Basal-like Research Articles

Abstract P4-06-03: Unravelling triple-negative breast cancer tumor microenvironment heterogeneity using an integrative multiomic analysis

Abstract Introduction: Triple negative breast cancer (TNBC) constitute 10-20% of all breast cancers and is associated with a worse prognosis and limited treatment options. Recent trials evaluating immune checkpoint blockade in TNBC demonstrated encouraging results for a subset of patients. TNBC is highly heterogeneous and its tumour microenvironment (TME) has been recognized as a critical determinant of its behavior and clinical outcome. Genome-wide gene expression profiling analyses have already improved our understanding of the complexity of this disease and have defined 6 different molecular subtypes namely Basal-like 1 (BL1), basal-like 2 (BL2), immunomodulatory (IM), mesenchymal (M), mesenchymal stem-like (MSL) and luminal androgen receptor (LAR), exhibiting distinct biological and clinical characteristic. In this study, we aim to dissect the molecular diversity of the TME and more specifically to assess the immune landscape according to TNBC molecular subtypes. Methods: A cohort of 485 TNBC patient with publicly available data (RNA-Seq and Illumina HT-12 v3) from the METABRIC and the TCGA consortia were used in the gene expression analysis. Gene signatures reflecting different features or cellular components (immune, stromal, angiogenesis, lymphangiogenesis, hypoxia, metabolism) of the TME were used to evaluate multiple biological processes known to contribute to tumorogenesis. A compendium of 17 immune specific gene signatures and T cell localisation classification were used to evaluate the immune composition and spatial pattern of immune infiltrates. All parameters were compared using a logistic regression model to evaluate their relative contribution according to each molecular subtype. Results: Our analyses demonstrated that each molecular subtype exhibits different TME profiles, as well as specific immune composition and localisation. IM tumors were associated with the highest expression of immune-related gene signatures, enriched with adaptive immune cells and with a fully inflamed spatial pattern. MSL tumors were mostly associated with the expression of Lymphangiogenesis and Stromal TME signatures. They also exhibited some immune activity through the expression of immune gene signatures capturing innate immune and adaptive immunosuppressive cells. This subtype was mainly associated with margin restricted and to some extent with fully inflamed spatial pattern. BL1 tumors were associated with the expression of Metabolism TME signatures, along with fully inflamed and stroma restricted spatial pattern. To a lesser extent, this subtype was also associated with activated DC and CD4 Tem cells. LAR and M tumors exhibited an immune cold phenotype. They were associated with Stromal and Metabolism TME signatures, enriched in margin restricted spatial pattern and negatively associated with every immune cells. Conclusions: Our results demonstrate for the first time the huge heterogeneity that characterizes the TME of TNBCs. Identification of specific TME profiles could help to design more rationale and appropriate synergistic therapeutic combinations targeting TME elements in this high-risk disease. Citation Format: Bareche Y, Buisseret L, Gruosso T, Girard E, Venet D, Dupont F, Desmedt C, Park M, Rothé F, Stagg J, Sotiriou C. Unravelling triple-negative breast cancer tumor microenvironment heterogeneity using an integrative multiomic analysis [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 P4-06-03.

Abstract P4-07-03: Immunomodulation of triple negative breast cancer by caffeic acid phenethyl ester (CAPE)

Abstract Background: CAPE is the major active component of propolis, a widely available, safe, honeybee natural product with anti-inflammatory, antioxidant, and antitumor properties. We have previously shown diverse effects of CAPE in breast cancer. We postulated that CAPE may be useful in primary or secondary prevention for triple-negative breast cancers (TNBC) and evaluated this in a Trp53 null mammary chimera model in which the tumor spectrum is shifted to TNBC when the host is irradiated (Nguyen, 2011). Bioinformatics species comparisons show that TNBC from this model exhibits a spectrum of tumors that is molecularly similar to that seen in human TNBC (Nguyen, 2013). Methods: The model consists of Trp53 null mammary epithelium transplanted to cleared mammary glands of wildtype mice. Mice irradiated with 1 Gy received transplants 3 days later and were placed on a CAPE or a control diet at 1month post transplantation that was maintained throughout the experiment. Tumor development was monitored by palpation for up to 18 months. Tumors were immunostained for estrogen receptor (ER) status and ER negative tumors were selected from all the groups for RNA sequencing. TNBC molecular subtypes were determined for control (n=5) and CAPE treated (n=9) samples using methods described by Lehman, 2011. Gene expression signatures consisting of 270 genes was analyzed for functional enrichment to identify patterns of signaling, as well as additional tumor characteristics, in each cohort of tumors. Results: Host irradiation significantly accelerated tumor growth rate compared to the control sham irradiated mice. CAPE treatment blocked this effect, but did not affect the control tumor growth rate. Resected tumors in CAPE treated mice recurred at significantly longer intervals (40 days in control group versus 90 days with CAPE) and much less frequently than tumors from mice on a control diet. Mean expression analysis showed that CAPE induces a distinct gene expression pattern in ER negative tumors from irradiated mice. As previously described (Illa-Bochaca, 2014), ER negative tumors from sham irradiated mice on a control diet were enriched in immune response genes. These genes were suppressed in tumors arising in irradiated host, but this effect of host irradiation was abrogated in mice on the CAPE diet. While untreated tumors were classified into the Basal-like 2 (BL2) or Mesenchymal TNBC molecular subtypes, we observed an increase in subtype diversity after CAPE treatment with tumors being classified as Immunomodulatory and Luminal Androgen Receptor (LAR) in addition to BL2 and Mesenchymal subtypes. Consistent with these data, functional enrichment analyses of RNA sequencing data identified increased immune signaling as well as upregulation of a number of signaling pathways including TGFβ, HGF, and EGFR in treated samples. Conclusion: These findings support the potential use of CAPE to modify the aggressive behavior of TNBC, which may be due to effects on the immune system in which CAPE acts to re-establish anti-tumor immunity. The finding that CAPE treatment shifts the tumor spectrum to Immunomodulatory subtypes suggests that it may be useful both for women at high risk for TNBC and to prevent or delay TNBC breast cancer recurrence, perhaps in combination with immunotherapy. Citation Format: Omene C, Patel M, Karagoz K, Gatza ML, Barcellos-Hoff MH. Immunomodulation of triple negative breast cancer by caffeic acid phenethyl ester (CAPE) [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 P4-07-03.

