Emerging pathways in treating human epidermal growth factor receptor-2-negative breast cancer

Breast cancer remains the leading cause of new cancer cases in women and is responsible for the most cancer-related deaths in women worldwide. The goals of breast cancer treatment are to maintain or improve quality of life, prolong survival, and increase disease-free progression. The majority of breast cancer cases are estrogen receptor (ER)-positive and human epidermal growth factor receptor-2 (HER-2)negative, and current treatment guidelines recommend multiple lines of endocrine therapy followed by chemotherapy in patients with locally recurrent or metastatic disease. Resistance to current therapies adds to the need for new therapeutic options. Translational research and preclinical data have provided insight into the identification of emerging signaling pathways for novel drug targets, and the development of a growing number of biologic targeted agents is currently underway to identify novel treatments. An alternative approach to improve patient benefit is to boost the efficacy and safety of existing agents by modifying their delivery or pharmacokinetics (ie, adding albumin to paclitaxel) as well as identifying new combination therapies. One combination therapy of interest is the addition of the 130 nm albumin-bound formulation of paclitaxel (nab-paclitaxel) to currently approved therapies or targeted agents in development. This review focuses on a number of key agents that are being investigated for the treatment of HER2-negative breast cancer and the utilization of these agents as combination therapy to achieve prolonged disease control.


Introduction
Breast cancer (BC) is responsible for an estimated 1.67 million new cases of cancer in women worldwide annually, accounting for 25% of the total new cancer cases, making it the most frequently diagnosed cancer in 2012. It is also the leading cause of cancer death in less-developed regions of the world and the secondleading cause of cancer death in more-developed regions [1]. In a review of 15,204 cases of BC, the National Comprehensive Cancer Network noted that 66% of patients were hormone receptorpositive (HR-positive)/human epidermal growth factor receptor-2 (HER-2)-negative, 17% were HER-2-amplified, and the remaining 17% had triple-negative breast cancer (TNBC) [2]. Receptor status is a key covariate as it determines the class/type of systemic therapy provided to BC patients, and this review will focus on HER-2negative BC, including TNBC.
Although advanced stage/metastatic BC is incurable, it is treatable, and the goals of treatment are to maintain or improve quality of life, prolong survival, and increase disease-free progression. Current treatment guidelines [3][4][5] recommend the use of endocrine therapy (ET) for all patients with early HR-positive disease, with the choice of agent primarily determined by the patient's menopausal status. Chemotherapy (CT) is recommended after progression or unacceptable toxicity and no clinical benefit after 3 sequential ET regimens. However, for patients with visceral crisis, CT is the recommended initial treatment [5]. Concomitant ET plus CT has shown no benefit for survival and should only be performed in a clinical trial [3,4]. For TNBC, single-agent CT is preferred, including taxanes and anthracyclines, as there is currently no compelling evidence that combination CT regimens are superior to sequential single agents for these patients [5,6]. A general schematic for current HER-2-negative BC treatment algorithms is presented in Fig. 1 [3][4][5]. Chemotherapy remains an essential component of systemic intervention in patients with HER-2-negative disease, including patients with advanced HRpositive disease who have progressed after multiple lines of endocrine therapy, patients with TNBC, and patients with symptomatic visceral disease in need of rapid symptomatic control. However, the efficacy of CT options is modest, particularly because second or later lines of therapy and combination CT regimens offer limited efficacy and increases in toxicity [7]. Targeted agents are under investigation for HER-2-negative BC; however, they are less effective in the treatment of advanced disease. Therefore, the combination of CT with a targeted agent is under intense investigation for the treatment of BC, including metastatic disease.

