HLA Class II Histocompatibility Antigen γ Chain (CD74) Expression Is Associated with Immune Cell Infiltration and Favorable Outcome in Breast Cancer

The triple-negative breast cancer (TNBC) subtype, defined as negative for ER, PgR, and HER2, is biologically more aggressive and with a poorer prognosis than the other subtypes, in part due to the lack of suitable targeted therapies. Consequently, identification of any potential novel therapeutic option, predictive and/or prognostic biomarker, or any other relevant information that may impact the clinical management of this group of patients is valuable. The HLA class II histocompatibility antigen γ chain, or cluster of differentiation 74 (CD74), has been associated with TNBCs, and poorer survival. However, discordant results have been reported for immunohistochemical studies of CD74 expression in breast cancer. Here we report validation studies for use of a novel CD74 antibody, UMAb231. We used this antibody to stain a TMA including 640 human breast cancer samples, and found no association with the TNBC subtype, but did find a positive correlation with outcome. We also found associations between CD74 expression and immune cell infiltration, and expression of programmed death ligand 1 (PD-L1). Given that CD74 may play a role in innate immune system responses and the potential of immunotherapy as a viable treatment strategy for TNBCs, CD74 expression may have predictive value for immune checkpoint therapies.

ERβ in Triple-Negative Breast Cancer: Emerging Concepts and Therapeutic Possibilities

Despite the improvements in diagnostic and therapeutic approaches, breast cancer still remains one of the world’s leading causes of death among women. Particularly, triple negative breast cancer (TNBC) is characterized by aggressiveness, metastatic spreading, drug resistance and a very high percentage of death in patients. Nowadays, identification of new targets in TNBC appears very compelling. TNBC are considered negative for the estrogen receptor alpha (ERα) expression. Nevertheless, they often express ERβ and its variants. As such, this TNBC subtype still responds to estrogens. While the ERβ1 variant seems to act as a tumor-suppressor, the two variants ERβ2 and 5 exhibit pro-oncogenic activities in TNBC. Thus, ERβ1 activation might be used to limit the growth and spreading as well as to increase the drug sensitivity of TNBC. In contrast, the pro-oncogenic properties of ERβ2 and ERβ5 suggest the possible development and clinical use of specific antagonists in TNBC treatment. Furthermore, the role of ERβ might be regarded in the context of the androgen receptor (AR) expression, which represents another key marker in TNBC. The relationship between AR and ERβ as well as the ability to modulate the receptor-mediated effects through agonists/antagonists represent a challenge to develop more appropriate therapies in clinical management of TNBC patients. In this review, we will discuss the most recent data in the field. Therapeutic implications of these findings are also presented in the light of the discovery of specific ERβ modulators.

Yuanhuacine Is a Potent and Selective Inhibitor of the Basal-Like 2 Subtype of Triple Negative Breast Cancer with Immunogenic Potential

The heterogeneity of triple negative breast cancer (TNBC) has led to efforts to further subtype this disease with the hope of identifying new molecular liabilities and drug targets. Furthermore, the finding that TNBC is the most inherently immunogenic type of breast cancer provides the potential for effective treatment with immune checkpoint inhibitors and immune adjuvants. Thus, we devised a dual screen to identify compounds from natural product extracts with TNBC subtype selectivity that also promote the expression of cytokines associated with antitumor immunity. These efforts led to the identification of yuanhuacine (1) as a potent and highly selective inhibitor of the basal-like 2 (BL2) subtype of TNBC that also promoted an antitumor associated cytokine signature in immune cells. The mechanism of action of yuanhuacine for both phenotypes depends on activation of protein kinase C (PKC), defining a novel target for the treatment of this clinical TNBC subtype. Yuanhuacine showed potent antitumor efficacy in animals bearing BL2 tumors further demonstrating that PKC could function as a potential pharmacological target for the treatment of the BL2 subtype of TNBC.