Abstract P2-08-19: Exploratory biomarker analyses of FAIRLANE, a double-blind placebo (PBO)-controlled randomized phase II trial of neoadjuvant ipatasertib (IPAT) + paclitaxel (PAC) for early triple-negative breast cancer (TNBC)

Abstract Background: The oral AKT inhibitor IPAT is being evaluated in cancers with a high prevalence of PI3K/AKT pathway activation. In the PBO-controlled randomized phase II FAIRLANE trial (NCT02301988), adding IPAT to PAC as neoadjuvant therapy for TNBC led to a numerical increase in pathologic complete response (pCR) in unselected patients (17.1% vs 13.3%), with a greater treatment effect in patients with PIK3CA/AKT1/PTEN-altered tumors (17.9% vs 11.8%). The addition of IPAT also led to an increase in complete response (CR) by MRI (27.6% vs 13.3%) that was enhanced in patients with PIK3CA/AKT1/PTEN-altered tumors (39.3% vs 8.8%) [Oliveira, AACR 2018]. We report an exploratory analysis performed to provide better understanding of potential biomarkers for response. Methods: Pretreatment tumor samples were evaluated for genomic alterations using the FoundationOne® (Foundation Medicine) assay (n=144) and gene expression by RNA-Seq (n=92). Samples were classified into TNBC subtypes based on the method developed by Lehmann and Pietenpol [Lehmann, J Clin Invest 2011]. Tumor-infiltrating lymphocytes (TILs) were quantified using the Salgado method [Salgado, Ann Oncol 2015] (n=135). Results: Of 62 patients (43%) with PIK3CA/AKT1/PTEN-altered tumors, 21 had an activating mutation in PIK3CA or AKT1 and 47 had an alteration in PTEN (6 [3 in each arm] had both PIK3CA mutation and PTEN alteration). Although only 3 patients with PIK3CA/AKT1-mutant tumors achieved a pCR, there was an increased rate of MRI CR with the addition of IPAT to PAC [Table]. In patients with PTEN alterations, both pCR rate and MRI CR rate were increased with IPAT. In patients treated with PBO + PAC, all 4 pCR patients evaluable by RNA-Seq were of the immunomodulatory (IM) subtype. However, in the IPAT + PAC arm, pCRs were also seen in patients with basal-like 1 (BL-1), mesenchymal (M), and mesenchymal stem-like (MSL) subtypes. Consistent with this observation, in the PBO + PAC arm, samples from patients achieving a pCR had significantly higher levels of stromal TILs than those from patients who did not have a pCR, while no difference was observed in the IPAT + PAC arm. Response, n (%)PIK3CA/AKT mutation (n=21)PTEN alteration (n=47) IPAT + PAC (n=11)PBO + PAC (n=10)IPAT + PAC (n=21)PBO + PAC (n=26)pCR1 (9%)2 (20%)4 (19%)3 (12%)CR by MRI5 (45%)1 (10%)8 (38%)2 (8%) Conclusions: This retrospective exploratory biomarker analysis of the phase II FAIRLANE trial of neoadjuvant IPAT for TNBC provides insight into the potential heterogeneity of response and resistance to taxane therapy. The results also hint that response to PAC alone is dependent on baseline immune infiltration and that this dependency might be relieved with the addition of AKT inhibition. Citation Format: Wongchenko MJ, Oliveira M, Saura C, Nuciforo P, Calvo I, Andersen J, Passos Coelho JI, Gil Gil M, Bermejo B, Patt DA, Ciruelos E, Singel SM, Maslyar DJ, Xu N, de la Peña L, Baselga J, Gendreau S, Isakoff SJ. Exploratory biomarker analyses of FAIRLANE, a double-blind placebo (PBO)-controlled randomized phase II trial of neoadjuvant ipatasertib (IPAT) + paclitaxel (PAC) for early triple-negative breast cancer (TNBC) [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 P2-08-19.

Abstract P2-06-09: Identification of oncogenic gene fusions in triple-negative breast cancer metastatic to the brain

Abstract BACKGROUND: As many as 24% of breast cancer (BC) patients develop metastasis to the brain and 27% of these occur in patients with triple negative (TN) BC. The brain (#2) and lung (#1) are the most common sites of TNBC metastasis. Half of women with metastatic (m) TNBC will die within one year. Novel therapies for women with mTNBC, especially to the brain, are desperately needed. TNBCs often exhibit genomic instability, resulting in gene fusions and other chromosomal rearrangements. Such alterations are long recognized as oncogenic drivers and effective drug targets in hematologic and other solid malignancies. We aimed to identify these alterations in mTNBC with Segmental Transcript Analysis (STA), a novel population-based algorithm ranking genes based on likelihood of harboring a gene rearrangement by identifying abrupt changes in transcript expression. RESULTS: We performed RNAseq on 25 mTNBC, 4 matched primary BC, 2 normal cerebellum and 2 normal cerebral cortex samples. TNBC subtype analysis showed an enrichment of basal-like (BL) 2 and mesenchymal (M) subtypes among mTNBC to the brain compared to primary (p) BC in the TCGA. A principal component analysis demonstrated mTNBC and their matched primaries clustered together, indicating the pairs were more closely related to each other genetically than to other mTNBCs. When the gene expression patterns of pBC were compared with their brain metastases following adjustment for contamination with normal brain tissue, an upregulation of mesenchymal genes (p63/p73 axis) and primitive/embryonic genes (NOTCH and PAX family) were noted in the brain mTNBC. We confirmed a modest to marked upregulation of p63 expression by immunohistochemistry across all TNBC subtypes, most notably in BL1 and BL2. STA found three gene rearrangements: EHF-WT1, NF1-VWDE and JAG1-Chr20. Two of these are potential driver gene rearrangements. The first, in a tumor of M subtype, results from fusion of the 5'UTR of EHF to WT1. The resulting fusion transcript is predicted to encode a truncated WT1 protein; loss of the N-terminal regulatory region of WT1 would likely cause constitutive activation. We also identified a truncating rearrangement in the Notch ligand JAG1 in a tumor of LAR subtype. JAG1 is a membrane bound ligand for the Notch receptor and the breakpoint occurs just distal to the DSL domain essential for Notch interaction. This rearrangement occurs within an intergenic chromosomal region, introduces an abrupt stop codon and generates a truncated protein lacking a transmembrane domain. An intact signal peptide should localize the protein to the endoplasmic reticulum; however, loss of the transmembrane domain would likely lead to secretion rather than membrane insertion. This alteration is predicted to constitutively stimulate Notch activity in a paracrine fashion. If the rearrangement functions in this manner, neutralizing antibodies to the N-terminal portion of JAG1 should have therapeutic efficacy. DISCUSION: EHF-WT1 and JAG1-Chr20 are potential driver rearrangements novel to brain mTNBC. The JAG1 rearrangement predicts constitutive production of Notch transcription factors and is potentially targetable. Continued interrogation of mTNBC for discovery of additional oncogenic fusions driving BC biology is warranted. Citation Format: Gonzalez-Ericsson P, Lehmann B, Mobley B, Chen Y-Y, Pietenpol J, Sanders ME. Identification of oncogenic gene fusions in triple-negative breast cancer metastatic to the brain [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 P2-06-09.

