Subset Of TNBC Research Articles

Abstract B008: KDM4C histone demethylase connects redox balance to chromatin remodeling via histone H3 tail clipping

Abstract Triple-negative breast cancer (TNBC) is a breast cancer subtype with inferior prognosis necessitating the discovery of novel therapeutic targets. In this study, we describe that KDM4C, a histone demethylase amplified and overexpressed in a subset of TNBC is a driver of TNBC tumor growth. Integrative multi-omic analysis using RNA-seq, ChIP-seq and ATAC-seq demonstrated that KDM4C inhibition triggers chromatin and transcriptomic remodeling without substantial changes of its canonical substrates H3K9me3 and H3K36me3 in TNBC breast cancer cells. Rather KDM4C loss surprisingly caused proteolytic cleavage at histone 3 N terminus (A21 site) identified by histone mass spectrometry. Protease inhibitor array pointed out cathepsin L (CTSL) as the endopeptidase mediating this procedure and CTSL proteomic interactome profiling further uncovered that grainyhead like transcription factor 2 (GRHL2) recruited CTSL to the chromatin. Metabolomic profiling showed reduced glutathione levels following KDM4C inhibition partly due to a decrease in glutamate-cysteine ligase (GCLC) triggered by CTSL-mediated histone H3 cleavage and subsequent increase in reactive oxygen species (ROS). Knockout of CTSL rescued KDM4C blockade-associated metabolic dysfunction and tumor suppression in vivo. The expression of KDM4C and GCLC correlated in TNBC and combined targeting of KDM4C and the GSH axis improved response to cisplatin in cell culture and xenograft models. Our study reveals a novel role for KDM4C in TNBC that links cellular redox regulation and epigenetics. Citation Format: Zheqi Li, Guillermo Peluffo, Laura Stevens, Xintao Qiu, Marco Seehawer, Xiao-Yun Huang, Shawn Egri, Malvina Papanastasiou, Natalie Kingston, Clive D'Santos, Eva Papachristou, Jun Nishida, Kyle Evans, Ji-Heui Seo, Kendell Clement, Daniel Temko, Muhammad Ekram, Matthew Rees, Melissa Ronan, Jennifer Roth, Anton Simeonov, Stephen Kales, Ganesha Rai, Madhu Lal-Nag, David Maloney, Ajit Jadhav, Franziska Michor, Alex Meissner, Justin Balko, Jason Carroll, Matthew Freedman, Henry Long, Jacob Jaffe, Kornelia Polyak. KDM4C histone demethylase connects redox balance to chromatin remodeling via histone H3 tail clipping [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B008.

A phase II study of weekly carboplatin and nab-paclitaxel with trastuzumab or bevacizumab as neoadjuvant therapy for patients with early-stage nonmetastatic breast cancer.

e12623 Background: Neo-Adjuvant chemotherapy (NAC) allows patients to undergo breast conserving surgery, treats micro metastatic disease and improves patient outcomes. Achieving a complete pathologic response to NACT has prognostic significance in HER2+ and triple negative breast cancer. Anthracyclines are commonly integrated to Neo-adjuvant regimens. Their use is limited by cardiac and hematologic toxicity. We proposed that weekly Carboplatin and Nab-Paclitaxel is a well-tolerated regimen and aim to demonstrate that this approach can be a selective anthracycline sparing regimen with comparable improvement in recurrence free survival (RFS) and pathologic complete response (pCR). Methods: We conducted a single institution, open label, prospective Phase II clinical trial for patients with non-metastatic breast cancer who were eligible to receive NACT. All Patients with stage I-III breast cancers were treated with weekly Nab-paclitaxel (90 mg/m2) and carboplatin (AUC 2) for 12 weeks. Patients with HER2+ breast cancer received trastuzumab (4 mg/kg induction, followed by 2 mg/kg/week) weekly for 12 weeks. Triple negative and HR + patients received bevacizumab (10 mg/kg) every other week for 5 doses. Results: We enrolled 125 patients from February 2008 to December 2015. 85 pts were HER2- and 42 pts were HER2+. The pCR rate was 17.6% (15 pts) in the HER2- cohort and 50% (21 pts) in the HER2+ cohort. In the subgroup analysis based on hormone receptor (HR) status, pts with HR- tumor had more favorable response with higher rate of pCR compared with pts with HR+ tumor. In the HER2- cohort, the total pCR rate was 34% in HR- tumors vs 8% in HR+ tumors, P = 0.0018. After a median follow up of 53 months, overall survival was 88.9% for pts who achieved pCR and 77.7% for those who did not. HR = 0.3664, CI = 0.06147 to 2.1843, P = 0.34.In the HER2+ cohort the pCR rate was 33% in pts with HR+ tumors vs 72% in pts with HR- tumor, P = 0.0137. After a median follow up of 53 months, overall survival was 100% for pts who achieved pCR vs 20% in those who did not. The relapse-free survival was 90% vs 25% respectively (HR = 0.0669, CI = 0.00454 to 0.9251 P = 0.0016). In the entire study population, 6 pts (4.7%) had treatment discontinuation and 12 pts (9.4%) had dose reduction due to side effects. The rate of treatment disruption was higher in the HER2- cohort. Nausea, vomiting, and neutropenia were the most common side effects in both cohorts (7.9%) followed by thrombocytopenia and anemia (4.7%). Hematologic toxicity was the most common toxicity grade 3 and 4. Conclusions: Combination of nab-paclitaxel and carboplatin plus targeted therapy as an anthracycline sparing neoadjuvant chemotherapy showed a good pCR rate in HER2+ subgroup and moderate activity in TNBC subset. High rate of pCR was associated with high survival outcomes in HER2+ cohort. The regimen was well tolerated among both cohorts. Clinical trial information: NCT00618657 .

