Invasive Triple-negative Breast Cancer Research Articles

Dihydrotestosterone regulates expression of CD44 via miR-328-3p in triple-negative breast cancer cells

Triple-negative breast cancer (TNBC) is an aggressive subtype that lacks effective targeted therapeutics strategy and has poor prognosis. Targeting androgen receptor (AR) in TNBC is thought to be a promising approach. We hypothesized that AR, functioning as a transcription factor, controls cell behavior via regulating the expression of microRNA molecules (miRNAs). The expression of 84 breast cancer-specific miRNAs in MDA-MB-231 cells, a highly invasive TNBC model system, was investigated using PCR arrays following treatment of cells with 5α-dihydrotestosterone (DHT). The expression of 33 miRNAs was changed by more than 2 folds including miR-328-3p, which was up-regulated by 13 folds. Transfection of cells with either miR-328-3p mimic or anti-sense molecules decreased cell motility. DHT-mediated effect on the expression and function of CD44, a target of miR-328-3p, was investigated. CD44 expression and cell adhesion to hyaluronic acid (HA) were down-regulated when cells were treated with DHT or transfection with a miR-328-3p mimic. On the other hand, the AR antagonist, bicalutamide, or transfection of cells with miR-328-3p anti-sense molecules had the opposite effect. Cells transfected with miR-328-3p anti-sense molecules reduced the negative effect of DHT on CD44 expression and cell adhesion to HA. In addition, DHT further reduced the expression of CD44 and cell adhesion to HA in cells transfected with miR-328-3p mimic. These results strongly suggest that miRNAs can mediate AR regulation of breast cancer cells and that AR controls the expression of CD44 via miRNA-dependent and independent mechanisms.

Tumor-infiltrating lymphocyte volume is a better predictor of neoadjuvant therapy response and overall survival in triple-negative invasive breast cancer

Triple-negative breast cancer (TNBC) has an aggressive behavior, limited therapeutic options, and high mortality rate. Neoadjuvant chemotherapy is a standard treatment for TNBC, and patients with pathological complete response (pCR) have a favorable outcome. We conducted a comprehensive evaluation of cancer clinicopathological parameters and correlated these parameters with the pCR rate and the overall survival. Fifty-eight patients with TNBC of no special type who underwent breast biopsy, neoadjuvant therapy, and mastectomy in our institution during 2005-2016 were included in this study. Among the 58 TNBC patients, 26 (45%) achieved pCR, and 32 (55%) had residual disease. The study parameters included age, histologic grade, clinical stage, mitotic count, Ki-67 proliferation index, stromal ratio, stromal type, tumor necrosis, stromal tumor-infiltrating lymphocytes (TILs), tumor intraepithelial lymphocytes, and tumor-infiltrating lymphocytes volume (TILV). Whereas most factors did not affect pCR, stromal TILs and TILV showed significant correlation with pCR (P = .01 and P = .0008, respectively). In the residual disease group, all factors showed no significant differences before and after neoadjuvant chemotherapy, except for tumor sizes. Lastly, pCR, TILs, and TILV were all significantly correlated with the overall survival, with P = .028, .029, and .015, respectively. In summary, we proposed a new concept of TILV to precisely evaluate the tumor immunity, and our data showed that TILV had a better predictive value than TILs for the pCR and the overall survival in TNBC.

Structural Diversity and Anticancer Activity of Marine-Derived Elastase Inhibitors: Key Features and Mechanisms Mediating the Antimetastatic Effects in Invasive Breast Cancer.

Three new 3-amino-6-hydroxy-2-piperidone (Ahp)-containing cyclic depsipeptides, named loggerpeptins A-C (1-3), along with molassamide (4), were discovered from a marine cyanobacterium, extending the structural diversity of this prevalent scaffold of cyanobacterial serine protease inhibitors. Molassamide, which contains a 2-amino-butenoic (Abu) unit in the cyclic core, was the most potent and selective analogue against human neutrophil elastase (HNE). Given the growing evidence supporting the role of HNE in breast cancer progression and metastasis, we assessed the cellular effects of compounds 3 and 4 in the context of targeting invasive breast cancer. Both compounds inhibited cleavage of the elastase substrate CD40 in biochemical assays; however, only 4 exhibited significant cellular activity. As CD40 and other receptor proteolytic processing culminates in NFκB activation, we assessed the effects of 4 on the expression of target genes, including ICAM-1. ICAM-1 is also a direct target of elastase and, in our studies, compound 4 attenuated both elastase-induced ICAM-1 gene expression and ICAM-1 proteolytic processing by elastase, revealing a potential dual effect on migration through modulation of gene expression and proteolytic processing. Molassamide also specifically inhibited the elastase-mediated migration of highly invasive triple-negative breast cancer cells.