High Glucose Represses the Anti-Proliferative and Pro-Apoptotic Effect of Metformin in Triple Negative Breast Cancer Cells.

Metformin, the most widely prescribed anti-diabetic drug, is shown to possess anti-cancer potential in treatment of cancers, including breast cancer; decreases breast cancer risk; and improves overall survival. However, reports suggest that higher glucose concentrations may negatively impact the anti-cancer efficacy of metformin. Therefore, we examined the anti-cancer potential of metformin in triple-negative breast cancer cells (TNBCs) exposed to different glucose (25 mM, 5.5 mM and zero glucose/glucose-starved) conditions. Our data indicates that a high glucose (25 mM) concentration (mimicking diabetes) significantly abrogated the effect of metformin on cell proliferation, cell death and cell cycle arrest in addition to loss of efficacy in inhibition of the mTOR pathway, a key metabolic pathway in TNBC cells. The mTOR pathway is activated in TNBCs compared to other subtypes of breast cancer, regulates the synthesis of proteins that are critical for the growth and survival of cancer cells and its activation is correlated to poor outcomes among TNBC patients, while also contributing to metastatic progression and development of resistance to chemotherapy/radiotherapy. Our studies were performed in two different types of TNBCs, MDA-MB-231 cells (mesenchymal stem cell-like (MSL)) and MDA-MB-468 (basal like-1 (BL-1)). Interestingly, lower concentrations of metformin (50, 100, 250, and 500 μM) significantly increased cell proliferation in 25 mM glucose exposed MDA-MB-231 cells, an effect which was not observed in MDA-MB-468 cells, indicating that the effective concentration of metformin when used as anti-cancer drug in TNBCs may have to be determined based on cell type and blood glucose concentration. Our data indicates that metformin treatment was most effective under zero glucose/glucose-starved conditions in MDA-MB-468 with a significant increase in the apoptotic population (62.3 ± 1.5%; p-value < 0.01). Under 5.5 mM glucose conditions in both MDA-MB-231 and MDA-MB-468 cells our data showed reduced viability of 73.56 ± 2.53%; p-value < 0.05 and 70.49 ± 1.68%; p-value < 0.001, respectively, along with a significant increase in apoptotic populations of both cell types. Furthermore, metformin (2 mM) inhibited the mTOR pathway and its downstream components under zero glucose/glucose-starved conditions indicating that using metformin in combination with agents that inhibit the glycolytic pathway should be more beneficial for the treatment of triple-negative breast cancers in diabetic individuals.

Rates of immune cell infiltration in patients with triple-negative breast cancer by molecular subtype.

In patients with triple-negative breast cancer (TNBC), tumor-infiltrating lymphocytes (TILs) are associated with improved survival. Lehmann et al. identified 4 molecular subtypes of TNBC [basal-like (BL) 1, BL2, mesenchymal (M), and luminal androgen receptor (LAR)], and an immunomodulatory (IM) gene expression signature indicates the presence of TILs and modifies these subtypes. The association between TNBC subtype and TILs is not known. Also, the association between inflammatory breast cancer (IBC) and the presence of TILs is not known. Therefore, we studied the IM subtype distribution among different TNBC subtypes. We retrospectively analyzed patients with TNBC from the World IBC Consortium dataset. The molecular subtype and the IM signature [positive (IM+) or negative (IM-)] were analyzed. Fisher’s exact test was used to analyze the distribution of positivity for the IM signature according to the TNBC molecular subtype and IBC status. There were 88 patients with TNBC in the dataset, and among them 39 patients (44%) had IBC and 49 (56%) had non-IBC. The frequency of IM+ cases differed by TNBC subtype (p = 0.001). The frequency of IM+ cases by subtype was as follows: BL1, 48% (14/29); BL2, 30% (3/10); LAR, 18% (3/17); and M, 0% (0/21) (in 11 patients, the subtype could not be determined). The frequency of IM+ cases did not differ between patients with IBC and non-IBC (23% and 33%, respectively; p = 0.35). In conclusion, the IM signature representing the underlying molecular correlate of TILs in the tumor may differ by TNBC subtype but not by IBC status.

SY27-2 - The development of TNBC treatment through overcoming heterogeneity

The treatment of TNBC is challenging due to the lack of effective treatments except for chemotherapies. Various research has evaluated TNBC's heterogeneity, establishing a consensus that TNBC is not a single unit but rather a biologically heterogeneous group. To seek optimal treatments, we have tried to divide the heterogeneous TNBC into homogeneous subtypes or identified mutations that have the potential to be therapeutic targets. These findings are reduced in the clinic to developing and conducting clinical trials focused on the molecularly targeted therapies. To overcome TNBC heterogeneity and seek optimal treatments, we cannot ignore biological features and must establish biology-oriented treatment strategies. Lehmann et al. identified seven molecular subtypes--basal-like 1 (BL1), basal-like 2 (BL2), immunomodulatory (IM), mesenchymal (M), mesenchymal stem-like (MSL), luminal androgen receptor (LAR), and unstable (UNS)--in 2011 on the basis of gene ontologies and differential gene expression and recently reported refinement TNBC 4 subtypes that removed IM and MSL subtypes because they confirmed these two subtypes represent tumors with substantial infiltrating lymphocytes and tumor-associated mesenchymal cells, respectively. We have confirmed these subtypes using the same methodology and indicated treatment strategies in each group. As a next step, to use this molecular information in a clinical setting, an identification tool for TNBC subtypes is under development. Further, evaluation of the mutations and mutation burden is an essential leading to the use of PARP inhibitor and immune checkpoint inhibitors. There are several new treatment agents based on antibody-drug conjugates have in TNBC. The learning objectives of this talk are as follows: 1. To understand the recent development of novel treatment approaches for TNBC 2. To acknowledge the importance of TNBC molecular subtyping and other new classification, which may impact the outcome of treatment developmet.