Identification of a minimum number of genes to predict triple-negative breast cancer subgroups from gene expression profiles

BackgroundTriple-negative breast cancer (TNBC) is a very heterogeneous disease. Several gene expression and mutation profiling approaches were used to classify it, and all converged to the identification of distinct molecular subtypes, with some overlapping across different approaches. However, a standardised tool to routinely classify TNBC in the clinics and guide personalised treatment is lacking. We aimed at defining a specific gene signature for each of the six TNBC subtypes proposed by Lehman et al. in 2011 (basal-like 1 (BL1); basal-like 2 (BL2); mesenchymal (M); immunomodulatory (IM); mesenchymal stem-like (MSL); and luminal androgen receptor (LAR)), to be able to accurately predict them.MethodsLehman’s TNBCtype subtyping tool was applied to RNA-sequencing data from 482 TNBC (GSE164458), and a minimal subtype-specific gene signature was defined by combining two class comparison techniques with seven attribute selection methods. Several machine learning algorithms for subtype prediction were used, and the best classifier was applied on microarray data from 72 Italian TNBC and on the TNBC subset of the BRCA-TCGA data set.ResultsWe identified two signatures with the 120 and 81 top up- and downregulated genes that define the six TNBC subtypes, with prediction accuracy ranging from 88.6 to 89.4%, and even improving after removal of the least important genes. Network analysis was used to identify highly interconnected genes within each subgroup. Two druggable matrix metalloproteinases were found in the BL1 and BL2 subsets, and several druggable targets were complementary to androgen receptor or aromatase in the LAR subset. Several secondary drug–target interactions were found among the upregulated genes in the M, IM and MSL subsets.ConclusionsOur study took full advantage of available TNBC data sets to stratify samples and genes into distinct subtypes, according to gene expression profiles. The development of a data mining approach to acquire a large amount of information from several data sets has allowed us to identify a well-determined minimal number of genes that may help in the recognition of TNBC subtypes. These genes, most of which have been previously found to be associated with breast cancer, have the potential to become novel diagnostic markers and/or therapeutic targets for specific TNBC subsets.

Immunologically “cold” triple negative breast cancers engraft at a higher rate in patient derived xenografts

TNBC is a heterogeneous subtype of breast cancer, and only a subset of TNBC can be established as PDXs. Here, we show that there is an engraftment bias toward TNBC with low levels of immune cell infiltration. Additionally, TNBC that failed to engraft show gene expression consistent with a cancer-promoting immunological state, leading us to hypothesize that the immunological state of the tumor and possibly the state of the immune system of the host may be essential for engraftment.

A phase 1b/2 study of the BET inhibitor ZEN-3694 in combination with talazoparib for treatment of patients with TNBC without gBRCA1/2 mutations.

1023 Background: Metastatic triple negative breast cancer (mTNBC) is an aggressive and heterogeneous cancer with limited therapeutic options. PARP inhibitors (PARPi), approved to treat patients with HER2- breast cancer with a germline BRCA1/2 (gBRCA1/2) mutation, have not shown efficacy in homologous recombination repair (HRR) proficient tumors. In pre-clinical models, the BET inhibitor (BETi) ZEN-3694 sensitizes wild-type (WT) BRCA1/2 tumors to PARPi through downregulation of HRR gene expression, providing a rationale for combination therapy. We previously reported results from the Ph 1b portion of the trial evaluating the combination of ZEN-3694 plus talazoparib, in TNBC patients without gBRCA1/2 mutations; here we present results from the completed Ph 1b/2 study. Methods: A Ph 1b dose finding portion (n = 15) was followed by a single arm Ph 2 Simon 2-stage portion (n = 17+20 (37)). The primary endpoint of the Ph 1b portion of the study was safety and recommended Ph 2 dose (RP2D). The secondary endpoints were pharmacokinetics (PK), pharmacodynamics (PD), and clinical benefit rate (CBR = confirmed objective response rate (ORR) + stable disease > 16 weeks). Ph 2 measured CBR as the primary endpoint, ORR and duration of response (DOR) as key secondary endpoints. Eligibility criteria for Ph 1b included TNBC (ER/PR < 10%, HER2-), WT gBRCA1/2, and > 1 prior cytotoxic regimen for mTNBC, and in the Ph2 portion ER/PR < 1% and < 2 prior cytotoxic regimens for mTNBC. Patients were dosed daily in continuous 28 day cycles until disease progression or unacceptable toxicity. Adverse events, PK, and PD in whole blood and tissue biopsies were assessed. Response endpoints were assessed per RECIST 1.1 every 2 cycles. Results: RP2D was determined to be 48mg qd ZEN-3694 plus 0.75mg qd talazoparib. The most common AE for the Ph 1b/2 study was thrombocytopenia (TCP) (55% any grade, 34% G3/4), which was managed with dose holds and reductions. Dose intensity analysis showed average daily doses of ZEN-3694 and talazoparib could be maintained above 40mg and 0.5mg, respectively, over 8 cycles. Robust target engagement was demonstrated using BET-dependent and HRR transcripts assessed in paired tumor biopsies. Ph 2 portion of the trial met its primary endpoint with a CBR of 30% (11/37). For the Ph 1b/2 trial, investgator assessed ORR was 22% (11/50), including 2 CR, CBR was 35% (18/51) and the median DOR was 24 weeks. For the subset of TNBC at diagnosis patients (no history of HR+ disease), ORR was 32% (11/34), and CBR was 44% (15/34). Conclusions: Combination of ZEN-3694 and talazoparib demonstrated anti-cancer activity in pretreated mTNBC WT gBRCA1/2 patients. All confirmed responses were observed in TNBC at diagnosis patients, whose tumors are expected to be more sensitive to the combination due to their basal-like properties. The trial is being expanded to Ph. 2b to accrue an additional 80 TNBC at diagnosis patients. Clinical trial information: NCT03901469.