Insight from translational research
Traditionally, BC subtypes have been classified based on receptor status; however, the definitions for intrinsic subtypes were recently expanded [8]. Luminal A-like is estrogen receptor (ER)-positive and/or progesterone receptor-positive and HER-2negative with low expression of Ki-67, a marker for cell proliferation [8,9]. Luminal B is broken down into 2 types: Luminal B-like (HER-2-negative) is ER-positive and HER-2-negative with either high expression of Ki-67 or low/no expression of progesterone receptor, while Luminal B-like (HER-2-positive) is ER-positive with amplified expression of HER-2 [8]. HER-2-positive (non-luminal) BC is classified as having amplified expression of HER-2 and as absent for HR expression. Finally, TNBC (ductal) is negative for expression of all 3 receptors [8]. The differentiation of traditional clinical subsets into additional categories with varying prognoses suggests that the current treatment paradigm is suboptimal. Furthermore, systemic treatments often impose the selection of resistant phenotypes. In a recent study on the inference of tumor evolution during chemotherapy for BC treatment, phenotypic diversity had been altered before and after treatment, while a pathologic complete response was associated with lower pretreatment genetic diversity [10].
Resistance to treatment is an important aspect of BC therapy, as up to 50% of HR-positive BC patients are refractory to primary treatment, while the remainder will acquire resistance [11]. For example, resistance to tamoxifen can be due to a number of mechanisms including altered tamoxifen metabolism, and modification of ERα activity due to increased phosphorylation, activation of phosphatidylinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling, aberrant expression of ERα target genes, and even expression of a dominant-negative ERα isoform [12]. Furthermore, it has been demonstrated that ER and HER-2 status can change over time in patients with metastatic BC (MBC), and there is a growing need for repeat tumor biopsies to determine whether a change in Fig. 1. Current recommendations for the treatment of HER-2-negative breast cancer. For patients who have completed surgical resection of breast tumors, general systemic intervention is determined by receptor status. Patients who are HR-positive and HER-2-negative are treated with multiple lines of endocrine therapy; if a rapid response is required, chemotherapy is the recommended treatment. After progression on endocrine therapy, patients can be treated with exemestane plus everolimus; otherwise, chemotherapy is recommended. Patients who are HR-negative and HER-2-negative (TNBC) are treated with sequential lines of single-agent chemotherapy. Combination chemotherapy regimens are only recommended if patients require a rapid response or in the case of rapid disease progression. If patients progress after chemotherapy, palliative care is provided. AI, aromatase inhibitor; HER-2, human epidermal growth factor receptor-2; HR, hormone receptor; TNBC, triple-negative breast cancer. a -Cardoso et al. [3], b -Senkus et al. [4], and c -National Comprehensive Cancer Network [5]. therapy is required [13]. Patients with TNBC can experience resistance to chemotherapeutics mainly due to increased efflux of drugs through ABCB1 transporters, thereby lowering the effective intracellular drug concentrations [14]. Many of these resistance pathways have become targets for BC treatment.
To overcome the many obstacles of drug resistance and disease progression, a number of emerging pathways are under investigation for the treatment of HER-2-negative BC. Furthermore, the field of genomics is currently playing an important role in our understanding of the genetic differences between normal and malignant tissues. For example, The Cancer Genome Atlas has documented the genetic diversity of luminal/ER-positive, HER-2-positive, and TNBC, and these data have provided the rationale for recently approved and emerging treatments [15]. Inhibition of multiple pathways using combination approaches with CT and targeted agents is also under investigation to improve the duration of response to initial treatment and to provide new options to overcome resistance.

Agents in development
Inhibition of a number of physiologic pathways by targeted agents is currently under investigation for the treatment of HER-2negative BC (Fig. 2). These agents include inhibitors of the extracellular receptor tyrosine kinase vascular endothelial growth factor receptor (VEGFR) and its ligand VEGF, the mammalian target of rapamycin (mTOR) and PI3K signaling pathways, cell cycle progression through cyclin-dependent kinase (CDK) 4 and 6, epigenetic regulation through histone deacetylase (HDAC), and poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP)mediated DNA repair. Furthermore, the therapeutic potential of novel targets for modulating immune checkpoint regulation, including cytotoxic T-lymphocyte antigen-4 (CTLA-4), programmed death-1 (PD-1), and programmed death ligand-1 (PD-L1), is also being investigated for patients with BC. Many of these agents are being investigated as combination therapies and are a rational means of achieving prolonged disease control.