Abstract 3698: Unraveling triple-negative breast cancer molecular heterogeneity using an integrative multiomic analysis

Abstract Introduction : Triple negative breast cancers (TNBCs) represent about 10-20% of all breast cancers and show worse prognosis compared to other molecular subtypes with increased likelihood of early distant recurrence and death. Recent efforts of genome-wide gene expression profiling analyses have improved our understanding of the biological complexity and diversity of TNBCs reporting, at least 6 different molecular subtypes of TNBC namely Basal-like 1 (BL1), basal-like 2 (BL2), immunomodulatory (IM), mesenchymal (M), mesenchymal stem-like (MSL) and luminal androgen receptor (LAR). However, little is known regarding the potential driving molecular events within each subtype, their difference in survival and response to therapy. Further insight into the underlying genomic alterations is therefore needed. Aims : Here, we aimed to study the genomic aberrations that drive each of these TNBC subtypes by applying an integrated analysis of somatic mutation, copy number and gene expression of 550 TNBC derived from Molecular Taxonomy of Breast Cancer International (METABRIC) and The Cancer Genome Atlas (TCGA) consortia. Methods : The METABRIC &amp; the TCGA datasets were used, with available data for copy-number aberrations, somatic mutations and gene expression. ER, HER2 and PR status were inferred based on the bimodality of their mRNA expression level. TNBC samples (n=550) were classified according to Lehmann's molecular subtypes using the TNBCtype online subtyping tool (http://cbc.mc.vanderbilt.edu/tnbc). Results : We were able to globally reproduce Lehmann's classification with Basal-like 1 (BL1), Immunomodulatory (IM), Androgen-Receptor-like (LAR), Mesenchymal-like (M) and Mesenchymal Stem Cell-like (MSL) being the most stable TNBC subtypes. Each subtype showed significant clinic-pathological characteristic differences. Using a multivariate model, the IM and LAR subtypes showed to be associated with a better prognosis (HR=0.68; CI=0.46-0.99; p=0.043) and a worst prognosis (HR=1.47; CI=1.0-2.14; p=0.046), respectively. BL1 subtype was found to be most genomically instable subtype with high TP53 mutation (92%) and copy-number deletion in genes involved in DNA repair mechanism (BRCA2, MDM2, PTEN, RB1 &amp; TP53). LAR tumours were associated with higher mutational burden with significantly enriched mutations in PI3KCA (55%), AKT1 (13%) and CDH1 (13%) genes. M and MSL subtypes were associated with higher signature score for angiogenesis. IM showed high expression levels of immune signatures and check-point inhibitor genes such as PD1, PDL1 and CTLA4. Conclusions : Our findings highlight for the first time the substantial genomic heterogeneity that characterize TNBC molecular subtypes, allowing for a better understanding of the disease biology as well as the identification of several candidate targets paving novel approaches for the development of anti-cancer therapeutics for TNBC. Citation Format: Yacine Bareche, David Venet, Philippe Aftimos, Michail Ignatiadis, Martine Piccart, Francoise Rothe, Christos Sotiriou. Unraveling triple-negative breast cancer molecular heterogeneity using an integrative multiomic analysis [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 3698.

Abstract 5800: Vorinostat and indole-3-carbinol modulate microRNAs and histone deacetylase activity in subtypes of triple-negative breast cancer

Abstract Triple-negative breast cancer is a highly heterogeneous and aggressive subtype of breast cancer. Epigenetic drugs are continuing to emerge as promising therapies for triple-negative breast cancer (TNBC). These drugs, including the one used in this study, vorinostat, a histone deacetylase inhibitor (HDAC), are capable of multiple actions and are considered multifunctional drugs. Epigenetic mechanisms have been shown to play a role in the etiology of this cancer, through modulation of DNA methylation, histone modification or microRNAs. We have shown in this study that vorinostat and indole-3-carbinol (I3C) modulated the expression of microRNAs 221 and 222 differently in basal-like-2 (BL2), MSL, basal-like1 (BL1) and M subtypes of TNBC cells. Additional studies demonstrated that combination of tamoxifen and vorinostat significantly decreased expression of miRNA 221 in TNBC cell lines. MiRNAs are short noncoding RNAs that regulate the function of target genes post-transcriptionally. In addition, breast cancer microarrays further showed modulation of a number of other miRNAs that play a role in cell proliferation, drug resistance, cell invasion and metastasis. MicroRNAs are epigenetic targets for drug development. Using a global HDAC activity assay, it was also shown that subtypes of TNBC cells respond differently to vorinostat and I3C treatment, depending on their HDAC activity. HCC70, an African American cell line and a BL2 subtype, had the highest HDAC activity and its activity was significantly decreased by vorinostat treatment compared to BT549 (M), HCC 1806 (BL2), MCF7 (ER positive) and a basal nonmalignant cell line, MCF10A. Both vorinostat and I3C show great promise alone and in combination as effective agents in triple-negative breast cancer. Histone deacetylase inhibitors and dietary agents are emerging as therapy for triple-negative breast cancer and are currently in clinical trials. Citation Format: Beverly Lyn-Cook, Fatemeh NouriEmamzaden, Anfernee Hawkins, George Hammons, Beverly Word. Vorinostat and indole-3-carbinol modulate microRNAs and histone deacetylase activity in subtypes of triple-negative breast cancer [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 5800.

Abstract 157: Targeting cancer stem-like cells in triple negative breast cancer

Abstract Triple Negative Breast Cancer (TNBC), an aggressive, heterogeneous subtype of breast cancer, and immunohistochemically negative for estrogen receptor α (ER-), progesterone receptor (PR-), and lacking amplification of the human epidermal growth factor receptor 2 locus (HER2-). TNBC patients have high risk of recurrence and metastasis, and current treatment options remain limited. Cancer stem-like cells (CSCs) have been linked to cancer initiation, progression and chemotherapy resistance. Therefore CSC-targeted therapies are keenly sought. There is strong evidence for the involvement of Notch signaling in TNBC. Notch1 is highly expressed in Basal-like 1 (BL1) and especially Mesenchymal-Stem-Like (MSL) TNBCs. Expression of Notch1 and its ligand Jagged1 correlates with poor prognosis. Moreover, strong evidence supports key roles of different Notch paralogs in breast CSCs. Moreover the role of non-canonical Notch signaling in TNBC remains unknown. Here, we demonstrate that Notch1 promotes cell survival in MDA-MB-231 cells, representative of MSL TNBC, in part by activating NF-κB. Notch activation by Jagged1-expressing stromal cells enhances transcription of the anti-apoptotic gene cIAP-2 (BIRC3), a known NF-κB target. This event is dependent on recruitment of NF-κB subunits, IKKα and Notch1 to the cIAP-2 promoter. Short term exposure of MDA-MB-231 cells (MSL, PTEN wild-type), but not MDA-MB-468 cells (BL1, PTEN-null) to recombinant Jagged1 leads to AKT phosphorylation. This is suppressed by dual mTORC1/2 inhibitors, AKT inhibitors and IKKα inhibitors but not Everolimus (mTORC1-selective inhibitor). These observations support a model where canonical and non-canonical mechanisms downstream of Notch1 trigger AKT phosphorylation and NF-κB activation in PTEN wild type TNBC cells. This suggests a bidirectional crosstalk between the IKKα and AKT arms of this Jagged1-activated pathway. We also demonstrate that recombinant Jagged1 increases the cellular metabolism of TNBC cells and knockdown of Notch1 or IKKα by siRNA decreases mitochondrial respiration and glycolysis. We have found that CSCs derived from MDA-MB-231 cells have increased Notch1, p-AKT, and mitochondrial respiration. AKT inhibition or NF-κB inhibition decreases both mitochondrial respiration and glycolysis of TNBC derived CSCs. Pharmacological inhibition of Notch cleavage by gamma secretase inhibitor (PF-03084014) in combination with AKT inhibitor (MK-2206) or NF-κB inhibitor (Bay11-7082) blocks CD90hi or CD44+CD24low sorted secondary mammospheres formation. These data suggest that combination treatments affecting Notch, NF-kB or AKT pathways have potential therapeutic importance in targeting CSCs of TNBC. Citation Format: Fokhrul Hossain, Ayse D Ucar Bilyeu, Claudia Sorrentino, Judy Crabtree, Antonio Pannuti, Margarite Matossian, Matthew Burow, Todd Golde, Barbara Osborne, Lucio Miele. Targeting cancer stem-like cells in triple negative breast cancer [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 157.

