excluded Hoechst 33342 dye, exhibited characteristic CD44+/CD24-/low/EpCAMlow phenotype, and generated progenitors in presence of serum, a CSC trait responsible for regenerating tumor mass. CAPE inhibited CSC self-renewal, progenitors’ generation, and growth of CSC-derived tumor cells. Treatment of CSC with CAPE for 4.5 days decreased CD44 levels by ∼95%, while another cell population containing 10-100-fold lower CD44 content concurrently increased. Incubation of CSC with different CAPE doses for 4 days caused dose-dependent cell growth inhibition. After pre-treating cells with CAPE, the same numbers of live cells were taken from each well and plated on soft agar. Clonal growth was also inhibited in a dose-dependent fashion, but apoptosis was not induced as determined by Annexin V/PI assay. Instead, CSC were noted to significantly progress from a quiescent cell cycle state in G0/G1 (82%), S phase (12%) to a cycling state with an increase in S phase (41%) and subsequent decrease in G0/G1 (54%). Conclusions: These results suggest that CAPE causes pronounced changes in CSC characteristics manifested by inhibition of clonal growth in soft agar and concurrent significant decrease in CD44 content, a sign of decreased potential of malignancy. This conclusion is strengthened by the observation that the CSC are induced into a more differentiated state that is less malignant after CAPE treatment. Our results strongly suggest that CAPE could be an inhibitor of CSC making them more susceptible to chemotherapy. [grants BCTR0600476 & ES00260] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3344. doi:10.1158/1538-7445.AM2011-3344

Recent studies in our laboratory have described the development of the SV40-transformed thymic nurse cell (TNC) line SVT-II2, that maintains the ability to internalize thymocytes in vitro. SVT-II2 cells were shown to bind and internalize a subset of the αβ TCR+, CD4+CD8+ thymocyte population exclusively. Also, SVT-II2 cells express cell surface class I and class II MHC antigens. These data are consistent with reports that suggest that TNCs may have a role in thymic education. In this report, we used scanning electron microscopy, transmission electron microscopy, and long-term video microscopy to study binding, internalization, and release of thymocytes by TNCs. The results of these experiments showed the internalization event to be selective and dynamic. The process appears to involve programmed cooperation between the two cell types that terminates with the release of selected thymocytes. Although no changes in thymocyte cell surface phenotype were detected as a result of their interaction with TNCs in vitro, over 90% remained viable after a 48-hr incubation period.

e13587 Background: TNBC is a high mortality disease with a paucity of therapeutics. Our preclinical data used CAPE, a main component of propolis, a honeybee product credited with antiinflammatory, antioxidant, and antitumor properties. CAPE, which is innocuous to normal human mammary epithelial cells, inhibits growth of MDA-MB-231 (231) cells, MDR gene expression, NF-kB, EGFR and VEGF. We used 231 cells, a model of human TNBC, to show that CAPE can combat TNBC as a therapeutic, and/or adjuvant to chemotherapeutic drugs like Taxol. Cancer stem cells (CSC) are implicated in tumor metastasis and recurrence, thus, agents impairing their self-renewal, could be invaluable as novel cancer therapeutics. We show that CAPE could be such an agent, because it inhibits CSC growth and self renewal. We propose that the mechanism for this inhibition is due to the induction of CSC apoptosis. Methods: Nude mice bearing 231 xenografts were fed CAPE diets and/or treated topically with CAPE and tumors measured 2x/week. bCSC w...