Abstract OT2-07-03: IMpassion031: A phase III study comparing neoadjuvant atezolizumab vs placebo in combination with nab-paclitaxel–based chemotherapy in early triple-negative breast cancer (TNBC)

Abstract Background Atezolizumab is an anti–programmed death-ligand 1 (PD-L1) monoclonal antibody that blocks the binding of PD-L1 to PD-1 and B7.1 receptors, thereby restoring tumor-specific immunity. TNBC is characterized by PD-L1 expression on tumor-infiltrating immune cells (IC), high levels of tumor-infiltrating lymphocytes (TILs), and a higher mutation rate compared with other breast cancers suggesting a therapeutic opportunity for atezolizumab. Atezolizumab alone and in combination with nab-paclitaxel is well tolerated, with promising clinical activity in metastatic TNBC. Furthermore, cytotoxic chemotherapies like nab-paclitaxel can enhance anti-tumor immune responses via neoantigen release. Taken together, this supports the investigation of atezolizumab in combination with nab-paclitaxel in early-stage TNBC. IMpassion031 is a global Phase III, double-blind, randomized, multicenter, placebo-controlled study being conducted to evaluate the efficacy and safety of neoadjuvant treatment with nab-paclitaxel → doxorubicin + cyclophosphamide and either atezolizumab or placebo in invasive stage II/III early TNBC. The selection and sequence of chemotherapy has been chosen to maximize the opportunity for a robust immune response. Methods: Eligible patients are those with previously untreated, central laboratory–confirmed invasive TNBC with primary tumor size > 2 cm and ECOG PS 0-1. Exclusion criteria include history of invasive breast cancer, stage IV disease, bilateral breast cancer, prior systemic therapy for the treatment or prevention of breast cancer, prior immunotherapy and a history of autoimmune disease. Approximately 204 patients will be randomized 1:1 to receive atezolizumab (840 mg q2w) or placebo with nab-paclitaxel (125 mg/m2 qw) for 12 weeks. Subsequent atezolizumab (840 mg q2w) or placebo with doxorubicin (60 mg/m2 q2w) + cyclophosphamide (600 mg/m2 q2w) will be given for 4 cycles prior to surgery. Post-surgery, patients will be unblinded. Patients in the atezolizumab arm will continue to receive atezolizumab (1200 mg q3w × 11 doses) post-surgery. Stratification factors include stage II vs III TNBC at diagnosis and PD-L1 expression on tumor-infiltrating IC (IC0 < 1% vs IC1/2/3 ≥ 1% with the VENTANA SP142 IHC assay). The primary endpoint is pathologic complete response (pCR); key secondary endpoints include pCR according to PD-L1 IC status, patient-reported outcomes, event-free survival and overall survival. Tumor samples will be taken at baseline, on treatment (optional), at surgery and post-recurrence for the assessment of biomarkers associated with treatment response and immune escape. (NCT pending). Citation Format: Mittendorf E, Barrios CH, Harbeck N, Miles D, Saji S, Zhang H, Duc A-N, Rafii S, Lai C. IMpassion031: A phase III study comparing neoadjuvant atezolizumab vs placebo in combination with nab-paclitaxel–based chemotherapy in early triple-negative breast cancer (TNBC) [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr OT2-07-03.

Inhibition of growth and metastasis of triple-negative breast cancer targeted by Traditional Chinese Medicine Tubeimu in orthotopic mice models

Triple-negative breast cancer (TNBC) is highly invasive and metastatic, which is in urgent need of transformative therapeutics. Tubeimu (TBM), the rhizome of Bolbostemma paniculatum (Maxim.) Franquet, is one of the Chinese medicinal herbs used for breast diseases since the ancient times. The present study evaluated the efficacy, especially the anti-metastatic effects of the dichloromethane extract of Tubeimu (ETBM) on TNBC orthotopic mouse models and cell lines. We applied real-time imaging on florescent orthotopic TNBC mice model and tested cell migration and invasion abilities with MDA-MB-231 cell line. Digital gene expression sequencing was performed and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis applied to explore the pathways influenced by ETBM. Moreover, quantitative real-time polymerase chain reactions (qRT-PCR) and Western blot were delivered to confirm the gene expression changes. ETBM exhibited noticeable control on tumor metastasis and growth of TNBC tumors with no obvious toxicity. In compliance with this, it also showed inhibition of cell migration and invasion in vitro. Its impact on the changed biological behavior in TNBC may be a result of decreased expression of integrin β1 (ITGβ1), integrin β8 (ITGβ8) and Rho GTPase activating protein 5 (ARHGAP5), which disabled the focal adhesion pathway and caused change in cell morphology. This study reveals that ETBM has anti-metastatic effects on MDA-MB-231-GFP tumor and may lead to a new therapeutic agent for the integrative treatment of highly invasive TNBC.

Half-sandwich Os(ii) and Ru(ii) bathophenanthroline complexes: anticancer drug candidates with unusual potency and a cellular activity profile in highly invasive triple-negative breast cancer cells.