Abstract 2598: In silico gene expression analysis of PTHrP and its association with molecular subtypes and organ-specific metastasis in human triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) represents 10-20% of all BC cases, and is characterized by an aggressive clinical course with high risk of metastasis and lack of targeted therapy. Gene expression profiling revealed the heterogeneity of TNBC enabling its classification into distinct molecular subtypes with distinct susceptibilities to chemotherapies; including basal-like (BL), mesenchymal (M), and luminal androgen receptor (LAR) subtypes. Overexpression of the parathyroid hormone-related protein (PTHrP) in breast cancer has been extensively linked to its progression with increased propensity for bone metastasis. However, its expression and implication in organ-specific metastasis in the TNBC subtype remains largely unknown. In this study, we conducted in silico analyses to examine the association of PTHrP with various molecular BC subgroups and with organ-specific metastasis in TNBC. Breast Cancer Gene-Expression Miner Version 4.0 (bc-GenExMiner v4.0) online database was used to evaluate the relative mRNA expression levels of the PTHLH gene (which encodes PTHrP) with respect to other markers. This microarray-based tool uses 36 public datasets and 5861 patients, and allows the expression, correlation, and prognostic analyses of genes in BC. Using the gene expression correlation analysis module of bc-GenExMiner v4.0, we assessed Pearson's correlation coefficient between PTHLH expression and gene signatures characteristic of TNBC molecular subtypes or representative of organ-specific metastasis in BC. We found that PTHLH expression displays significant positive correlations with components of signalling pathways enriched in the mesenchymal subtype, and with key luminal markers and AR signalling genes characteristic of the LAR subtype. While PTHLH expression presents significant positive correlations with signature genes involved in bone and lung metastases in all BC subtypes, we identified for the first time a correlation between PTHLH expression and brain metastasis specifically in TNBC patients. Interestingly, PTHLH correlates with the brain metastatic genes HBEGF (Heparin-binding EGF-like growth factor) and ANGPTL4 (Angiopoietin-like 4) selectively in both TNBC and BL subtypes as opposed to other BC subtypes, which is in line with studies reporting their common morphological and genetic features and increased rate of brain metastasis. In conclusion, our in silico analysis reveals for the first time a strong association between PTHrP expression and specific TNBC molecular subtypes' markers, as well as its potential role in the mechanisms of TNBC brain metastasis and the identification of a novel gene signature with a prognostic value for brain progression in TNBC. Funding: Department of Defense (DoD, USA) Award No. W81XWH-15-1-0723 Citation Format: Gloria Assaker, Anne Camirand, Siham Sabri, Richard Kremer. In silico gene expression analysis of PTHrP and its association with molecular subtypes and organ-specific metastasis in human triple-negative breast cancer [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 2598.

Clinicopathological, immunohistochemical and molecular correlation of neural crest transcription factor SOX10 expression in triple-negative breast carcinoma

The transcription factor SOX10 mediates the differentiation of neural crest-derived cells, and SOX10 by immunohistochemistry (IHC) is used primarily for the diagnosis of melanoma. SOX10 expression has been previously documented in benign breast myoepithelial cells. However there is limited literature on its expression in triple-negative breast carcinoma (TNBC). The aim was to study the clinical, pathologic and molecular profiles of SOX10+ tumors in TNBC. Tissue microarrays of TNBC were evaluated for SOX10 expression in 48 cases. SOX10 expression was correlated with clinical and pathologic features such as age, grade, and stage. Gene expression was analyzed on RNA extracted from formalin-fixed paraffin-embedded (FFPE) specimens with Affymetrix 2.0 HTA. Co-expression of SOX10 with androgen receptor (AR), WT1, gross cystic disease fluid protein-15 (GCDFP-15), mammaglobin, epidermal growth factor receptor (EGFR), CK5/6 and GATA transcription factor 3 (GATA3) were also assessed. The mean age was 59.38 (range, 28-90 years). Overall, 37.5% cases (18/48) were SOX10+. There was no association between SOX10 expression and age, grade or stage of patients; 6 of 10 (60%) cases of basal-like 1 (BL1), and 5 of 8 cases of unstable (UNS) molecular subtype were SOX10+. One of 5 basal-like-2 (BL2), 1 of 6 immunomodulatory (IM), 1 of 4 mesenchymal (M), 1 of 5 luminal androgen receptor (LAR) and 2 of 8 mesenchymal stem cell (MSL) showed lower frequencies of SOX10 expression. There was negative correlation between SOX10 and AR+ subtypes (P < .002). SOX10 was positively correlated with WT1 (P = .05). SOX10 did not show significant correlation with mammaglobin, GCDFP15, EGFR, CK5/6 and GATA3. SOX10 expression in the basal-like and unstable molecular subtypes supports the concept that these neoplasms show myoepithelial differentiation.

Triple negative breast cancer subtypes and pathologic complete response rate to neoadjuvant chemotherapy.