Genomic features of rapid versus late relapse in triple negative breast cancer

BackgroundTriple-negative breast cancer (TNBC) is a heterogeneous disease and we have previously shown that rapid relapse of TNBC is associated with distinct sociodemographic features. We hypothesized that rapid versus late relapse in TNBC is also defined by distinct clinical and genomic features of primary tumors.MethodsUsing three publicly-available datasets, we identified 453 patients diagnosed with primary TNBC with adequate follow-up to be characterized as ‘rapid relapse’ (rrTNBC; distant relapse or death ≤2 years of diagnosis), ‘late relapse’ (lrTNBC; > 2 years) or ‘no relapse’ (nrTNBC: > 5 years no relapse/death). We explored basic clinical and primary tumor multi-omic data, including whole transcriptome (n = 453), and whole genome copy number and mutation data for 171 cancer-related genes (n = 317). Association of rapid relapse with clinical and genomic features were assessed using Pearson chi-squared tests, t-tests, ANOVA, and Fisher exact tests. We evaluated logistic regression models of clinical features with subtype versus two models that integrated significant genomic features.ResultsRelative to nrTNBC, both rrTNBC and lrTNBC had significantly lower immune signatures and immune signatures were highly correlated to anti-tumor CD8 T-cell, M1 macrophage, and gamma-delta T-cell CIBERSORT inferred immune subsets. Intriguingly, lrTNBCs were enriched for luminal signatures. There was no difference in tumor mutation burden or percent genome altered across groups. Logistic regression mModels that incorporate genomic features significantly outperformed standard clinical/subtype models in training (n = 63 patients), testing (n = 63) and independent validation (n = 34) cohorts, although performance of all models were overall modest.ConclusionsWe identify clinical and genomic features associated with rapid relapse TNBC for further study of this aggressive TNBC subset.

Abstract PO008: Stromal Tumor Infiltrating Lymphocytes (sTILs) as a putative prognostic marker to identify a responsive subset of TNBC in an Indian Breast Cancer Cohort

Abstract Objectives: Prognostic significance of stromal tumor infiltrating lymphocytes; sTILs is evaluated to identify a responsive subset of TNBC in an Indian cohort of breast cancer patients. Methods: A retrospective cohort of breast cancer patients from a single onco-surgeon breast cancer clinic treated with uniform treatment strategy across is evaluated for sTILs. FFPE tissue of primary tumor of invasive breast carcinoma are collected with ethical approvals. Tumor sections blinded for subtypes are stained with H&E and scored for sTILs by a pathologist following Immuno-Oncology TILs working groups scoring guidelines. Results: Analysis of 144 primary breast tumors for sTILs scores re-enforces significantly higher infiltration in TNBC tumors than HER2+ve and ER+ve tumors. Higher sTILs scores co-relate with gradually incremental pathological response to therapy specifically in TNBC subset and with better disease-free survival outcomes. Within TNBC, older and post-menopausal patients harbor higher scores of sTILs. Conclusion: Despite a small cohort of breast cancer patients, TNBC subtype reflected significantly higher scores of sTILs with better response to therapy and disease-free outcomes as compared to other breast cancer subtypes. A larger number of breast cancer patients from an Indian cohort will strengthen the findings to establish sTILs as a marker to identify a responsive subset of TNBC. Citation Format: Madhura Kulkarni, Pooja M. Vaid, Anirudha Puntambekar Puntambekar, Rituja A. Banale, Ruhi Reddy, Rohini Unde, Namrata Namewar, Devaki Kelkar, L.S. Shashidhara, C.B. Koppiker. Stromal Tumor Infiltrating Lymphocytes (sTILs) as a putative prognostic marker to identify a responsive subset of TNBC in an Indian Breast Cancer Cohort [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO008.

Abstract B02: Elevated YAP expression associates with EMT, stemness, and angiogenic properties of TNBC cell lines and recurrence in TNBC patients

Abstract Triple-negative breast cancer (TNBC) is considered one of the aggressive breast cancer subtypes with higher risk of recurrence and mortality compared to other subtypes. There is a need to identify molecular markers and drivers that could distinguish metastatic subset of TNBC. Based on our previous study (Kulkarni et al., Oncotarget 2018), we investigated for association of elevated YAP expression specifically in TNBC tissue samples and cell lines. Six TNBC cell lines with different degrees of YAP expression were selected for the study. Cell lines were grouped based on high, intermediate, or low nuclear YAP expression. After stable knockdown of YAP expression using retrovirus, these three groups of TNBC cell lines were assessed for migration, mammosphere formation, and angiogenic properties. Further, detailed characterization of media supernatants was carried out to identify angiogenic markers altered after YAP knockdown. Metastatic potential of the cell lines after YAP knockdown was assessed using mouse tail vein injection model for lung metastasis nodules and lung Mets were analyzed for angiogenic marker. With appropriate ethical approvals, frozen TNBC tumors, along with adjacent normal tissue, were analyzed for YAP and its angiogenic target expression. The expressions were co-related with clinical parameters such as age at diagnosis, grade, lymph node positivity, and recurrence. TNBC cell lines with high YAP and intermediate YAP expression showed significantly reduced migration and mammosphere formation after YAP knockdown. Similarly, media supernatants from high YAP and medium-YAP cell lines showed severely affected angiogenic potential after YAP knockdown. Fluidigm analysis for YAP-signature (Kulkarni et al., Oncotarget 2018) gene expression showed significantly reduced expression of YAP-target genes involved in EMT and stemness in these cell lines. Proteomic analysis of media supernatants revealed expression of two angiogenic factors to be downregulated after YAP knockdown in all 4 cell lines. Metastatic potential of high-YAP expressing cell line in mouse tail vein injection model was severely affected after YAP knockdown. The two TNBC cell lines with low YAP expression did not show any effect on mammosphere formation or angiogenic properties after YAP knockdown; neither had any effect on expression of the angiogenic targets. Patient samples with elevated YAP expression in the tumor compared to adjacent normal tissue showed strong association with younger age at diagnosis, high grade, and lymph node positivity. Our analysis of six TNBC cell lines has uncovered YAP to be significantly associated with increased mesenchymal properties of TNBC cell lines. Analysis of TNBC tissue samples strengthened this association of elevated YAP expression with aggressive clinical parameters within TNBC patients. With further validation, YAP expression can be employed as a prognostic marker to predict aggressive and recurrent TNBC for close follow-up and aggressive treatment planning. Citation Format: Madhura Kulkarni, Nurfarhanah Bte Syed Sulaiman, Supriya Srivastava, Tuan Zea Tan, Thomas Choudary Putti, Xiaomeng Wang, Wanjin Hong, Soo Chin Lee, Yoshiaki Ito. Elevated YAP expression associates with EMT, stemness, and angiogenic properties of TNBC cell lines and recurrence in TNBC patients [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B02.