Introduction. Cancer stem cells (CSC) are implicated in tumor metastasis, recurrence, and high patient’ mortality, therefore, substances impairing their activity, e.g., self-renewal, could be invaluable as novel cancer therapeutics. We propose that CAPE (caffeic acid phenethyl ester), a component of propolis produced by honeybees, could be such a novel anti-CSC agent. We previously showed that CAPE, which is innocuous to normal human mammary epithelial cells, inhibits growth of MDA-MB-231 (231) cells, multidrug resistance gene expression, NF-kB transcription factors, EGFR, its phosphorylation, and VEGF formation. These cells are often used as a model of human triple-negative breast cancer, hence, we hypothesized that CAPE could also affect CSC-mediated effects. Methods. We isolated CSC from 231 cells and from nude mouse xenografts induced by 231 cell inoculation, and propagated them in low adherence tissue culture plates in the absence of serum. CSC characteristics, e.g., exclusion of Hoechst 33342 dye, a measure of drug resistance, and the presence of CD44+/CD24−/low/EpCAMlow phenotype, were assessed by flow cytometry, while clonal growth was assayed in soft agar. Results. CSC were grown as mammospheres and, when dissociated into single cells, formed mammospheres again. This process was repeated for many generations and is considered a sign of self-renewal. These cells excluded Hoechst 33342 dye, exhibited characteristic CD44+/CD24−/low/EpCAMlow phenotype, and generated progenitors in presence of serum, a CSC trait responsible for regenerating tumor mass. CAPE inhibited CSC self-renewal, progenitors’ generation, and suppressed growth of CSC-derived tumor cells. When mammosphere-derived single cells were treated with CAPE for 4.5 days, CD44 levels were decreased by ∼95%, while another cell population containing lower CD44 content concurrently increased by ∼10-fold. This decrease in CD44 levels is probably responsible for the following results. An incubation of CSC (as single cells) with different CAPE doses (0-40 microM, one treatment only) for 4 days caused cell growth inhibition in a dose-dependent fashion. After pre-treating cells with CAPE, the same numbers of live cells were taken from each well and plated on soft agar. Clonal growth was also inhibited in a dose-dependent fashion depending on CAPE dose used for the pre-treatment. Conclusions. These results suggest that CAPE causes pronounced changes in CSC characteristics manifested by inhibition of clonal growth in soft agar, a sign of decreased potential of malignancy, and this conclusion is strengthened by the concurrent decrease in CD44 content. Cumulatively, our results strongly suggest that CAPE could be a powerful inhibitor of cancer stem cell growth and functions that are responsible for CSC-mediated recurrence of breast cancer and increased patient mortality. [This work was supported in part by grants BCTR0600476 & ES00260] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3555.

Introduction Rapamycin acts synergistically with platinum agents to induce apoptosis and inhibit proliferation in breast cancer cell lines. Combination of everolimus also known as RAD001 (oral mammalian target of rapamycin (mTOR) inhibitor) and carboplatin may have activity in metastatic triple-negative breast cancer (TNBC). Methods The primary objective of this study was to determine clinical benefit rate (CBR), that is (complete remission (CR) + partial remission (PR) + stable disease (SD) lasting ≥6 months) and the toxicity of everolimus/carboplatin in women with metastatic TNBC. Prior carboplatin was allowed. Treatment consisted of intravenous carboplatin area under the curve (AUC) 6 (later decreased to AUC 5 and subsequently to AUC 4) every 3 weeks with daily 5 mg everolimus. Results We enrolled 25 patients in this study. Median age was 58 years. There were one CR, six PRs, seven SDs and eight PDs (progression of disease). CBR was 36% (95% confidence interval (CI) 21.1 to 57.4%). One SD was achieved in a patient progressing on single agent carboplatin. The median progression free survival (PFS) was 3 months (95% CI 1.6 to 4.6 months) and overall survival (OS) was 16.6 months (95% CI 7.3 months to not reached). There were seven patients (28%) with ≥ grade 3 thrombocytopenia; three (12%) with grade 3 neutropenia (no bleeding/febrile neutropenia) and one (4%) with grade 3 anemia. Greater hematological toxicity was seen in the first seven patients treated with carboplatin AUC5/6. After the amendment for starting dose of carboplatin to AUC 4, the regimen was well tolerated with only one out of 18 patients with grade 3 neutropenia and two patients with grade 3 thrombocytopenia. There was only one case of mucositis. Conclusion Everolimus-carboplatin was efficacious in metastatic TNBC. Dose limiting hematological toxicity was observed when AUC5/6 of carboplatin was combined with everolimus. However, carboplatin AUC 4 was well tolerated in combination with everolimus with continuing responses. Trial registrations ClinicalTrials.gov NCT01127763.