Antiangiogenic agents
Studies have demonstrated that BC is angiogenesis-dependent; elevated expression of VEGF is common in BC and associated with a higher incidence of recurrence or death [16]. Therefore, circulating VEGF and VEGFR, along with other receptor tyrosine kinases such as platelet-derived growth factor receptor, have become important targets for the development of therapeutic agents in BC. Bevacizumab is a fully humanized monoclonal antibody (mAb) VEGF-A inhibitor that was approved by the United States Food and Drug Administration in 2008 for first-line treatment of HER-2negative metastatic BC in combination with paclitaxel based on results from a phase 3 trial [17] (Table 1) . Median progression-free survival (PFS) was increased in patients with MBC treated with bevacizumab and paclitaxel compared with paclitaxel alone (11.8 vs 5.9 months; P o.001), although there was no significant difference in overall survival (OS) between the 2 groups [17]. Grade 3 or 4 neuropathy, infection, and fatigue were more frequent with bevacizumab combination therapy compared with paclitaxel alone (Table 1) [17]. However, the United States Food and Drug Administration recommended the removal of this indication from its label in 2010 based on its interpretation that safety concerns outweighed the improvement in PFS; the use of bevacizumab for the treatment of BC continues in Europe [48]. A number of ongoing clinical trials are evaluating bevacizumab as Emerging targets for the treatment of HER-2-negative breast cancer. Multiple emerging pathways are currently under investigation for the treatment of HER-2negative breast cancer. Inhibition of these physiologic pathways with targeted agents as combination therapy with chemotherapeutic agents, notably with nab-paclitaxel, is a rational means of prolonged disease control. CDK, cyclin dependent kinase; CTLA-4, cytotoxic T-lymphocyte antigen-4; HDAC, histone deacetylase; HER-2, human epidermal growth factor receptor-2; mTOR, mammalian target of rapamycin; PARP, poly(adenosine diphosphate [ADP]-ribose) polymerase; PD-1, programmed death-1; PDGFR, platelet-derived growth factor receptor; PD-L1, programmed death ligand-1; PI3K, phosphatidylinositide 3-kinase; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor.  combination therapy for HER-2-negative BC ( Table 2). Several small-molecule, multi-targeted tyrosine kinase inhibitors that block VEGFR and platelet-derived growth factor receptor signaling have also been investigated for firstor second-line treatment of HER-2-negative MBC, including sorafenib and sunitinib. Sorafenib has been evaluated in a handful of phase 2 studies, mostly in combination with chemotherapeutic agents, and has demonstrated efficacy in firstor second-line treatment of MBC (Table 1). Of note, increased incidence of grade 3 or 4 hand-foot syndrome is common in most studies of sorafenib (Table 1). Sunitinib has failed to show significant efficacy either alone or in combination with CT for the treatment of HER-2-negative BC, including TNBC, and two of four phase 3 studies have been terminated for futility (Table 1). Sorafenib is currently being evaluated in a number of ongoing phase 2 trials, and one phase 3 trial as combination therapy for firstor second-line treatment of HER-2-negative MBC, while sunitinib is currently being investigated in combination with CT for neoadjuvant treatment of TNBC in a phase 1/2 trial (Table 2).