Abstract P5-12-09: Patient-reported outcomes (PROs) during one year of adjuvant enzalutamide for the treatment of early stage androgen receptor positive (AR+) triple negative breast cancer

Abstract BACKGROUND: A subset of TNBC expresses a luminal signature and demonstrates AR-dependence. Enzalutamide (ENZA), an AR-antagonist, has activity with metastatic AR+ TNBC, with a clinical benefit rate of 33% (Traina et al, JCO 2018). We recently demonstrated that one year (y) of adjuvant ENZA is feasible in early stage, AR+ TNBC, with no new safety signals observed (Traina ASCO 2019). Fatigue was the most common, treatment (tx)-related, adverse event of any grade according to investigator assessment (24%). Here we report per-protocol planned secondary analyses of PROs during one year of ENZA including quality of life (QoL), fatigue, and self-reported physical activity (NCT02750358). METHODS: Eligible patients have Stage I-III, ER/PR <1%, HER2(-), AR ≥1% breast cancer and completed all planned standard of care surgery, chemotherapy and radiation within 6 months of study start. Tx = ENZA 160mg orally daily for 1y with the option to extend tx to 2y as per patient preference. Clinician-reported toxicities (NCI CTCAEv4) were assessed every 4 weeks for 12 weeks, then every 3 months. Total study N=50 patients. PROs were assessed every 12 weeks. We examined serial changes in overall QoL (functional assessment of cancer therapy – breast (FACT-B), fatigue (FACIT-fatigue) and exercise behavior (total exercise minutes/week over past 7 days) from pre-treatment and every 3 months thereafter until month 12. All analyses are exploratory and consist of descriptive summaries. RESULTS: Between 5/2016 and 6/2018, 50 patients were enrolled. As of 6/27/19, 34 patients completed 1y of ENZA; 15 patients are off tx: recurrence (3), toxicity (5), nonadherence (4), and consent withdrawal (3). Patients evaluable for PRO (N): baseline (50), 12 weeks (43) and 52 weeks (34). Median scores on FACT-B and fatigue scales remained stable over the course of adjuvant ENZA in the 1st year of therapy (Table). Self-reported exercise increased during this period. Table. Median scores (range) for PROs during 1 year of adjuvant ENZATime PointBaselineWeek 12Week 52FACT-G88.0 (53.5-105.0)82.0 (36.4-105.0)89.5 (58.0-105.0)Breast Cancer Subscale28.5 (15.6-39.0)29.0 (9.0-40.0)29.0 (19.0-40.0)Total118.0 (77.0-141.0)111.0 (45.4-145.0)121.0 (83.0-145.0)FACT-B trial outcome index73.0 (43.0-92.0)73.0 (25.7-96.0)76.9 (51.0-96.0)FACIT43.0 (17.0-52.0)39.0 (10.0-52.0)43.0 (11.0-52.0)Total Exercise (min/week)120 (0-1260)152 (0-3120)180 (0-2580) CONCLUSIONS: Patient reported outcomes of health related QOL remain stable when ENZA is administered for 1 year in the adjuvant setting. Self-reported time spent engaged in physical exercise increased numerically during 1y of therapy. During presentation, additional analyses will be reported including QOL at additional time points, relationship of FACT-B score with FACIT fatigue score and exercise, and QOL changes in relation to relative dose intensity of ENZA, toxicity and off study reason. Citation Format: Tiffany A Traina, Lee W Jones, Victoria Blinder, Jessica Scott, Leigh Ann Boyle, Artavazd Arumov, Tina Alano, Sujata Patil, Patricia DeFusco, Nicholas Lamparella, Mark Robson, Ayca Gucalp. Patient-reported outcomes (PROs) during one year of adjuvant enzalutamide for the treatment of early stage androgen receptor positive (AR+) triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-12-09.

A Predictor of Pathological Complete Response to Neoadjuvant Chemotherapy Stratifies Triple Negative Breast Cancer Patients with High Risk of Recurrence

We developed a test to predict which patients will achieve pathological complete response (pCR) to neoadjuvant chemotherapy (NAC) and which will have residual disease (RD). Gene expression data from pretreatment biopsies of patients with all breast cancer subtypes were combined into a 519-patient cohort containing 177 TNBC patients. Two RNA classifiers of 16 genes each were sequentially applied to the total cohort, classifying patients into 3 distinct classes. The test performance was further validated in an independent 304-patient cohort. The test accurately identified 70.5% (79/112) of pCR and 83.5% (340/407) of RD patients in the total population, and 75.0% (45/60) of pCR and 75.2% (88/117) of RD patients in the TNBC subset. For the independent cohort, the test identified 91.5% RD patients in the total population and 86.2% RD patients in the TNBC subset. However, the identification of pCR in both total and TNBC population are as low as 21.1% and 30%, respectively. The TNBC RD patients were subdivided by our classifiers, with one class showing significantly higher levels of Ki67 expression and having significantly poorer survival rates than the other classes. This stratification of patients may allow predicted residual disease classes to be assigned an alternative therapy.

Adjuvant enzalutamide for the treatment of early-stage androgen receptor-positive (AR+) TNBC.