There is an urgent need to discover new, selective compounds to add to the limited arsenal of chemotherapeutics displaying selective toxicity for aggressive triple-negative breast cancer (TNBC) cells. The effect of two, recently developed metal-based half-sandwich complexes [Os(η6-pcym)(bphen)(dca)]PF6 (Os-dca) and [Ru(η6-pcym)(bphen)(dca)]PF6 (Ru-dca) [pcym = 1-methyl-4-(propan-2-yl)benzene (p-cymene); bphen = 4,7-diphenyl-1,10-phenanthroline (bathophenanthroline); dca = dichloroacetate] on triple-negative breast cancer cells MDA-MB-231 is reported. The complexes display selective toxicity in several tumor cells (at submicromolar concentrations), and a prominent effect is observed against highly progressive triple negative breast cancer MDA-MB-231 cells for Os-dca. The lower potency of Ru-dca in comparison with Os-dca is apparently connected with a relatively quick release of the dca ligand due to the hydrolysis of Ru-dca before this complex enters the cells. Remarkably, both Os-dca and Ru-dca reduce successfully metastasis-related properties of the triple-negative breast cancer cells such as migration, invasion, and re-adhesion. The anti-metastatic effects of Os-dca and Ru-dca are associated with their ability to suppress matrix metalloproteinase activity and/or production and reduce the expression of aquaporins. Further detailed mechanistic studies reveal that Os-dca reverses Warburg's effect and oncosis seems to be a prominent mode of cell death that predominates over apoptosis. As such, Os-dca can efficiently overcome the resistance of cancer cells to clinically-used apoptotic inducers cisplatin and carboplatin. The cytostatic and anti-metastatic properties of Os-dca in MDA-MB-231 provide a strong impetus for the development of new metal-based compounds to target hardly treatable human TNBC cells and displaying different modes of action compared to the antitumor metallodrugs in clinical use.

Invasive triple negative breast cancer with basal and non-basal-like immunophenotypes: prognostic implications

Invasive triple negative breast cancer with basal and non-basal-like immunophenotypes: prognostic implications

115P - Triple negative breast cancer: Survival and age at time of diagnosis among the Lebanese population

Background: Invasive triple-negative breast cancer accounts for 10-20% of breast cancers and is responsible for a high percentage of breast cancer-related deaths. It is indeed a very aggressive tumor that is associated with a poor prognosis with high risk of relapse and a short disease-free survival. It is treated by chemotherapy only and does not benefit from targeted therapy until now. The purpose of this study was to analyze progression-free survival (PFS) and overall survival (OS) after the change of therapeutic modalities, and the age at diagnosis of triple-negative breast cancer in Lebanese women and to compare the age distribution with a preceding Lebanese study and a recent American study of 38,813 patients nationwide. Methods: Data were collected from hospitals and pathology laboratories across Lebanon. The collection was based on the following inclusion criteria: female diagnosed with an invasive TNBC between 2010 and 2016. PFS was studied in 193 cases, the overall survival analysis was done on 63 cases and the age at time of diagnosis on 387 cases. The statistical analysis was done using Student t tests. Results: PFS improved from 19 to 35 months after the change of therapeutic protocols and the OS increased from 19 to 23 months (p < 0.001). The average age at time of diagnosis was approximately 56 years, compared with 52 years in the previous Lebanese study and there was a clear age disparity of the age at time of diagnosis between and Lebanese and American study when comparing patients of identical age groups. The age at diagnosis does not depend on the geographical area. Conclusions: The new therapeutic protocols proved to be more effective, but screening programs must be done at an earlier age. The findings may be pertinent for the Lebanese patients and warrant further evaluation in different ethnic groups and populations. Legal entity responsible for the study: Holy Spirit University of Kaslik Funding: None Disclosure: All authors have declared no conflicts of interest.

Cancer-Associated Fibroblasts Autophagy Enhances Progression of Triple-Negative Breast Cancer Cells.

BackgroundCancer-associated fibroblasts (CAFs) are key factors in malignant tumor initiation, progression, and metastasis. However, the effect of CAFs autophagy on triple-negative breast cancer (TNBC) cells is not clear. In this study, the growth effect of TNBC cells regulated by CAFs autophagy was evaluated.Material/MethodsCAFs were obtained from invasive TNBC tumors and identified by Western blot and immunofluorescence staining assay. CAFs were co-cultured with TNBC cells, and migration and invasion were evaluated by Matrigel-coated Transwell and Transwell inserts. TNBC cells growth was detected by MTT assay, and epithelial-mesenchymal transition (EMT) regulated by CAFs was evaluated by Western blot assay.ResultsCAFs were identified by the high expression of α-smooth muscle actin (α-SMA) protein. Autophagy-relevant Beclin 1 and LC3-II/I protein conversion levels in CAFs were higher than those in NFs (P<0.05). TNBC cells migration, invasion, and proliferation levels were significantly improved in the CAFs-conditioned medium (CAFs-CM) group, compared with the other 3 groups (P<0.05). TNBC cells vimentin and N-cadherin protein levels were upregulated and E-cadherin protein level was downregulated in the CAFs-CM group compared with the control group (P<0.05). Further study indicated β-catenin and P-GSK-3β protein levels, which are the key proteins in the Wnt/β-catenin pathway, were upregulated in the CAFs-CM group compared with the control group (P<0.05).ConclusionsOur data demonstrated CAFs autophagy can enhance TNBC cell migration, invasion, and proliferation, and CAFs autophagy can induce TNBC cells to engage in the EMT process through the Wnt/β-catenin pathway.