PI3K and mTOR inhibitors
Hyperactivation of the PI3K/AKT/mTOR pathway is frequently observed in BC, leading to cancer pathogenesis, progression, and resistance to endocrine treatment [49]. Therefore, the addition of PI3K and mTOR inhibitors to ET or CT may enhance efficacy or delay resistance. A number of PI3K and mTOR inhibitors are being developed for the treatment of HER-2-negative BC, including buparlisib, BEZ235, and everolimus. Buparlisib is a pan-class I PI3K inhibitor that is effective in a number of different cancer types and cellular systems, irrespective of their level of PI3K addiction [50]. BEZ235 is a dual PI3K/mTOR inhibitor that induced downregulation of VEGF, cell cycle arrest, and autophagy in a preclinical study [51]. Clinical activity of either buparlisib or BEZ235 in HER-2-negative BC has yet to be published; however, a number of clinical trials are underway for buparlisib either alone or as combination therapy in HER-2-negative BC and TNBC ( Table 2). Everolimus (EVE) is an mTOR inhibitor approved in combination with exemestane for the treatment of ER-positive, HER-2-negative advanced BC (ABC) in patients whose disease has progressed after a nonsteroidal aromatase inhibitor (AI) [52]. In the pivotal phase 3 trial, EVE was evaluated in combination with exemestane (EXE) compared with placebo (PBO) plus EXE in 724 post-menopausal women with ER-positive, HER-2-negative ABC with recurrence or progression following nonsteroidal AI therapy. Median PFS was 7.8 months in the EVE group and 3.2 months in the PBO group (Po .0001) by independent review [52], and the most common grade 3 or 4 adverse events (AEs) were stomatitis (8% for EVE vs 1% for PBO), anemia (6% vs o1%), dyspnea (4% vs 1%), hyperglycemia (4% vs o 1%), fatigue (4% vs 1%), and pneumonitis (3% vs 0%) ( Table 1) [40]. Everolimus is also currently being investigated in a number of clinical trials for HER-2-negative BC or TNBC, often as combination therapy with another targeted therapy ( Table 2).

CDK4/6 inhibitors
Dysregulated cell cycle progression due to uncontrolled cellular growth is another hallmark of cancer, and disruption of cell cycle progression through inhibition of CDKs is a therapeutic strategy undergoing intense evaluation in multiple cancers [53]. Cyclindependent kinase 4 is a key regulator of the transition from G 1 to S phase of the cell cycle, which, when inhibited, causes cell cycle arrest and apoptosis of dividing cells. Furthermore, resistance to ET is often caused by upregulation of signaling pathways that modify cell cycle control [54]. A number of selective CDK4/6 inhibitors are under investigation in HER-2-negative BC, including palbociclib, abemaciclib, and LEE011. Palbociclib has been investigated as single-agent treatment for HR-positive/HER-2-negative ABC, HR-positive/HER-2-positive ABC, and advanced TNBC, and in combination with letrozole as first-line therapy for ER-positive/ HER-2-negative MBC. In a phase 2 study in 165 women with ERpositive/HER-2-negative MBC who were treated with first-line palbociclib plus letrozole (LET) or LET alone, median PFS with palbociclib treatment was 20.2 months vs 10.2 months with LET alone (P ¼.0004) ( Table 1) [42]. The most common AEs in the palbociclib treatment group were neutropenia, leukopenia, fatigue, and anemia (Table 1). A phase 3 study in the same population is ongoing (NCT01740427; Table 2). Another CDK4/6 inhibitor, LEE011, is also under investigation in a number of ongoing clinical trials as combination therapy for HER-2-negative BC ( Table 2).