Triple negative breast cancer (TNBC) is a heterogeneous disease with distinct molecular subtypes that differentially respond to chemotherapy and targeted agents. The purpose of this study is to explore the clinical relevance of Lehmann TNBC subtypes by identifying any differences in response to neoadjuvant chemotherapy among them. We determined Lehmann subtypes by gene expression profiling in paraffined pre-treatment tumor biopsies from 125 TNBC patients treated with neoadjuvant anthracyclines and/or taxanes +/- carboplatin. We explored the clinicopathological characteristics of Lehmann subtypes and their association with the pathologic complete response (pCR) to different treatments. The global pCR rate was 37%, and it was unevenly distributed within Lehmann’s subtypes. Basal-like 1 (BL1) tumors exhibited the highest pCR to carboplatin containing regimens (80% vs 23%, p=0.027) and were the most proliferative (Ki-67>50% of 88.2% vs. 63.7%, p=0.02). Luminal-androgen receptor (LAR) patients achieved the lowest pCR to all treatments (14.3% vs 42.7%, p=0.045 when excluding mesenchymal stem-like (MSL) samples) and were the group with the lowest proliferation (Ki-67≤50% of 71% vs 27%, p=0.002). In our cohort, only tumors with LAR phenotype presented non-basal-like intrinsic subtypes (HER2-enriched and luminal A). TNBC patients present tumors with a high genetic diversity ranging from highly proliferative tumors, likely responsive to platinum-based therapies, to a subset of chemoresistant tumors with low proliferation and luminal characteristics.

Incidence of PI3K pathway aberrations and their impact on response to neoadjuvant chemotherapy (NACT) in triple-negative breast cancer (TNBC) subtypes.

588Background: TNBC cell lines characterized as mesenchymal (M) and luminal androgen receptor (LAR) commonly have aberrations in the PI3K pathway. However, the incidence in human tumors and impact on response to therapy is less clear. Methods: Pre-treatment biopsies were collected from TNBC patients (pts) prior to NACT. Tumors were categorized into 5 groups using the Pietenpol criteria (Lehmann JCI 2011): basal-like (BL) comprised of BL-1 and BL-2, M comprised of M and mesenchymal stem-like, immunomodulatory (IM), LAR, or unspecified (UNS). Using RNAseq data, variants were identified in 16 PI3K pathway genes: AKT1, AKT2, AKT3, CRKL, IRS2, MTOR, PIK3CA, PIK3CG, PIK3R1, PTEN, RICTOR, RPTOR, RNF43, TSC1, TSC2. Results: Data was available in 63 pts (N = 4 stage I; N = 41 stage II; N = 18 stage III). There was no significant association between stage and subtype. 67 PI3K pathway variants were identified in 39 (62%) tumors. The incidence of mutated tumors and their differential response to NACT defined by resid...

Characterization and Targeting Cancer Stem‐like Cells in Triple Negative Breast Cancer

Triple negative breast cancer (TNBC) is a heterogeneous group of clinically aggressive diseases. TNBC patients have high risk of recurrence and metastasis, and current treatment options remain limited. Cancer stem‐like cells (CSCs) have been linked to cancer initiation, progression and chemotherapy resistance. Therefore, CSC‐targeted therapies are keenly sought. There is strong evidence for the involvement of canonical‐Notch signaling in TNBC, however the role of non‐canonical Notch signaling in TNBC remains unknown. Notch1 is highly expressed in Basal‐like 1 (BL1) and especially Mesenchymal (M) TNBCs. Expression of Notch1 and its ligand Jagged1 correlate with poor prognosis. Moreover, strong evidence supports key roles of different Notch paralogs in breast CSCs. Here, we demonstrate that Notch activation by Jagged1‐expressing stromal cells enhances transcription of the anti‐apoptotic gene cIAP‐2 (BIRC3), a known NF‐κB target. This event is dependent on recruitment to the cIAP‐2 promoter of NF‐κB subunits, IKKα and Notch1. Short term exposure of MDA‐MB‐231 cells (M, PTEN wild‐type), but not MDA‐MB‐468 cells (BL1, PTEN‐null) to recombinant Jagged1 leads to AKT phosphorylation. This is suppressed by AKT inhibitors, IKK inhibitors, and dual mTORC1/2 inhibitors but not an mTORC1‐selective inhibitor. These observations support a model where canonical and non‐canonical mechanisms downstream of Notch1 trigger AKT phosphorylation and NF‐κB activation in PTEN wild type TNBC cells. Importantly, we find co‐localization of Notch1 with Mitochondria in MDA‐MB‐231 cells by confocal microscopy and Western blot of isolated mitochondrial fractions. We demonstrate that recombinant Jagged1 increases the metabolism of TNBC cells. Knockdown of Notch1 or IKKα by siRNA decreases mitochondrial respiration and glycolysis. CSCs derived from MDA‐MB‐231 cells have increased Notch1, p‐AKT, and oxidative metabolism compared to non‐stem cells. AKT inhibition or IKK inhibition decreases both mitochondrial respiration and glycolysis of TNBC derived CSCs. Pharmacological inhibition of Notch cleavage by gamma secretase inhibitor (PF‐03084014) in combination with AKT inhibitor (MK‐2206) or IKK inhibitor (Bay11‐7082) blocks CD90hi or CD44+CD24low sorted secondary mammospheres formation. Notably, we find similar results in TNBC patient derived xenograft (PDX) models. However, Notch inhibition may have adverse effects on tumor immunity, due to the role of Notch in CD4 and CD8 T‐cell activation. Thus, we explored an immunocompetent tumor mouse model to study the effects of these pharmacological interventions in that setting. We developed an immunocompetent TNBC mouse model by injecting TNBC mouse cells (C0321) into mammary fat pads of isogenic FVB female mice. Combination treatments with GSI (PF‐03084014) and AKT inhibitor (MK‐2206) significantly delayed tumor growth as well as decreased CSCs number in that model. These data suggest that combination treatments affecting the intersection of Notch and AKT pathways have potential therapeutic applications in targeting CSCs in TNBC cases with high Notch1 expression. The possible immunological effects of such combination treatments require additional investigation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Unravelling triple-negative breast cancer molecular heterogeneity using an integrative multiomic analysis