Role of transforming growth factor-β1 in triple negative breast cancer patients

BackgroundWe aimed to demonstrate the prognostic value of TGF-β1 in triple negative breast cancer (TNBC) and its association with clinicopathological characteristics of TNBC. Materials and methodsA total of 180 women were randomly selected from non-metastatic invasive TNBC patients diagnosed at two hospitals between 2003 and 2012. Lmmunohistochemistry was performed to semi-quantify the expression of TGF-β1. Relationship between TGF-β1 expression and clinicopathological features was performed by Chi-square test. Univariate and multivariate survival analyses were performed to identify the prognostic role of TGF-β1 expression on survival outcomes. ResultsOf the 180 women included in this study, 67 (37.2%) patients expressed high level of TGF-β1. High expression of cytoplasmic TGF-β1 was correlated with higher histologic tumor grade (P < 0.001) and lymph node status (P < 0.001), and more axillary lymph node dissection (P = 0.029). High cytoplasmic TGF-β1 expression was associated with reduced disease-free survival (DFS) and overall survival (OS) by log-rank test (PDFS<0.001, POS = 0.045). However, multivariate survival analyses showed that high TGF-β1 was marginally correlated with unfavorable DFS (hazard ratio (HR) 1.796, 95% CI 0.995–3.242, P = 0.052), while it was not significantly associated with OS (HR 0.747, 95% CI 0.367–1.522, P = 0.422). ConclusionsThis multi-centered retrospective study highlights the high expression of cytoplasmic TGF-β1 in TNBC is associated with higher histologic grade and lymph node status, more axillary lymph node dissection, as well as reduced DFS. Our observation that the prognostic role of TGF-β1 in TNBC suggests potential rationale for using therapeutic strategies based on targeting TGF-β1 in advanced tumors.

Phase 3 study evaluating efficacy and safety of veliparib (V) plus carboplatin (Cb) or Cb in combination with standard neoadjuvant chemotherapy (NAC) in patients (pts) with early stage triple-negative breast cancer (TNBC).

520 Background: Clinical studies suggest that TNBC is sensitive to DNA-damaging agents, including Cb. V is a potent PARP inhibitor that may enhance the antitumor activity of such agents. We present primary response data from a phase 3 randomized, placebo-controlled study (NCT02032277) evaluating the addition of V + Cb or Cb to neoadjuvant paclitaxel (P) followed by doxorubicin + cyclophosphamide (AC). Methods: Pts with histologically confirmed, invasive TNBC (T2–T4 N0–2 or T1 N1–2) amenable to surgical resection were randomized 2:1:1 to (Arm A) P 80 mg/m2 weekly + Cb AUC 6 mg/mL/min q3 weeks + V 50 mg PO BID; (Arm B) P + Cb + PO placebo; or (Arm C) P + IV placebo + PO placebo, for 12 weeks followed by AC (60 mg/m2 or 600 mg/m2 q2 or 3 weeks) × 4. Primary endpoint was pathologic complete response (pCR) in breast and nodes with &gt; 80% power at 2-sided α of 0.05 using pair-wise comparisons for A vs B and A vs C to detect significant treatment effects using Χ2 test; secondary endpoint was rate of conversion to eligibility for breast conservation surgery (BCS). Adverse events (AEs) were assessed with NCI CTCAE V4.0. Results: Six hundred thirty-four pts (median age 50 years; range 22–79) were randomized to Arms A (n = 316), B (n = 160), or C (n = 158). Baseline characteristics were well balanced. No pCR difference was observed between Arms A and B (53.2% vs 57.5% p = 0.36), but pCR in Arm A was higher than Arm C (53.2% vs 31.0% p &lt; 0.001). In non-prespecified analysis, pCR in Arm B was also higher than Arm C (57.5% vs 31.0% p &lt; 0.001). Among pts ineligible for BCS at screening (n = 141), 62% were eligible after NAC in Arm A vs 44% each in Arms B (p = 0.13) and C (p = 0.14). Grade 3–4 AEs (Arms A/B/C, 86%/85%/45%) and serious AEs (30%/27%/14%) neutropenia, thrombocytopenia, anemia, nausea, and vomiting were increased with the addition of Cb; V did not impact toxicity. Median cycles of NAC were not reduced with V + Cb + P or Cb + P vs P. Conclusions: Addition of V to neoadjuvant Cb + P followed by AC did not increase pCR rate in breast and nodes in stage II–III TNBC, while addition of V + Cb or Cb alone to P followed by AC did. Cb (+/– V) increased toxicity but did not impact delivery of NAC. Clinical trial information: NCT02032277.

Final efficacy and updated safety results of the randomized phase III BEATRICE trial evaluating adjuvant bevacizumab-containing therapy in triple-negative early breast cancer