Risk factors and prognostic factors for inflammatory breast cancer with bone metastasis: A population-based study

Purpose: Inflammatory breast cancer (IBC) is a rare type of breast cancer with poor prognosis. IBC patients with bone metastasis (BM) often suffer from many complications. This study was performed to identify risk factors with strong capability of predicting high BM risk for IBC patients and find prognostic factors for those patients. Methods: The Surveillance, Epidemiology and End Results (SEER) database was used to collect the clinicopathological and survival information of IBC patients. 966 IBC patients diagnosed between 2010 and 2015 were included to study the risk factors for developing BM by using Multivariable logistic regression. A total of 194 and 176 patients were included to analyze independent prognostic factors for overall survival (OS) and cancer specific survival (CSS) of IBC patients with BM respectively. Results: Of the 966 IBC patients, 194 (20.1%) patients were with BM. The risk factors for developing BM in IBC patients included unmarried marital status, double breast tumor, N1 stage, N3 stage, and liver metastases had higher risk of BM, while those of uninsured status and triple negative breast cancer (TNBC) were less likely to have BM. Analysis of prognostic factors for OS and CSS of IBC patients with BM showed that TNBC subtype and liver metastases were independently significantly associated with poorer OS and CSS of BM patients, while chemotherapy could serve as an independent prognostic factor for better OS and CSS of BM patients. Conclusion: Marital status, double breast tumor, N1 stage, N3 stage, and liver metastases should be considered for prediction of BM in IBC patients. TNBC subtype and liver metastases may indicate poor survival and chemotherapy can indicate improved survival for IBC patients with BM.

High Expression of microRNA-223 Indicates a Good Prognosis in Triple-Negative Breast Cancer

PurposeMicroRNAs can influence many biological processes and have shown promise as cancer biomarkers. Few studies have focused on the expression of microRNA-223 (miR-223) and its precise role in breast cancer (BC). We aimed to examine the expression level of miR-223 and its prognostic value in BC.MethodsTissue microarray (TMA)-based miRNA detection in situ hybridization (ISH) with a locked nucleic acid (LNA) probe was used to detect miR-223 expression in 450 BC tissue samples. Overall survival (OS) and disease-free survival (DFS) were compared between two groups using the Kaplan-Meier method and Cox regression model.ResultsOS and DFS were prolonged in the high miR-223 expression group compared to the low miR-223 expression group (p < 0.0001 and p = 0.017, respectively), especially in patients with the triple-negative breast cancer (TNBC) subtype (p = 0.046 and p < 0.001, respectively). Univariate and multivariate Cox regression analyses revealed that TNM stage (p = 0.008), the molecular subtype (p = 0.049), and miR-223 (p < 0.001) were independently associated with OS and DFS. External validation was performed with the METABRIC and The Cancer Genome Atlas (TCGA) databases via online webtools and was consistent with the data described above.ConclusionsThis study provides evidence that high miR-223 expression at diagnosis is associated with improved DFS and OS for BC patients, especially those with the TNBC subtype. miR-223 is a valid and independent prognostic biomarker in BC.

Triple negative aggressive phenotype controlled by miR-135b and miR-365: new theranostics candidates

Triple negative breast cancer (TNBC) accounts for about a fifth of all breast cancers and includes a diverse group of cancers. The heterogeneity of TNBC and the lack of target receptors on the cell surface make it difficult to develop specific therapeutic treatments. These aspects cause the high negative prognosis of patients with this type of tumor. The analysis of the molecular profiles of TNBC samples has allowed a better characterization of this tumor, supporting the search for new reliable diagnostic markers. To this end, we have developed a bioinformatic approach to integrate networks of genes differentially expressed in basal breast cancer compared to healthy tissues, with miRNAs able to regulate their expression. We studied the role of these miRNAs in TNBC subtype cell lines. We therefore identified two miRNAs, namely miR-135b and miR-365, with a central role in regulating the altered functional pathways in basal breast cancer. These two miRNAs are differentially expressed in human TNBC immunohistochemistry-selected tissues, and their modulation has been shown to play a role in the proliferation of tumor control and its migratory and invasive capacity in TNBC subtype cell lines. From the perspective of personalized medicine, we managed to modulate the expression of the two miRNAs in organotypic cultures, suggesting their possible use as diagnostic and therapeutic molecules. miR-135b and miR-365 have a key role in TNBC, controlling proliferation and invasion. Their detection could be helpful in TNBC diagnosis, while their modulation could become a new therapeutic tool for TNBC.