BackgroundRecent efforts of genome-wide gene expression profiling analyses have improved our understanding of the biological complexity and diversity of triple-negative breast cancers (TNBCs) reporting, at least six different molecular subtypes of TNBC namely Basal-like 1 (BL1), basal-like 2 (BL2), immunomodulatory (IM), mesenchymal (M), mesenchymal stem-like (MSL) and luminal androgen receptor (LAR). However, little is known regarding the potential driving molecular events within each subtype, their difference in survival and response to therapy. Further insight into the underlying genomic alterations is therefore needed. Patients and methodsThis study was carried out using copy-number aberrations, somatic mutations and gene expression data derived from the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) and The Cancer Genome Atlas. TNBC samples (n=550) were classified according to Lehmann’s molecular subtypes using the TNBCtype online subtyping tool (http://cbc.mc.vanderbilt.edu/tnbc/). ResultsEach subtype showed significant clinic-pathological characteristic differences. Using a multivariate model, IM subtype showed to be associated with a better prognosis (HR=0.68; CI=0.46–0.99; P=0.043) whereas LAR subtype was associated with a worst prognosis (HR=1.47; CI=1.0–2.14; P=0.046). BL1 subtype was found to be most genomically instable subtype with high TP53 mutation (92%) and copy-number deletion in genes involved in DNA repair mechanism (BRCA2, MDM2, PTEN, RB1 and TP53). LAR tumours were associated with higher mutational burden with significantly enriched mutations in PI3KCA (55%), AKT1 (13%) and CDH1 (13%) genes. M and MSL subtypes were associated with higher signature score for angiogenesis. Finally, IM showed high expression levels of immune signatures and check-point inhibitor genes such as PD1, PDL1 and CTLA4. ConclusionOur findings highlight for the first time the substantial genomic heterogeneity that characterize TNBC molecular subtypes, allowing for a better understanding of the disease biology as well as the identification of several candidate targets paving novel approaches for the development of anticancer therapeutics for TNBC.

Abstract P2-13-01: Differential efficacies of DNA damaging agents in basal-like TNBC subtypes

Abstract New approaches are needed to improve long term survival for patients with metastatic triple negative breast cancer (TNBC). Women with metastatic TNBC have a worse 3-year survival compared to women with metastatic HER2+ or ER/PR+ disease.1 Identifying the molecular vulnerabilities of TNBC subtypes could provide for optimal treatment strategies. Gene expression analyses of TNBC patient samples by Lehmann and Bauer, et al. identified 6 subtypes of TNBC.2 Based on the analysis of laser dissected tumor samples, the number of tumor subtypes was reduced from 6 to 4.3 Both analyses identified two types of basal-like cancers, basal-like 1 (BL1) and basal-like 2 (BL2).2,3 The BL1 subtype is defined by the amplified expression of genes involved in cell division and DNA damage response, and RNA polymerase.2 BL2 cancers express high levels of genes involved in growth factor signaling, glycolysis, and gluconeogenesis.2 They show that cell lines representative of the BL1 and BL2 subtypes of TNBC are more sensitive to cisplatin than cell lines representing the other TNBC subtypes. The BL1 or BL2 categorization, however, failed to predict sensitivity to olaparib.2 BL1 and BL2 representative cell lines responded differently when treated with veliparib; BL1 cells were more sensitive than BL2 cells.2 We investigated the response of BL1 and BL2 cells to a variety of DNA damaging agents to determine if the BL1 or BL2 subtype was predictive of sensitivity to a particular drug. DNA damaging agents, including alkylating agents, anti-metabolites, topoisomerase inhibitors, and PARP inhibitors were tested in cells resenting the BL1 and BL2 subtypes. HCC1806 cells (BL2) were found to be selectively sensitive to gemcitabine when compared to HCC1937 cells (BL1). The panel of drugs and cell lines was expanded to further understand the sensitivity of BL1 and BL2 subtypes to a variety of DNA damaging agents. Our goal is to provide preclinical evidence for the informed use of specific DNA damaging agents in the treatment of patients with BL1 or BL2 subtypes of TNBCs. 1. Li, X. et al. Breast Cancer Res. Treat. 161, 279–287 (2017). 2. Lehmann, B. D. et al. J. Clin. Invest. 121, 2750–2767 (2011). 3. Lehmann, B. D. et al. PLoS One 11, e0157368 (2016). Citation Format: Shaffer CV, Robles AJ, Risinger AL, Mooberry SL. Differential efficacies of DNA damaging agents in basal-like TNBC subtypes [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 P2-13-01.

Abstract PD8-02: Mechanisms of recurrence: Paired analysis of primary and metastatic triple negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC) is the most aggressive subtype of invasive breast cancer that lacks ER, PR, and HER2 expression. It is a heterogeneous disease with several molecular subtypes: basal-like1 (BL-1), basal-like 2 (BL-2), mesenchymal (M), and luminal androgen receptor (LAR). Treatment for TNBC is normally limited to chemotherapy, and relapse is common. Here we report molecular alterations associated with TNBC metastasis by analyzing the genomic profiles of paired primary and metastatic TNBCs. Methods:50 paired TNBCs were identified through an IRB-approved protocol via the City of Hope (COH) Biospecimen Repository. DNA mutation and mRNA expression profiles of 10 paired primary and metastatic TNBCs were analyzed. DNA mutations were identified using exome sequencing by FoundationOne®. Affymetrix Human Genechip 2.0 was used for mRNA expression profiling. Raw data were normalized and processed using Expression Console, and linear regression was performed using Limma to identify the differentially expressed genes between primary and metastatic TNBCs. Results: DNA mutation profiling showed that multiple mutations occurred within genes covering pathways of PI3K/AKT/mTOR, DNA repair, RAS/MAPK, cell cycle, and growth factor receptor signaling, reconfirming genomic heterogeneity of TNBCs. Gene expression profiles were analyzed for Lehmann's TNBC molecular subtypes (BL-1, BL-2, M, and LAR). Six of ten TNBCs showed phenotype shift between the primary and metastatic TNBCs. Several unique gene expression patterns were identified by comparing the paired TNBCs. CCNE1 and TPX2 were co-overexpressed in metastatic TNBCs compared to paired primaries. This mirrored prior studies in ovarian cancer, where co-overexpression of CCNE1 and TPX2 were found related to clonal resistance against chemotherapy. Splicing factors TRA2B and SRSF7 were also over-expressed in metastatic TNBCs compared with primaries. The analysis studying the association of CCNE1 and TPX2 with overall survival is ongoing using TCGA. Conclusion: Overall, these results show the comparative changes between primary and relapsed TNBCs and indicate the heterogeneity of molecular mechanisms of recurrence. CNNE1 and TPX2 may represent important genes involved in TNBC metastasis. Further analyses including a total of 50 paired TNBCs are currently underway. Study Contact: Yuan Yuan MD PhD, City of Hope Comprehensive Cancer Center; Duarte, CA 91030; Email: yuyuan@coh.org Citation Format: Zhao Z-M, Yost S, Yuan Y-C, Warden C, Chuang J, Yuan Y. Mechanisms of recurrence: Paired analysis of primary and metastatic triple negative breast cancer [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 PD8-02.