The purpose of this analysis was to assess the long-term impact of adding bevacizumab to adjuvant chemotherapy for early triple-negative breast cancer (TNBC). Patients eligible for the open-label randomized phase III BEATRICE trial had centrally confirmed triple-negative operable primary invasive breast cancer (pT1a-pT3). Investigators selected anthracycline- and/or taxane-based chemotherapy for each patient. After definitive surgery, patients were randomized 1:1 to receive ≥4 cycles of chemotherapy alone or with 1 year of bevacizumab (5 mg/kg/week equivalent). Stratification factors were nodal status, selected chemotherapy, hormone receptor status, and type of surgery. The primary end point was invasive disease-free survival (IDFS; previously reported). Secondary outcome measures included overall survival (OS) and safety. After 56 months' median follow-up, 293 of 2591 randomized patients had died. There was no statistically significant difference in OS between treatment arms in either the total population (hazard ratio 0.93, 95% confidence interval [CI] 0.74-1.17; P = 0.52) or pre-specified subgroups. The 5-year OS rate was 88% (95% CI 86-90%) in both treatment arms. Updated IDFS results were consistent with the primary IDFS analysis. Five-year IDFS rates were 77% (95% CI 75-79%) with chemotherapy alone versus 80% (95% CI 77-82%) with bevacizumab. From 18 months after first study dose to study end, new grade ≥3 adverse events occurred in 4.6% and 4.5% of patients in the two arms, respectively. Final OS results showed no significant benefit from bevacizumab therapy for early TNBC. Late-onset toxicities were rare in both groups. Five-year OS and IDFS rates suggest that the prognosis for patients with TNBC is better than previously thought. NCT00528567.

Abstract P2-04-16: CD103+/PD-1+ T-cells identify a subset of triple-negative breast cancer eligible for targeted checkpoint inhibition

Abstract Background: The presence of tumor-infiltrating lymphocytes (TILs) is a prognostic factor in triple-negative breast cancer (TNBC). In particular, cytotoxic CD8+ cells are associated with favorable outcome in TNBC. Recently, the integrin CD103 have been suggested as a potential prognostic marker in a small cohort of triple-negative/basal-like breast cancer (Wang et al, Clin Cancer Res 2016). In this study, we aimed to evaluate the functional status and clinical relevance of CD8+ and CD103+ TILs in TNBC. Methods: A large collection of formalin-fixed, paraffin-embedded tissues was retrospectively collected from 210 patients with primary invasive ductal TNBC. The presence of CD8, CD103 and the checkpoint receptor PD-1 was assessed by immunohistochemistry. Whole tumor slides were independently assessed by two pathologists blinded for patient characteristics and outcome. Cases where ≥5% of TILs expressed CD103 or PD-1 were considered positive. Statistical analyses were performed using Spearman's correlation, Kaplan-Meier and Cox regression analyses. Results: We found that CD103+ cells mostly co-expressed the CD8 marker, and were preferentially distributed within tumor epithelium. No consistent correlation was found between the presence of CD103+ lymphocytes and the expression of its ligand E-cadherin by TNBC cells (rs=0.365). CD8+ lymphocytes were consistently associated with better relapse-free survival (RFS) and overall survival (OS) in both univariate (HR=0.61; P=1.06E-02 for RFS; HR=0.59; P=1.93E-02 for OS) and multivariate analysis (HR=0.65; P=2.45E-02 for RFS; HR=0.63; P=3.96E-02 for OS). The presence of CD103+ lymphocytes significantly correlated with prolonged RFS and OS in univariate analysis only (HR=0.82; P=4.12E-02 for RFS; HR=0.85; P=4.93E-02 for OS), and a trend for longer RFS was also observed in univariate analysis for PD-1 (HR=0.71; P=5.22E-02). Interestingly, a subset of TNBC showed co-expression of CD103+/PD-1+ in lymphocytes localized to the intraepithelial areas of the tumor. Conclusions: The expression of CD103 on CD8+ T cells in direct contact with cancer cells may identify a subpopulation of cells with potent cytolytic activity. The interaction between CD103+ cytotoxic lymphocytes and cancer cells is mediated by mechanisms other than the binding to E-cadherin. Even though CD103 has a role in mediating an effective anti-tumor immune response, its presence alone may not be sufficient to impact the outcome of TNBC patients. Immunomodulatory therapies may be useful to boost the anti-tumor activity of potentially quiescent CD103+/PD-1+ cells in a subgroup of TNBC. Citation Format: Bottai G, Loi S, Sotiriou C, Roncalli M, Pusztai L, Reis-Filho JS, Santarpia L. CD103+/PD-1+ T-cells identify a subset of triple-negative breast cancer eligible for targeted checkpoint inhibition [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 P2-04-16.

Abstract OT3-02-05: NCI-2016-00367: A phase IIB study of neoadjuvant ZT regimen (enzalutamide therapy in combination with weekly paclitaxel) for androgen receptor (AR)-positive triple-negative breast cancer (TNBC)