PD-L1 expression in tumor lesions and soluble PD-L1 serum levels in patients with breast cancer: TNBC versus TPBC

BACKGROUND: Block of programmed cell death protein 1 (PD-1) interaction with its ligand, PD-L1, enhances anti-tumor activity. OBJECTIVES: We aimed to assess the association between PD-L1 expression in tumor cells and CD8+ tumor infiltrating T cells (TILs) as well as soluble (s)PD-L1 serum levels in patients with triple negative breast cancer (TNBC) compared to triple positive (TPBC). METHODS: A total of 113 tumor sections and 133 serum samples were available from 144 patients with breast cancer (72 TNBC and 72 TPBC). Dual immunohistochemistry staining was applied to determine differential PD-L1 expression in tumor cells and CD8+ TILs. Soluble PD-L1 serum levels were also evaluated in patients compared to 40 healthy women by ELISA method. RESULTS: Despite TPBC patients which were mostly grades 1/2, TNBC patients were grade 3 (72% versus 66.7%, P < 0.001). Most of the TNBC patients were stages I/II, whereas most of the TPBC patients were stages III/IV (57.3% versus 68.3%,P = 0.005). There was no difference in tumor size and metastasis between TNBC and TPBC patients, although the number of involved lymph nodes was significantly more in TPBC patients (P = 0.0012). PD-L1 expression was detected in 11.5% of samples mostly in TNBC subtype and was associated with advanced grades (P = 0.039). There was no relationship between PD-L1 expression and tumor stage. PD-L1 expression in CD8+ TILs was nonsignificantly higher than tumor cells. Serum levels of sPD-L1 showed no difference between patients and healthy women. We found no correlation between PD-L1 expression in tumor lesions and serum levels of sPD-L1 in patients. CONCLUSION: PD-L1 expression was more detected in our patients with TNBC. It seems that, these patients who are resistant to standard chemotherapy regimens may get benefit from PD-L1 inhibition therapy and because of its low serum levels, sPD-L1 cannot interfere with this therapy.

Targeting the Id1-Kif11 Axis in Triple-Negative Breast Cancer Using Combination Therapy

The basic helix-loop-helix (bHLH) transcription factors inhibitor of differentiation 1 (Id1) and inhibitor of differentiation 3 (Id3) (referred to as Id) have an important role in maintaining the cancer stem cell (CSC) phenotype in the triple-negative breast cancer (TNBC) subtype. In this study, we aimed to understand the molecular mechanism underlying Id control of CSC phenotype and exploit it for therapeutic purposes. We used two different TNBC tumor models marked by either Id depletion or Id1 expression in order to identify Id targets using a combinatorial analysis of RNA sequencing and microarray data. Phenotypically, Id protein depletion leads to cell cycle arrest in the G0/G1 phase, which we demonstrate is reversible. In order to understand the molecular underpinning of Id proteins on the cell cycle phenotype, we carried out a large-scale small interfering RNA (siRNA) screen of 61 putative targets identified by using genomic analysis of two Id TNBC tumor models. Kinesin Family Member 11 (Kif11) and Aurora Kinase A (Aurka), which are critical cell cycle regulators, were further validated as Id targets. Interestingly, unlike in Id depletion conditions, Kif11 and Aurka knockdown leads to a G2/M arrest, suggesting a novel Id cell cycle mechanism, which we will explore in further studies. Therapeutic targeting of Kif11 to block the Id1–Kif11 axis was carried out using small molecular inhibitor ispinesib. We finally leveraged our findings to target the Id/Kif11 pathway using the small molecule inhibitor ispinesib in the Id+ CSC results combined with chemotherapy for better response in TNBC subtypes. This work opens up exciting new possibilities of targeting Id targets such as Kif11 in the TNBC subtype, which is currently refractory to chemotherapy. Targeting the Id1–Kif11 molecular pathway in the Id1+ CSCs in combination with chemotherapy and small molecular inhibitor results in more effective debulking of TNBC.