Abstract P3-05-02: Immune profiling of paired primary and recurrent triple negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC) is a heterogeneous disease with several molecular subtypes: basal-like1 (BL-1), basal-like 2 (BL-2), mesenchymal (M), and luminal androgen receptor (LAR). Molecular evolution of TNBC through chemotherapy selection pressure is well recognized but poorly understood. In addition, approximately 20% of TNBCs respond to PD-1 or PD-L1 inhibitors. It has been observed that heavily pre-treated patients may not respond well to immunotherapy. This study was carried out to perform immune profiling of paired primary and recurrent TNBC. Here we report the result of the first 10 paired tissue pilot analysis. Methods: Twenty specimens were identified through an IRB-approved protocol via the City of Hope Biospecimen Repository (2002-2015). Two brain and one bone metastasis specimens were not included due to technical difficulty. Formalin-fixed paraffin embedded (FFPE) sample blocks were cut into 5-mm thick slides and labeled with the following antibodies: CD4, CD8, CD3, FOXP3, CD20, CD33, Pan-CK, and PD-1 using the multiplex IHC opal method. Image acquisition and cell counting were carried out using PerkinElmer Vectra automated quantitative pathology imaging system and inForm software analysis (PerkinElmer, Waltham, MA). mRNA expression profiling was performed using Affymetrix Human Genechip 2.0. Raw data were normalized and processed using Expression Console. Using Vanderbilt TNBC sub-classification tool, we have sub-classified the 20 primary and recurrent TNBC specimens. Tumor mutation burden (TMB) was generated through FoundationOne® platform. Result: A total of 17 samples were analyzed (M, 5; LAR, 3; BL-1, 4; BL-2, 5). M-subtype had a significantly lower tumor-infiltrating CD3+ T cells (p=0.005), CD8+ T cells (p=0.024), CD4+ T cells (p=0.065) and CD4+FOXP3+ Treg cells (p=0.054), irrespective of the site of metastasis. CD20+ B cells were particularly enriched in BL-1 subtype (p=0.0013, 23.5% of 17 samples). Of 17 samples, 8 had TMB. Seven had low TMB (&amp;lt;10 mut/Mb) and one had intermediate TMB (11 mut/Mb, LAR subtype). The tumor with intermediate TMB had the highest quantity of tumor-infiltrating CD3+ T cells, CD8+ T cells, CD8+PD1+ T cells, and CD4+FOXP3+PD1+ Treg cells compared to the 7 tumors with low TMB. Compared with recurrent tumors, primary tumors had a significantly higher percentage of tumor-infiltrating T cells (TIL). To validate multiplexed IHC results, these samples were evaluated by a licensed pathologist at City of Hope using the International TILs Working Group 2014 guidelines, and there was a good correlation between percent of TILs and CD3+ T cells by IHC approach. Conclusion: To our knowledge, this is the first study linking tumor immune cell profiles with the TNBC 4 subtypes. Distinctive immune cell patterns were observed among 4 TNBC subtypes. M subtype had significantly lower TILs, which may indicate poor response to checkpoint inhibitors. Further analysis of a total of 50 paired TNBCs is currently underway. Contact information: Yuan Yuan MD PhD, Email: yuyuan@coh.org Citation Format: He T-F, Yost S, Schmolze D, Wang R, Rosario A, Tu T, Chu P, Lee P, Yuan Y. Immune profiling of paired primary and recurrent triple negative breast cancer [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 P3-05-02.

Abstract P6-07-06: Targeting cancer stem-like cells metabolism via non-canonical notch signaling pathways in triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is a heterogeneous group of clinically aggressive diseases. TNBC patients have high risk of recurrence and metastasis, and current treatment options remain limited. Cancer stem-like cells (CSCs) have been linked to cancer initiation, progression and chemotherapy resistance. Therefore CSC-targeted therapies are keenly sought. There is strong evidence for the involvement of Notch signaling in TNBC. Notch1 is highly expressed in Basal-like 1 (BL1) and especially Mesenchymal-Stem-Like (MSL) TNBCs. Expression of Notch1 and its ligand Jagged1 correlate with poor prognosis. Moreover, strong evidence supports key roles of different Notch paralogs in breast CSCs. Here, we demonstrate that Notch activation by Jagged1-expressing stromal cells enhances transcription of the anti-apoptotic gene cIAP-2 (BIRC3), a known NF-κB target. This event is dependent on recruitment to the cIAP-2 promoter of NF-κB subunits, IKKα and Notch1. Short term exposure of MDA-MB-231 cells (MSL, PTEN wild-type), but not MDA-MB-468 cells (BL1, PTEN-null) to recombinant Jagged1 leads to AKT phosphorylation. This is suppressed by AKT inhibitors, IKK inhibitors, and dual mTORC1/2 inhibitors but not an mTORC1-selective inhibitor. These observations support a model where canonical and non-canonical mechanisms downstream of Notch1 trigger AKT phosphorylation and NF-κB activation in PTEN wild type TNBC cells. Rapid AKT phosphorylation downstream of Notch1 requires mTORC2, PI3K and IKKα, and contributes to NF-κB activation. This suggests a bidirectional crosstalk between the IKKα and AKT arms of this Jagged1-activated pathway. Importantly, we find co-localization of Notch1 with Mitochondria in MDA-MB-231 cells by confocal microscopy and Western blot of isolated mitochondrial fractions. We demonstrate that recombinant Jagged1 increases metabolism of TNBC cells. Knockdown of Notch1 or IKKα by siRNA decreases mitochondrial respiration and glycolysis. CSCs derived from MDA-MB-231 cells have increased Notch1, p-AKT, and oxidative metabolism compared to non-stem cells. AKT inhibition or IKK inhibition decreases both mitochondrial respiration and glycolysis of TNBC derived CSCs. Pharmacological inhibition of Notch cleavage by gamma secretase inhibitor (PF-03084014) in combination with AKT inhibitor (MK-2206) or IKK inhibitor (Bay11-7082) blocks CD90hi or CD44+CD24low sorted secondary mammospheres formation. Notably, we find similar results in TNBC patient derived xenograft (PDX) models. These data suggest that combination treatments affecting the intersection of Notch, NF-kB and AKT pathways have potential therapeutic importance in targeting CSCs in TNBC cases with high Notch1 expression. Citation Format: Hossain F, Sorrentino C, Ucar Bilyeu AD, Matossian M, Crabtree J, Pannuti A, Burow M, Golde T, Osborne B, Miele L. Targeting cancer stem-like cells metabolism via non-canonical notch signaling pathways in triple negative breast cancer [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 P6-07-06.