Abstract BACKGROUND: Approximately 50% of TNBC expresses AR by immunohistochemical (IHC) staining. Luminal androgen receptor (LAR) subtype is heavily enriched in hormonally regulated genes, yet negative for ER by IHC. LAR is associated with low pCR rates and long survival. Preclinical data have shown that taxanes inhibit translocation of AR from the cytoplasm to the nucleus where AR is activated. Combining paclitaxel with enzalutamide may inhibit the AR pathway synergistically thereby increasing pCR rates. We hypothesized that patients with AR-positive TNBC who have chemo-insensitive disease (CID) after initial anthracycline-based chemotherapy treated with ZT would have higher RCB-0 and RCB-I rates than those who receive conventional taxane-based chemotherapy. Our team developed a clinical trial to identify patients with CID (ARTEMIS: A Randomized, TNBC Enrolling trial to confirm Molecular profiling Improves Survival). In the ARTEMIS trial, treatment-naïve patients with localized TNBC undergo a pretreatment biopsy and then begin anthracycline-based chemotherapy. Molecular testing results and radiographic response assessment are used to identify CID and will guide the second phase of neoadjuvant chemotherapy (NACT) to overcome CID. PRIMARY OBJECTIVE: To determine RCB-0 and RCB-I rates of patients with TNBC who have CID to initial anthracycline-based chemotherapy and who received ZT. TRIAL DESIGN AND STATISTICAL METHODS: Patients with CID from the ARTEMIS trial can enroll in the 12-week ZT (paclitaxel, 80 mg/m2 intravenously per week; enzalutamide, 160 mg orally per day). We will define pCR (RCB-0) or RCB-I as a response, using a Simon optimal 2-stage design with alpha=beta=10% and then setting the threshold for an acceptable pCR or RCB-I rate at 20%. We will enroll 12 patients into the first stage. If no patients experience pCR or RCB-I, we will stop the study after the first stage. If at least 1 patient experiences pCR or RCB-I, we will enroll 25 more patients for a total of 37 patients. We would declare the treatment worthy of further study if at least 4 of the 37 patients experience pCR or RCB-I. This design has a 54% probability of early termination after the first stage if the true pCR or RCB-I probability is 5%. Because patients with CID have a very low chance (5%) of achieving pCR with additional chemotherapy, improving pCR rates to 20% in this patient population would be clinically meaningful. BRIEF ELIGIBILITY CRITERIA: Inclusion criteria: Primary invasive TNBC patients who have CID under the ARTEMIS trial; AR+ ≥1% nuclear staining by IHC; and adequate physical, organ, bone marrow, and cardiac functions. Exclusion criteria: Pregnant or lactating patients, history of colitis or absorption abnormality, known or suspected brain metastasis or leptomeningeal disease, or history of seizure. CORRELATIVE SCIENCE: Enumeration of circulating tumor cells (CTCs) and expression of CTC-related gene transcripts will be measured to correlate CTC characteristics and/or gene profiles related to the AR pathway and treatment response to ZT. Citation Format: Fujii T, Lim B, Helgason T, Hess KR, Gilcrease MZ, Willey JS, Tripathy D, Litton JK, Moulder S, Krishnamurthy S, Yang W, Reuben JM, Symmans WF, Ueno NT. NCI-2016-00367: A phase IIB study of neoadjuvant ZT regimen (enzalutamide therapy in combination with weekly paclitaxel) for androgen receptor (AR)-positive triple-negative breast cancer (TNBC) [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 OT3-02-05.

Analysis of the factors influencing adjuvant chemotherapy decisions for triple negative breast cancer

Objective: To analyze adjuvant chemotherapy decisions for triple negative breast cancer (TNBC), and explore the influencing factors in the multidisciplinary treatment (MDT) modality. Methods: A retrospective analysis was performed. The cases with invasive TNBC who underwent surgery and MDT discussion for adjuvant treatment in Ruijin Hospital, from April 2013 to June 2015, were recruited. The patients' clinicopathological characteristics were analyzed and adjuvant treatment suggestions from MDT were obtained. Here the chemotherapy decision alteration was defined as a disagreement in chemotherapy or not, or inconsistence in regimens between the attending doctor and the multidisciplinary team. Results: A total of 194 patients aged ≤70 years old were enrolled in the multidisciplinary discussion, and 187 patients (96.4%) were suggested to receive chemotherapy. When compared the opinions of the attending doctor to suggestions of the multidisciplinary team, we found that the percentage of chemotherapy decision alteration reached 22.7% (39/172), of which 94.9% (37/39) were inconsistence in chemotherapy regimens. There were 119 patients who were recommended to receive epirubicin plus cyclophosphamide (EC) followed by docetaxel (T) or weekly paclitaxel (wP) regimens. Before the announcement of results for the E1199 trial, EC-T accounted for 62.5% (55/88), and EC-wP accounted for 37.5% (33/88) for this group of patients. After that, the proportion of EC-T was decreased to 22.6% (7/31) and proportion of EC-wP increased to 77.4%(24/31) (P<0.001). In addition, a total of 20 patients were suggested to receive platinum based chemotherapy. The proportions were 9.3% in cases with invasive ductal carcinoma, and 33.3% in cases with metaplastic carcinoma, respectively (P=0.016). Conclusions: The adjuvant chemotherapy decision for TNBC patients is altered in 22.7% of the patients after MDT discussion. After the announcement of SABCS E1199 results, more patients are suggested to receive EC followed by weekly paclitaxel. There is a lack of detailed evidence for platinum based adjuvant chemotherapy for TNBC, and more patients with metaplastic carcinoma receive platinum based adjuvant chemotherapy.

GINS2 regulates matrix metallopeptidase 9 expression and cancer stem cell property in human triple negative Breast cancer.