Development of an absolute assignment predictor for triple-negative breast cancer subtyping using machine learning approaches

Triple-negative breast cancer (TNBC) heterogeneity represents one of the main impediment to precision medicine for this disease. Recent concordant transcriptomics studies have shown that TNBC could be splitted into at least three subtypes with potential therapeutic implications. Although, a few studies have been done to predict TNBC subtype by means of transcriptomics data, subtyping was partially sensitive and limited by batch effect and dependence to a given dataset, which may penalize the switch to routine diagnostic testing. Therefore, we sought to build an absolute predictor (i.e. intra-patient diagnosis) based on machine learning algorithm with a limited number of probes. To this end, we started by introducing probe binary comparison for each patient (indicators). We based predictive analysis on this transformed data. Probe selection was first performed by combining both filter and wrapper methods for variable selection using cross validation. We thus tested three prediction models (random forest, gradient boosting [GB] and extreme gradient boosting) using this optimal subset of indicators as inputs. Nested cross-validation allowed us to consistently choose the best model. Results showed that the 50 selected indicators highlighted biological characteristics associated with each TNBC subtype. The GB based on this subset of indicators has better performances as compared to the other models.

Comprehensive profiling of androgen receptor-positive (AR+) triple-negative breast cancer (TNBC) patients (pts) treated with standard neoadjuvant therapy (NAT) +/- enzalutamide.

517 Background: The luminal androgen receptor (LAR) subtype of TNBC has a low pathologic complete response (pCR) rate after NAT. We determined the pCR rate of the enzalutamide and paclitaxel (ZT) regimen for pts with anthracycline-insensitive AR+ TNBC (NCT02689427), and related biomarkers. Methods: ARTEMIS (NCT02276443) is a non-randomized trial to determine if NAT can be used to personalized therapy. Pts received 4 cycles of doxorubicin-based NAT (AC). Pts with insensitive disease by imaging were offered clinical trials as the second phase of NAT based upon molecular profiling of pre-treatment biopsies. Immunohistochemistry (IHC) of AR+≥10% was the threshold for selecting ZT (enzalutamide 160 or 120 mg PO qD + paclitaxel 80 mg/m2 qW for 12 cycles). pCR was determined by surgery after NAT. Trial had two-stage Phase II design, and we report the completed first stage. We evaluated the concordance between Vanderbilt LAR subtype by molecular profiling (microarray and RNAseq) and IHC %AR+ cells. Frequency of PI3K pathway alterations within the LAR subtype was assessed. Results: 267 pts had tumors profiled by IHC, 220 by microarray, 187 by RNAseq and 197 by whole exome sequencing. 96 pts had post-AC RNAseq. LAR scores from both RNAseq and microarray profiling (n = 139) were highly concordant (R = 0.89, P < 0.001) and identified ~10% of TNBCs tested as LAR. The %AR+ cells from IHC correlated with LAR subtype scores according to RNAseq (R = 0.6, P < 0.001), with a cut-point of ≥30% AR+ having the best concordance with LAR subtype. Unlike other subtypes, by serial profiling, LAR TNBCs did not change subtype signatures after exposure to AC. LAR TNBCs had low rates of pCR (23%) and high rates of PI3K pathway activating aberrations (85%); however PI3K aberrations did not correlate with pCR. Seventeen patients with AC-insensitive TNBC received ZT. Five of 15 patients (33.3%) had responses (pCR or RCB-I). Toxicities are Grade (Gr) 4 syncope (n = 1), Gr3 abnormal liver function (n = 2), Gr3 neutropenia (n = 4). IHC & LAR subtype scores did not statistically associate with response to ZT (P = 0.8, P = 0.9). However, all responders to ZT had an upregulated androgen response pathway (ssGSEA Z > 1) as measured by transcriptomic analysis in pre-treatment biopsies analysis (P = 0.05, ppv = 0.56, npv = 1). Conclusions: The LAR TNBC subtype has a low pCR rate to NAT. Among pts with AC-insensitive TNBC, baseline upregulated androgen response pathway and LAR subtype may benefit from the ZT regimen, potentially by PI3K targeting. Clinical trial information: NCT02689427 .