546 Background: A subset of TNBC is dependent on AR signaling. Enzalutamide (ENZA), an AR-antagonist, has activity in patients (pts) with metastatic AR+ TNBC, with a clinical benefit rate of 33%. This study tests the feasibility of adjuvant ENZA for the treatment (tx) of early stage, AR+ TNBC. We now report the primary endpoint (endpt) and safety. Methods: Eligible pts have centrally confirmed, Stage I-III, ER/PR < 1%, HER2(-), AR ≥1% BC and completed all planned surgery, chemotx and radiation (RT) < 6 months of tx start. AR testing by IHC per MSK methods. Tx consists of ENZA 160mg daily for 1 year (y) with the option to extend tx to 2y. Toxicity per NCI CTCAEv4 every (q) 4 weeks (wk) for 12 wk, then q3 months. Primary endpt: feasibility of 1y ENZA defined as the discontinuation rate due to toxicity, consent withdrawal or tolerability. 50 pts are enrolled to have 46 evaluable pts required to discriminate between feasibility of 50% and 70%, with type I error 5% and 88% power. Pts who have disease progression (PD) or die during 1st y of ENZA and do not have tx discontinuation due to the above will not be included in the primary analysis. If 29 pts complete 1y, adjuvant ENZA will be deemed feasible. Secondary endpts: safety and 3y DFS and OS. Exploratory endpts: PROs and biomarker development. Results: Between 5/2016-6/2018, 50 pts were enrolled. Pt and tumor characteristics (N = 50): Median age 55y (33-81); Stage: I 20 (40%), II 23 (46%), III 7 (14%); Grade (gr): 2 = 26%, 3 = 74%. AR > 10% = 35 (70%), AR ≤10% = 15 (30%). Chemotx 47/50 (94%): Neoadjuvant (neo) 40%, Adjuvant (adj) 60%; Anthracycline/Taxane-based 38/47 (81%), Platinum 1/47 (2%), Docetaxel/Cyclophosphamide 3/47 (6%), other 5/47 (11%). 13/19 who received neo tx failed pCR; 9/13 (69.2%) received adj capecitabine. RT: 38/50 (76%). 27 pts completed 1y of tx. 7 pts will be evaluable by 6/1/19. 1 pt to complete 1y 6/21/19. 15 pts are off tx: PD (3), toxicity (5), noncompliance (4), withdrawal of consent (3). Tx-related AEs, any gr, > 10% (N = 50): fatigue (48%), hot flashes (22%), headache (18%), hyperglycemia (18%), nausea (18%), WBC decreased (16%), dizziness (14%), arthralgia (12%), dyspnea (12%). Tx-related, gr 3 AEs: fatigue (6%), hyperglycemia (2%), hypertension (2%). No gr 4/5 AEs or seizures. 11 pts had dose reduction. Conclusions: Feasibility of adjuvant ENZA will be fully evaluable in 4/2019 and is anticipated to meet the prespecified statistical expectations for primary endpt. ENZA is well tolerated following locoregional tx and standard of care systemic tx. Secondary analyses and correlatives are ongoing to define the role of AR in TNBC. Clinical trial information: NCT02750358.

Protein arginine methyltransferase 5: A novel therapeutic target for triple-negative breast cancers.

TNBC is a highly heterogeneous and aggressive breast cancer subtype associated with high relapse rates, and for which no targeted therapy yet exists. Protein arginine methyltransferase 5 (PRMT5), an enzyme which catalyzes the methylation of arginines on histone and non‐histone proteins, has recently emerged as a putative target for cancer therapy. Potent and specific PRMT5 inhibitors have been developed, but the therapeutic efficacy of PRMT5 targeting in TNBC has not yet been demonstrated. Here, we examine the expression of PRMT5 in a human breast cancer cohort obtained from the Institut Curie, and evaluate the therapeutic potential of pharmacological inhibition of PRMT5 in TNBC. We find that PRMT5 mRNA and protein are expressed at comparable levels in TNBC, luminal breast tumors, and healthy mammary tissues. However, immunohistochemistry analyses reveal that PRMT5 is differentially localized in TNBC compared to other breast cancer subtypes and to normal breast tissues. PRMT5 is heterogeneously expressed in TNBC and high PRMT5 expression correlates with poor prognosis within this breast cancer subtype. Using the small‐molecule inhibitor EPZ015666, we show that PRMT5 inhibition impairs cell proliferation in a subset of TNBC cell lines. PRMT5 inhibition triggers apoptosis, regulates cell cycle progression and decreases mammosphere formation. Furthermore, EPZ015666 administration to a patient‐derived xenograft model of TNBC significantly deters tumor progression. Finally, we reveal potentiation between EGFR and PRMT5 targeting, suggestive of a beneficial combination therapy. Our findings highlight a distinctive subcellular localization of PRMT5 in TNBC, and uphold PRMT5 targeting, alone or in combination, as a relevant treatment strategy for a subset of TNBC.

GSK3\u03b2 regulates epithelial-mesenchymal transition and cancer stem cell properties in triple-negative breast cancer

BackgroundTriple-negative breast cancers (TNBCs), which lack receptors for estrogen, progesterone, and amplification of epidermal growth factor receptor 2, are highly aggressive. Consequently, patients diagnosed with TNBCs have reduced overall and disease-free survival rates compared to patients with other subtypes of breast cancer. TNBCs are characterized by the presence of cancer cells with mesenchymal properties, indicating that the epithelial to mesenchymal transition (EMT) plays a major role in the progression of this disease. The EMT program has also been implicated in chemoresistance, tumor recurrence, and induction of cancer stem cell (CSC) properties. Currently, there are no targeted therapies for TNBC, and hence, it is critical to identify the novel targets to treat TNBC.MethodsA library of compounds was screened for their ability to inhibit EMT in cells with mesenchymal phenotype as assessed using the previously described Z-cad reporters. Of the several drugs tested, GSK3β inhibitors were identified as EMT inhibitors. The effects of GSK3β inhibitors on the properties of TNBC cells with a mesenchymal phenotype were assessed using qRT-PCR, flow cytometry, western blot, mammosphere, and migration and cell viability assays. Publicly available datasets also were analyzed to examine if the expression of GSK3β correlates with the overall survival of breast cancer patients.ResultsWe identified a GSK3β inhibitor, BIO, in a drug screen as one of the most potent inhibitors of EMT. BIO and two other GSK3β inhibitors, TWS119 and LiCl, also decreased the expression of mesenchymal markers in several different cell lines with a mesenchymal phenotype. Further, inhibition of GSK3β reduced EMT-related migratory properties of cells with mesenchymal properties. To determine if GSK3β inhibitors target mesenchymal-like cells by affecting the CSC population, we employed mammosphere assays and profiled the stem cell-related cell surface marker CD44+/24− in cells after exposure to GSK3β inhibitors. We found that GSK3β inhibitors indeed decreased the CSC properties of cell types with mesenchymal properties. We treated cells with epithelial and mesenchymal properties with GSK3β inhibitors and found that GSK3β inhibitors selectively kill cells with mesenchymal attributes while sparing cells with epithelial properties. We analyzed patient data to identify genes predictive of poor clinical outcome that could serve as novel therapeutic targets for TNBC. The Wnt signaling pathway is critical to EMT, but among the various factors known to be involved in Wnt signaling, only the higher expression of GSK3β correlated with poorer overall patient survival.ConclusionsTaken together, our data demonstrate that GSK3β is a potential target for TNBCs and suggest that GSK3β inhibitors could serve as selective inhibitors of EMT and CSC properties for the treatment of a subset of aggressive TNBC. GSK3β inhibitors should be tested for use in combination with standard-of-care drugs in preclinical TNBC models.