GINS2, a subunit of GINS complex, is critical for the initiation of DNA replication and DNA replication fork progression. The expression of GINS2 is misregulated in many malignant tumors, such as leukemia, breast cancer and melanoma. However, the role of GINS in breast cancer remains poorly characterized. We investigate the possible effect and particular mechanism of GINS in breast cancer cells. We showed that expression of GINS2 is enriched in triple negative breast cancer (TNBC) cell lines. Furthermore, GINS2 knockdown decreased the growth, invasive ability and stem-like property of TNBC cells. Mechanistically, silencing of GINS2 in TNBC cells caused dramatic decrease of matrix metalloproteinase-9 (MMP9). Finally, the abundance of GINS2 correlated with the advance stages of tumor in human TNBC patients. Our studies provided insight into the molecular regulation of TNBC progression and invasion. More importantly, our data suggest that GINS2 could be an outstanding therapeutic target for inhibiting invasive TNBC growth and metastasis.

Abstract A118: CD103+/PD-1+ T-cells identify a subset of triple-negative breast cancer eligible for targeted checkpoint inhibition

Abstract The presence of tumor-infiltrating lymphocytes (TILs) is a prognostic factor in triple-negative breast cancer (TNBC). Specifically, cytotoxic CD8+ cells are associated with favorable outcome in TNBC. Recently, the integrin CD103 has been suggested as a potential prognostic marker in breast cancer. In this study, we aimed to evaluate the functional status and clinical relevance of CD8+ and CD103+ TILs in TNBC. A retrospective cohort of 210 formalin-fixed, paraffin-embedded tissues from patients with primary invasive ductal TNBC was assessed for the presence of CD8, CD103 and the checkpoint receptor PD-1 by immunohistochemistry assay. Whole tumor slides were assessed. Cases where ≥5% of TILs expressed CD103 or PD-1 were considered positive. Statistical analyses were performed using Spearman's correlation, Kaplan-Meier and Cox regression analyses. We found that CD103+ cells mostly co-expressed the CD8 marker, and were preferentially distributed within tumor epithelium. No consistent correlation was found between the presence of CD103+ lymphocytes and the expression of its ligand E-cadherin by TNBC cells (rs = 0.365). CD8+ lymphocytes were consistently associated with better relapse-free survival (RFS) and overall survival (OS) in both univariate (HR = 0.61; P = 1.06E-02 for RFS; HR = 0.59; P = 1.93E-02 for OS) and multivariate analysis (HR = 0.65; P = 2.45E-02 for RFS; HR = 0.63; P = 3.96E-02 for OS). The presence of CD103+ lymphocytes significantly correlated with prolonged RFS and OS in univariate analysis only (HR = 0.82; P = 4.12E-02 for RFS; HR = 0.85; P = 4.93E-02 for OS), and a trend for longer RFS was also observed in univariate analysis for PD-1 (HR = 0.71; P = 5.22E-02). Interestingly, a subset of TNBC showed co-expression of CD103+/PD-1+ in lymphocytes localized to the intraepithelial areas of the tumor. The expression of CD103 on CD8+ T cells in direct contact with cancer cells may identify a subpopulation of cells with potent cytolytic activity. The interaction between CD103+ cytotoxic lymphocytes and TNBC cells is mediated by different mechanisms rather than the binding to E-cadherin. Even though CD103 has a role in mediating an effective anti-tumor immune response, its presence alone may not be sufficient to impact the outcome of TNBC patients. Immunomodulatory therapies may be useful to boost the anti-tumor activity of potentially quiescent CD103+/PD-1+ cells in a subgroup of TNBC patients. Citation Format: Giulia Bottai, Massimo Roncalli, Libero Santarpia. CD103+/PD-1+ T-cells identify a subset of triple-negative breast cancer eligible for targeted checkpoint inhibition [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A118.

KR-33028, a potent inhibitor of the Na+/H+ exchanger NHE1, suppresses metastatic potential of triple-negative breast cancer cells

Hyper-activation of the Na+/H+ exchanger NHE1 occurs at the onset of oncogenic transformation and plays a critical role in breast cancer carcinogenesis. Dysregulation of NHE1 activity results in intracellular alkalinization and the acidification of the extracellular tumor microenvironment that promotes metastasis. Hence, the use of chemical inhibitors of NHE1 as chemotherapeutic agents is an alluring prospect. We previously demonstrated that two structurally different NHE1 inhibitors, EMD87580 [(2-methyl-4,5-di-(methylsulfonyl)-benzoyl)-guanidine], and HMA [5-(N,N-hexamethylene)-amiloride], were effective as co-adjuvants to potentiate paclitaxel-mediated cytotoxic chemotherapy in triple-negative breast cancer (TNBC) cells. Both these drugs, however, had reduced or minimal anti-cancer effects when used alone. Here, we tested KR-33028 (4-cyano (benzo[b]thiophene-2-carbonyl)guanidine), a potent and selective inhibitor of NHE1, to determine its efficacy in inhibition of metastatic potential of TNBC cells. In highly invasive MDA-MB-231, moderately invasive MDA-MB-468, and lowly invasive Hs578T TNBC cells, KR-33028 considerably reduced rates of cell migration and anchorage-independent colony growth. Invasion of MDA-MB-231 and MDA-MB-468 cells through extracellular matrix was also dramatically decreased in response to KR-33028. We further tested the effect of KR-33028 on MDA-MB-231 cells lacking NHE1 expression (231koNHE1); no differences were observed between untreated control and KR-33028-treated 231koNHE1 cells. Taken together, our results highlight the in vitro efficacy of KR-33028-mediated NHE1 inhibition on limiting cellular functions that are predictive of metastasis in vivo. We suggest that targeting NHE1 in the development of novel chemotherapeutics could be highly effective in combatting triple-negative breast cancer and that KR-33028 is potentially useful in prevention of metastasis.