HDAC inhibitors
The recent discovery that alterations in histone proteins and DNA can lead to tumorigenesis has led to the evaluation of HDAC inhibitors in solid tumors [55]. Histone deacetylases catalyze the deacetylation of histones, leading to the coiling of chromatin and     No clinical trial information available the blockade of transcription of affected genes [56]. Histone deacetylases are critical in the regulation of expression of numerous genes involved in cell survival, proliferation, and differentiation [57]. A number of HDAC inhibitors have been investigated in various cancers, and 2 agents (entinostat and vorinostat) are under investigation for HER-2-negative BC. Entinostat, a class-specific HDAC inhibitor, has been investigated in combination with EXE in postmenopausal women with ER-positive ABC after they had progressed on a nonsteroidal AI in a phase 2 trial [43]. Entinostat was associated with increased median OS (28.1 months for entinostat plus EXE vs 19.8 months for PBO plus EXE; P ¼.036) and the most common grade 3 or 4 AEs were neutropenia (14% vs 0) and fatigue (13% vs 3%) ( Table 1) [43]. A second phase 2 trial has evaluated entinostat in combination with 5-azacitidine in women with advanced TNBC or hormone-resistant BC. No responses were observed in the first 13 TNBC subjects and this cohort was closed; 27 patients were enrolled in the hormone-resistant cohort, and median PFS was 1.8 months at a median follow-up of 6.3 months (Table 1) [44,47]. A phase 3 trial is currently underway for entinostat plus EXE in HER-2-negative ABC, while a phase 2 trial is underway for vorinostat, a pan-HDAC inhibitor, in combination with CT for first-line treatment of HER-2-negative BC and TNBC ( Table 2).

PARP inhibitors
There is intense interest in DNA repair pathways in oncology. Dysregulation of homologous recombination can be caused by mutations to BRCA1 or BRCA2, which are responsible for 5-10% of BCs, most notably TNBC [58]. These tumors may be susceptible to lethality if another DNA repair mechanism, such as base excision repair, is also inhibited. Several groups have demonstrated that BRCA-deficient cells are sensitive to inhibition of PARP [59,60], which is involved in a variety of cellular processes, including homologous recombination repair of DNA double-strand breaks (reviewed by Calvert and Azzariti [61] and Shah et al. [62]). The PARP inhibitors veliparib and rucaparib are currently being investigated in TNBC or in BC patients with known BRCA mutations. In phase 2 studies, veliparib [45] has demonstrated modest efficacy in MBC patients with BRCA mutations, while rucaparib [46] was observed to have similar 1-year OS in patients with TNBC or BC with known BRCA mutations receiving rucaparib plus cisplatin compared with cisplatin monotherapy (Table 1). Veliparib is currently being investigated in combination with CT in a number of clinical trials, including 2 phase 3 trials (Table 2).

Immunotherapies
The immune system plays an important role in cancer, both in the promotion of tumorigenesis through inflammatory pathways and suppression of adaptive immunity, and in the prevention of tumor formation through immune surveillance [63]. Tumor-infiltrating lymphocytes have been associated with improved outcome in BC and were recently shown to be a predictive marker of response to neoadjuvant CT [64]. An immune response is initiated by antigen recognition, but the magnitude and quality of the response is regulated by additional immune checkpoint molecules [65]. These molecules include PD-1, PD-L1, and CTLA-4, and inhibition of these novel targets is actively being investigated in a number of cancers, including non-small cell lung cancer, renal carcinoma, melanoma, ovarian cancer, and others. Nivolumab, pembrolizumab, and pidilizumab are mAb inhibitors of PD-1, and MPDL3280A is a mAb inhibitor of PD-L1; ipilimumab is a CTLA-4 inhibitor. No clinical trial data with these agents in BC have been published to date, and none of these agents has progressed to phase 2 trials for the treatment of HER-2-negative BC.