Estrogen receptor beta increases sensitivity to enzalutamide in androgen receptor-positive triple-negative breast cancer.

Androgen receptor (AR) is playing an important role in the progression of a subset of TNBC. We evaluated the impact of ERβ expression along with anti-AR drugs in AR-positive TNBC. ERβ expression was examined in AR-positive TNBC cell line using MTT assay, scratch and Annexin V-FITC assay in the presence or absence of anti-androgens. Protein levels of involved molecules were assessed using Western blot. Receptors' localization was detected by immunofluorescence and their physical association was examined using proximity ligation assay (PLA), which enables the visualization of interacting proteins in fixed cells and tissues. Transient transfection of ERβ in MDA-MB 453 AR-positive TNBC cell line significantly inhibited cell proliferation, metastatic potential and induced apoptosis. ERβ expression reversed the aggravating role of AR in both indirect and direct ways. Indirectly, ERβ decreased AR activation through the inhibition of PI3K/AKT signaling pathway. Directly, ERβ formed heterodimers with AR in MDA-MB 453 cells and in human tissue samples impeding AR from forming homodimers. Enzalutamide is a more potent anti-androgen in AR + TNBC compared to bicalutamide. ERβ expression increased the sensitivity of MDA-MB 453 cells to anti-androgens and especially to enzalutamide. The administration of enzalutamide enhanced AR:ERβ heterodimers formation increasing the anti-tumor capacity of ERβ. Collectively, our results provide evidence for a novel mechanism by which ERβ exerts oncosuppressive effect in AR-positive TBNC through direct and indirect interactions with AR. Moreover, ERβ expression may identify a new subset of TNBC that would respond more favorable to anti-androgens.

Abstract P5-05-08: Molecular cross-talk between retinoic acid and NOTCH1 signaling pathways: Role in triple negative breast cancer

Abstract Triple Negative Breast Cancer (TNBC) represents 10-20% of all breast cancers and it is characterized by poor prognosis and high recurrence rate. The heterogeneity of the disease and the absence of well-defined molecular targets have so far challenged successful therapeutic strategies. NOTCH1 has been found to act as a driver oncogene in a small subset of TNBC characterized by constitutive activation of the protein, acting as a transcription factor. Preclinical studies support an anti-tumor activity of All-Trans Retinoic Acid (ATRA) in specific subsets of breast cancer. By screening a large panel of breast cancer cell lines recapitulating the heterogeneity of TNBC we identify a specific subset sensitive to the anti-proliferative activity of ATRA. These cell lines (N-TNBC cell lines) are characterized by a Notch1 intragenic fusion transcript conferring gain of function activity to the protein. Indeed, sequence analysis reveals that the cell lines harbor an interstitial deletion in the NOTCH1 gene encompassing the negative regulatory region (NRR) domain. These cell lines depend on Notch active signaling for their proliferation since their cell growth is impaired by Notch inhibitors (γ-secretase inhibitors, e.g. DAPT, PF-3084014). Proliferation assays reveal that ATRA and γ-secretase inhibitors act synergistically in inhibiting cancer cell growth in N-TNBC cell lines suggesting the existence of a cross talk between the molecular pathways engaged by retinoic acid and NOTCH1. By using retinoic acid receptors (RARs) agonists and antagonists as well as RAR specific silencing experiments we identify RARα as the retinoic acid receptor responsible of the anti-proliferation activity of ATRA in N-TNBC cell lines. In particular N-TNBC cell lines respond to RARα activation by inducing high amounts of the onco-supressor protein RARβ. This feature is unique in ATRA sensitive TNBC cell lines and does not occur in ATRA sensitive luminal cell lines arguing for the existence of a retinoic acid specific mechanism of action in N-TNBC. Since RARs act as transcription factors inside the cells, to gain insights into the molecular pathway at the basis of the observed ATRA/NOTCH1 cross talk, we performed RNAseq analysis of ATRA and/or DAPT treated N-TNBC cells. Gene set enrichment analysis reveal that ATRA is able to directly affect NOTCH1 transduction pathway by modulating the expression of NOTCH1 target genes. In particular, in two out of three N-TNBC cell lines ATRA directly inhibits the NOTCH1 expression at a transcriptional level and its downregulation is increased by ATRA/DAPT combinations. Pathway analysis has allowed the identification of putative molecular hubs responsible for the synergistic effects observed and therefore likely at the basis of the crosstalk between ATRA/NOTCH pathways. These findings are of clinical interest since both the retinoid and NOTCH signaling display crucial physiologic activities and their pleiotropic effects could impinge on the success of therapeutic options based on their pathway modulation. The newly discovered specificity of ATRA action in the context of NOTCH1 addicted TNBC provides new tools for the identification of patients candidates benefitting from strategies targeting the ATRA/NOTCH axis. Citation Format: Paroni G, Zanetti A, Bolis M, Vallerga A, Troiani M, Fratelli M, Kurosaki M, Terao M, Garattini E. Molecular cross-talk between retinoic acid and NOTCH1 signaling pathways: Role in 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 P5-05-08.