Abstract 3981: In situ mass spectrometry using desorption electrospray ionization mass spectrometry (DESI-MS) can detect specific lipid profiles across breast cancer molecular subtypes

Abstract INTRODUCTION Lipid profiling as a source for biomarkers identification is an evolving field with some enthusiastic results in cancer. Alterations on lipid metabolism have been increasingly recognized as a hallmark of cancer cells and may help to understand breast cancer carcinogenesis. Analytical chemistry and Pathology have been recently combined in the development of chemical microscopy using in situ mass spectrometry (MS) and may help to decipher breast cancer molecular initiation and progression. OBJECTIVE We aimed to use in situ Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) to investigate lipid profiles across invasive breast cancer (IBC) molecular subtypes and related normal parenchyma (NBP) and ductal carcinoma in situ (DCIS). METHODS: We selected frozen samples from 9 IBC with various ER/PR/HER2 status (ER 0 to 100%, PR 0 to 90%, HER2 0 and 3+, Ki67 10 to 70%). Unstained slides obtained from frozen tissue at 10μm were submitted to DESI-MS analysis with no pretreatment and spatial resolution of 100μm. Molecular profiles (mass to charge [m/z] ratio versus signal intensity) were generated in triplicate directly from tissues to detect changes from areas of interest: IBC (and adjacent normal breast parenchyma and DCIS whenever present) regions were identified by a breast pathologist on HE-stained sequential slides. Experiment was conducted in full-scan mode and m/z range of 100-1200. DESI mass spectra were obtained using a Thermo ScientificTM Q ExactiveTM Hybrid Quadrupole Orbitrap Mass Spectrometer in the positive ion mode. The Biomap software was used to visualize the images and to export the spectrums to metaboanalyst, where the data were statiscally analyzed. RESULTS: The images obtained by lipid profiling presented a direct correlation with HE stained slides, with some ions m/z clearly overlaying areas of normal parenchyma, DCIS and invasive breast cancer. The unsupervised hierarchical clustering analysis showed all technical triplicates remained grouped. All NBP but one clustered together. Triple negative IBC and its related NBP formed a distinct cluster from all other ER+ samples. The ER+/HER2 + tumor segregated from other ER+/HER2- tumors. DCIS and IBC from ER+/PR+/HER2- tumor clustered together. The Ions with m/z 186.9 and m/z 303.2 were consistently related to NBP and IBC respectively. CONCLUSIONS: Chemical microscopy using DESI-MS is a powerful tool to identify specific lipid profiles across molecular subtypes of IBC and can contribute to understand breast cancer carcinogenesis. Citation Format: Victor P. Andrade, Adriana L. Santoro, Sabrina T. Moretti, Nicolas V. Schwab, Marcos N. Eberlin. In situ mass spectrometry using desorption electrospray ionization mass spectrometry (DESI-MS) can detect specific lipid profiles across breast cancer molecular subtypes. [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 3981.

Abstract 4400: Hematopoietic age at onset of breast cancer dictates disease aggressiveness and progression

Abstract Aging is strongly associated with increased risk of developing breast cancer. Young age at diagnosis is typically associated with more aggressive disease, while breast cancer is generally more indolent in older women. Among the different breast cancer subtypes, triple negative breast cancer (TNBC) accounts for ∼15% of invasive breast cancer cases and TNBC patients have a poor prognosis due to lack of targeted therapies and limited sensitivity to chemotherapy. Emerging studies indicate that older women with TNBC have a better outcome than younger women for reasons that are unclear. Nevertheless, older patients tend to suffer co-morbidities associated with chemotherapy and often forego treatment, resulting in disease recurrence and a poor outcome for these patients. Treatment of TNBC therefore presents age-specific challenges. Here, we investigated how age affects the composition and function of pro-tumorigenic bone marrow cells using TNBC models that enable us to interrogate the impact of bone marrow derived cells on both tumor cells and the tumor microenvironment. Consistent with clinical observations, we found that disease progression was significantly more aggressive in young mice relative to aged mice. We identified age- and tumor-dependent molecular and functional changes to bone marrow derived hematopoietic cells that impact TNBC progression. A specific population of Sca1+/cKit- bone marrow cells that expresses CSF1R and secretes the growth factor granulin to support stromal activation and robust tumor growth in young mice, fails to promote tumor outgrowth in aged mice, resulting in maintenance of disease indolence. Importantly, bone marrow cells from young mice are sufficient to activate a tumor-supportive microenvironment and rescue tumor progression in aged mice. The results presented here indicate that hematopoietic age is an important determinant of TNBC aggressiveness and provide rationale for investigating age-stratified therapies designed to prevent activation of tumor-promoting bone marrow cells. Citation Format: Jaclyn Sceneay, Timothy Marsh, Irene Wong, Yuanbo Qin, Andreas Sjödin, Björn Nilsson, Sheila A. Stewart, Sandra S. McAllister. Hematopoietic age at onset of breast cancer dictates disease aggressiveness and progression. [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 4400.