Combination regimens with chemotherapeutic agents
Taxanes, such as paclitaxel, are important chemotherapeutic agents for the treatment of HER-2-negative BC. Paclitaxel is indicated for the treatment of MBC after failure of combination CT for metastatic disease or relapse within 6 months of adjuvant CT, with prior therapy including an anthracycline unless clinically contraindicated [66]. It is also being investigated in numerous clinical trials as combination therapy for HER-2-negative and HER-2-positive BC. Due to the hydrophobicity of taxanes, synthetic solvents are used to enable parenteral administration; polyethylated castor oil and ethanol are used as vehicle for paclitaxel [66]. Recently, a 130 nm albumin-bound formulation of paclitaxel (nabpaclitaxel) has been developed to improve the chemotherapeutic The HER-2-positive patient subpopulation will receive TRA þnab-PAC þ CAR therapy. c The HER-2-positive patient subpopulation will receive TRAþ DOCþ CAR therapy. d The HER-2-positive subpopulation will receive DOX þCYCþ GM-CSF, followed by CARþ nab-PACþ TRA therapy.
effects of paclitaxel while avoiding the toxicities associated with polyethylated castor oil [67]. Albumin is a natural carrier of lipophilic molecules allowing nab-paclitaxel to be safely infused at higher doses, with shorter infusion times, and with no need for pre-medication [68]. A phase 3 trial comparing paclitaxel with nab-paclitaxel in women with MBC demonstrated a significant increase in response rate in the nab-paclitaxel group compared with the paclitaxel group (33% vs 19%; P ¼.001), as well as significantly longer time to tumor progression (23.0 weeks vs 16.9 weeks; P¼ .006) [69]. nab-Paclitaxel was also associated with a lower incidence of grade 4 neutropenia compared with paclitaxel (9% vs 22%; P o.001) [69]. Unlike paclitaxel, nab-paclitaxel is being investigated in a limited number of clinical trials in HER-2negative BC, mostly in combination with bevacizumab. Since their indications are similar [66,67], nab-paclitaxel may replace paclitaxel as combination therapy with targeted agents (bevacizumab, sorafenib, sunitinib, buparlisib, BEZ235, EVE, and veliparib) because of its improved efficacy and safety.

Conclusions
Breast cancer is the leading cause of cancer and cancer death in women worldwide. Although MBC is incurable, it is treatable, with prolonged survival as the ultimate goal of therapy. Many patients with BC progress after multiple lines of therapy or become resistant to treatment; therefore, there is a need for additional treatment strategies for this patient population and repeat biopsies to track change in receptor status to determine treatment modifications. Translational research, including The Cancer Genome Atlas, has generated a wealth of data that provides the rationale for the investigation of novel therapeutic targets, such as the identification of commonly mutated genes in BC. Inhibition of these emerging pathways, either alone or as combination therapy, may provide greater control of disease progression even in patients with resistance to ET. Clinical trial data for a number of these agents have demonstrated promising clinical activity, and further research is underway to develop novel treatment combinations for patients with HER-2-negative BC.

Executive summary
HER-2-negative breast cancer is incurable, but is treatable. New therapeutic options are needed to manage resistance to current therapies.
Breast cancer patients acquire resistance to therapy, and these pathways have become targets for treatment. The Cancer Genome Atlas has documented the frequency of common gene mutations in breast cancer subtypes, and a number of agents are under investigation to target these cellular pathways.
A number of targeted biologic therapies are currently under investigation for HER-2-negative breast cancer, and these include inhibitors of VEGF and VEGFR, mTOR and PI3K signaling pathways, CDK4 and 6, HDAC, PARP, CTLA-4, and PD-1 and PD-L1. Extensive descriptions of clinical efficacy and ongoing trials of key targeted agents from phase 2 or 3 trials for the treatment of HER-2-negative breast cancer are included in this review.
Combination regimens of a chemotherapeutic agent (such as nab-paclitaxel) and a biologic targeted agent are under investigation to achieve prolonged disease control in patients with HER-2-negative breast cancer, including triple-negative breast cancer.
Continued research is required to demonstrate the efficacy and safety of targeted agents and combination regimens in the treatment of HER-2-negative breast cancer.

Conflict of Interest
Source(s) of support (in the form of grants, equipment, drugs, or all of these): the author is employed by Celgene Corporation and received editorial support that was funded by Celgene Corporation.

Ethical approval
None Declared.

Role of the funding source
The author is employed by Celgene Corporation, and funding for medical editorial assistance was provided by Celgene.