Abstract 3337: Nuclear S1P binds PASB domain of HIFα to facilitate HIFα-HIF1β heterodimer formation in hypoxic TNBC cells

Abstract Triple-negative breast cancers (TNBCs) have increased expression of hypoxia-inducible factor 1α (HIF1α) and HIF-direct genes that associated with decreased overall survival in patients. Previously we have shown that nuclear sphingosine-1-phosphate (S1P), a lipophilic pro-cancer signal mediator, a product of the catalytic reaction by sphingosine kinase 2 (SphK2), directly binds to and inhibits histone deacetylases-1 and -2 (HDAC1 and 2), thus, altered epigenetic landscape in breast cancer cells. SphK2/S1P signaling is essential for metastatic TNBC cell growth and survival. We hypothesize that SphK2/S1P epigenetically regulates HIFα functions in TNBC cells; TNBC subset with elevated SphK2 and HIF1α would be a marker for SphK2-mediated HIFα targeted therapy. METABRIC cohort gene expression analysis revealed that the mRNA expression of HIF1α mRNA and downstream angiogenesis gene, vascular endothelial growth factor B (VEGFB) are associated with overall poor survival for TNBC patients compared to non-TNBC patients. Here, we demonstrate that a subset of TNBC patient tissue samples nuclei has elevated HIF1α and SphK2 protein expressions. SphK2/S1P markedly enhances hypoxia-induced HIFα proximal promoter histone acetylation. SphK2/S1P is found in the nuclear protein complexes with HIFα. Moreover, S1P is docked well at the PASB domain of HIFα and facilitate HIFα-HIF1β heterodimer formation in hypoxia. SphK2/S1P markedly augmented HIF1α direct target gene HIF2α and VEGFA promoter occupancy in hypoxia. Down-regulation of SphK2 with siRNA markedly attenuated transcription of HIFα-direct target genes associated with cancer stem cells, angiogenesis, and metastasis. In agreement with an oncogenic role of SphK2, selective inhibition of SphK2 markedly reduced nuclear histone acetylation, HIF1α expression, and in vivo human TNBC tumor growth in mice. Our data strongly suggested that TNBC subset with elevated SphK2 and HIF1α would be a good candidate for SphK2 targeted therapy. Supported by Health Research, Inc. (HRI) Grant 71-4084-01 (NCH). Citation Format: Nitai C. Hait, Aparna Maiti. Nuclear S1P binds PASB domain of HIFα to facilitate HIFα-HIF1β heterodimer formation in hypoxic TNBC cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3337.

Beta blockers and improved progression-free survival in patients with advanced HER2 negative breast cancer: a retrospective analysis of the ROSE/TRIO-012 study

Recent retrospective studies suggest that beta-adrenergic blocking drugs (BB) are associated with improved outcomes in patients with a range of cancers. Although limited and discordant data suggest that BB may increase overall survival (OS) in localized breast cancer (BC), there is no information on the effects of BB in women with advanced BC. To explore the association between BB use and BC outcomes, we retrospectively reviewed ROSE/TRIO-012, a double-blinded, multinational phase III trial that randomized 1144 patients with HER2-negative advanced BC to first-line docetaxel in combination with ramucirumab or placebo. We compared progression-free survival (PFS), OS, overall response rate, and clinical benefit rate in patients who received BB to those who did not. 153/1144 (13%) patients received BB; 62% prior to enrolment and 38% began after enrolment. Median PFS in BB treated patients was longer than in patients who did not receive them (10.3 versus 8.3 months; HR 0.81; 95% CI 0.66-0.99; P = 0.038). Patients treated with BB only after enrolment had even higher median PFS (15.5 versus 8.3 months, P < 0.001). In the TNBC subset, median PFS was 13.0 months with BB, compared to 5.2 months without BB (HR 0.52; 95% CI 0.34-0.79; P = 0.002). The benefit of BB intake in PFS was independent of treatment-emergent hypertension (P = 0.476) but associated with treatment arm (P = 0.037). The test for interactions between BB and treatment arm was not significant (P = 0.276). No differences were seen in OS, overall response rate, or clinical benefit rate. A validation dataset analysis had consistent but less substantial improved outcomes for women with node positive operable breast cancer receiving BB in the BCIRG-005 trial. In this exploratory analysis, BB intake was associated with significant improvement in PFS, particularly in patients with TNBC and patients not previously exposed to BB. NCT00703326.

Abstract 1236: Novel androgen receptor inhibitors in triple negative breast cancer

Abstract Triple negative breast cancer (TNBC), which lacks expression of hormone receptors and human epidermal growth factor receptor 2, has the worst prognosis of all breast cancer subtypes, due to inherent chemo-resistance and a lack of therapeutic targets. A subset of TNBC has been shown to express androgen receptor (AR) but despite promising pre-clinical data, clinical trials evaluating the use of approved anti-androgens (bicalutamide, enzalutamide) in AR-positive TNBC has shown modest efficacy to date. In prostate cancer, bicalutamide binds to AR with low affinity and in the presence of AR gene amplification or mutation, can exhibit partial agonist activity leading to resistance. In order to increase affinity for AR and to avoid AR antagonist to agonist conversion synthesized 7-substituted umbelliferones (UMB) derivatives with a distinct scaffold modified from available AR antagonists (Kandil et al Bioorganic &amp; Medicinal Chemistry Letters, 2016). These agents exhibited inhibitory activity in human prostate cancer cells lines at sub-micro molar level, a 50-fold and 30-fold improvement over bicalutamide and enzalutamide, respectively. We hypothesized that these novel AR antagonists would be effective in inhibiting the growth of AR-positive TNBC cells. Methods: TNBC AR negative mesenchymal MDA-MB-231 (as control) and AR positive MDA-MB-231 cells were utilized for determination of the efficacy of the UMB derivatives. For proliferation assays, viability of the cells was monitored over a period of 72 hours with redox dye Cell Titer-Blue. Standard Western blotting techniques were used for protein detection. For apoptosis analysis, standard flow cytometry reagents, FITC labelled Annexin V and Propidium Iodine(PI) mix were utilized. Results: UMB derivatives decreased the expression of AR in AR-positive MDA-MB-453 in a dose dependent manner. UMB derivatives, at micro Molar concentration decreased cell proliferation of AR-positive and interestingly AR-negative cell lines by 20 to 50%, also in a dose dependent manner. UMB derivatives induced apoptosis in both cell lines. Interestingly, UMB derivatives induced a change from a mesenchymal to epithelial phenotype in AR-negative MDA-MB-231 cells, which was confirmed by decreased expression of vimentin and other mesenchymal markers. Conclusions: Novel UMB derivatives inhibit growth and cause apoptosis in TNBC cells and induce mesenchymal-to-epithelial transition in an AR-negative mesenchymal TNBC cell line. Targeting AR with novel AR antagonists seem to be a promising therapeutic approach for AR-positive TNBC and further evaluation of AR antagonists in AR-negative mesenchymal TNBC is warranted. Citation Format: Saswati Bhattacharya, Yuanqi Cai, Steven Kurina, Pamela Westmark, Benjamin Hokanson, Matthew Anderson, Ruth O’Regan. Novel androgen receptor inhibitors in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1236. doi:10.1158/1538-7445.AM2017-1236

Abstract 420: Preclinical evaluation of the SF3B1 inhibitor E7107 in triple negative breast cancer

Abstract Triple-negative breast cancers (TNBC) are the most aggressive subtype of breast cancer and account for roughly 15% of human breast cancers. No effective targeted therapies are currently available against these tumors, at least in part due to their genetic and epigenetic heterogeneity. Yet, TNBCs are commonly enriched for cells arrested in a progenitor-like state. We therefore performed a genome-wide siRNA lethality screen to identify selective TNBC dependencies associated with this particular phenotype in vitro. These studies led to the identification of several spliceosome genes, including the core splicing factor SF3B1, as selectively and recurrently essential for progenitor-like (or basal-A) TNBC cell lines. On this basis, we examined the antitumor effect of E7107, a clinically tested SF3B1 inhibitor, on multiple TNBC cell lines. In vitro, treatment with E7107 for 24-48 hr selectively killed basal-A cell lines relative to differentiated luminal cell lines, decreasing viability by ≈40% or more in 5 of 6 basal-A lines tested. E7107-treated cells appeared to die of apoptosis based on immunoblotting for multiple apoptotic markers. In mice, E7107 (5 mg/kg i.v. for 4 consecutive days) suppressed the growth of preformed tumors by ≈80% and ≈50% in 2 different cell-line-derived xenograft models and was remarkably well-tolerated at this dose. Of note, tumor response in vivo closely mirrored cell sensitivity in vitro, suggesting that in vitro analysis provides a fair estimate of TNBC response to E7107 in mice. Amongst 2 additional patient-derived xenograft (PDX) models tested, E7107 inhibited tumor growth by ≈50% in one model. Thus, E7107 was therapeutically active against 3 of 4 xenograft models tested. Preliminary mechanistic studies in vitro and in vivo pointed to MCL1 inactivation as a major mechanism of action of E7107 in top responders. Together, these data suggest that the spliceosome is a druggable target in TNBC, and that E7107 or similar SF3B1 inhibitors could be effective in a TNBC subset that needs to be further defined. Citation Format: Praveen Sridhar, Stefanie Chan, Ying-Jie Lock, Fabio Petrocca. Preclinical evaluation of the SF3B1 inhibitor E7107 in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 420. doi:10.1158/1538-7445.AM2017-420

Abstract OT2-01-17: A Phase II randomized trial of pembrolizumab with carboplatin and gemcitabine for treatment of patients with metastatic triple-negative breast cancer (mTNBC)

Abstract Background: Treatment for mTNBC is limited, and significant challenges persist in treating this disease, as outcomes remain largely dependent on chemotherapy without any effective targeted treatment. Pembrolizumab (MK-3475) is a highly selective, humanized monoclonal antibody against PD-1, blocking the negative immune regulatory signaling of the PD-1 receptor that is usually expressed by T-cells. Recent data showed that some patients with mTNBC may benefit from immune-based therapies (PD-1 or PD-L1 antibodies). Cumulative evidence suggest that stromal tumor infiltrating lymphocytes (sTILs) have a prognostic and predictive role in response to treatment in subsets of TNBC, particularly in response to carboplatin use. Preclinical data revealed that blocking PD-1/PD-L1 pathway in combination with platinum containing cytotoxic therapy improved response rates and survival. High levels of sTILs and an increased PD-L1 expression make mTNBC a candidate for PD-1–targeted therapy. As studies showed that the subset of TNBC with better response rates to carboplatin are heavily infiltrated with sTILs, pembrolizumab, becomes a very attractive drug to be tested in combination with carboplatin, with the goal of improving outcomes in mTNBC. A Phase II multicenter, randomized, trial has been initiated to evaluate the efficacy and safety of combining pembrolizumab with carboplatin and gemcitabine in patients with mTNBC. Methods: A safety run-in will assess the safety and tolerability of combining pembrolizumab with carboplatin and gemcitabine in patients with mTNBC. Following the completion of the safety run-in, patients will be randomized 2:1 to receive pembrolizumab (200 mg IV) on day 1 along with carboplatin (AUC 2, day 1 and day 8, IV) plus gemcitabine (800 mg/m2, day 1 and day 8, IV) of a 21-day cycle, or carboplatin plus gemcitabine (same aforementioned dose) alone. Patients will have histologically documented unresectable mTNBC. Prior systemic therapy for mTNBC, for up to 2 lines is allowed, and patients will have ECOG PS 0–2 and measurable disease (RECIST v1.1). Prior carboplatin/gemcitabine or cisplatin therapy is allowed in the adjuvant or neoadjuvant setting, as long as it occurred more than 12 months from the beginning of their enrollment. Subjects whose tumors progressed while on treatment with carboplatin or cisplatin are excluded. Known CNS disease (except asymptomatic treated metastases), autoimmune disease or prior immune checkpoint blockade therapy is an exclusion to enrollment on this trial. Primary endpoint is assessing the objective response rate according to RECIST v1.1 . Other endpoints include clinical benefit rate (CBR), progression-free survival (PFS), overall survival (OS), duration of response (DOR), and safety. Tumor biopsies will be obtained at baseline and just prior to initiation of cycle 3 to assess biomarkers of response and immune escape. PD-L1 expression will be evaluated in exploratory analysis with a planned assessment of response based on PD-L1 status. This trial will enroll 6-12 patients in the safety run-in portion, and 75 patients in the randomized part, at 7 sites in the United States. Clinical trial information: NCT02755272 www.clinicaltrials.gov. Citation Format: Obeid E, Miller KD, Sparano JA, Blackwell K, Goldstein LJ. A Phase II randomized trial of pembrolizumab with carboplatin and gemcitabine for treatment of patients with metastatic triple-negative breast cancer (mTNBC) [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 OT2-01-17.