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Breast Cancer
May 23, 2018

Innovative Strategies: Targeting Subtypes in Metastatic Breast Cancer

Publication: American Society of Clinical Oncology Educational Book
Globally, breast cancer is the fifth highest cause for cancer death overall, accounting for more than 500,000 deaths annually.1 Metastatic breast cancer is the most frequent cause of cancer death in women in low-income regions of the world and second only to lung cancer in high-income regions.1 Current estimates suggest that up to 154,000 patients are living in the United States with metastatic breast cancer.2 Many patients with metastatic breast cancer are benefitting from targeted therapy to treat their disease; however, most will not achieve prolonged, durable remissions. Fortunately, novel targeted therapy strategies are currently being tested with the hopes of improving outcomes in patients with metastatic disease.


Although endocrine therapy remains an essential therapeutic option for those women whose tumors express the ER, intrinsic or acquired resistance inevitably emerges. Therefore, preventing and/or reversing resistance to endocrine therapy remain major research focuses for patients, clinicians, and researchers.
Cancer cells commonly exhibit loss of cell cycle control, resulting in uncontrolled cell growth. Cyclin-dependent kinases (CDKs) are a group of serine/threonine kinases that have an important role in the mammalian cell cycle. They exert their functions by interaction with regulatory subunits called cyclins. In G1 or growth phase, expression of D-type cyclins is promoted by mitogenic growth factors via multiple different signaling pathways. Cyclin D-CDK 4/6 complexes phosphorylate and inactivate retinoblastoma (Rb) tumor suppressor proteins, which cause dissociation of E2F transcription factors and the regulation of genes that trigger G1-S phase progression, DNA replication, DNA damage repair, and mitosis.3-6 Rb is considered to be the guardian of the restriction point gate in the mammalian cell cycle, because it has a fundamental role in G1-S phase transition.3
In the case of ER+ breast cancer, cyclin D1 is a major ER transcriptional target. Despite diverse mechanisms of endocrine resistance, many ER+ breast cancers resistant to hormone-based therapy remain dependent on cyclin D1 and CDK 4 to drive cell proliferation.7 Whereas Rb is functional in most luminal breast cancer, in contrast, many ER− breast cancers (e.g., basal subtype of TNBC) are characterized by the loss of RB1 activity.8-10 Consequently, basal-like breast cancer cell lines are insensitive to CDK 4/6 inhibition in vitro.11
Recently, multiple studies have shown that targeting CDK 4/6 resulted in substantial improvements in clinical response and PFS in women with metastatic ER+ breast cancer. Remarkably, three new CDK 4/6 inhibitors, including palbociclib (Ibrance, PD0332991; Pfizer, New York, NY), ribociclib (Kisqali, LEE011; Novartis, Basel, Switzerland), and abemaciclib (Verzenio, LY2834219; Lilly, Indianapolis, IN), received approvals from the FDA over a 3-year period from 2015 to 2018. These approvals were based on the initial randomized phase II study evaluating palbociclib in combination with letrozole12 and subsequent phase III studies showing that the addition of palbociclib,13 ribociclib,14 and abemaciclib in combination with aromatase inhibitors improved PFS in the first-line ER+ metastatic breast cancer setting. Additionally, FDA approval was gained based on phase III studies evaluating the combination of palbociclib and fulvestrant (the PALOMA III study) and abemaciclib plus fulvestrant (the Monarch 2 study) in patients with endocrine refractory ER+ breast cancer.15,16

Differences Between CDK 4/6 Inhibitors

In evaluating the three CDK 4/6 inhibitors, it should be noted that the most common side effects observed in the randomized trials were neutropenia/leukopenia and anemia. However, serious infection or febrile neutropenia was rare. Abemaciclib, in contrast, results in less neutropenia but greater incidence of diarrhea. However, diarrhea was effectively treated and/or prevented in most patients using antidiarrheal prophylaxis. In addition, thromboembolism has been reported with abemaciclib, with an incident rate of 4% to 5% in the Monarch 2 and 3 studies.16,17
In terms of efficacy, evaluation of the phase III registration trials has demonstrated that the relative improvement in PFS comparing combination CDK 4/6i plus endocrine therapy versus endocrine therapy alone is remarkably similar comparing the three different CDK 4/6 inhibitors. However, cross-trial comparisons suggest that response rates to abemaciclib as a single agent18 or in combination with endocrine therapy16,17 were higher than those reported with palbociclib or ribociclib. These differences in response may be important in certain clinical scenarios (e.g., visceral metastases)when choosing one CDK4/6 inhibitor over another.

Additional Mechanisms by Which CDK 4/6 Inhibitors Exert Their Anticancer Effects

Although Rb proficiency is considered essential for the function of CDK 4/6 inhibitors, there may be Rb-independent mechanisms by which CDK 4/6 inhibitors exert their anticancer effects. Recently, Liu et al19 identified a novel mechanism by which CDK 4/6 inhibitors alter epithelial to mesenchymal transition (EMT) and inhibit metastases in TNBC. Liu et al19 showed that overexpression of DUB3 increased Snail levels, whereas conversely, knockdown of DUB3 decreased Snail protein levels without affecting SNAIL messenger RNA levels. Liu et al19 went on to show that CDK 4/6 phosphorylates DUB3 at Ser41, thus activating DUB3. Treatment of cells with palbociclib inhibited DUB3 activity, decreased SNAIL stability and expression, and decreased cell migration. In a xenograft model derived from patients with highly metastatic TNBC, the administration of palbociclib did not alter primary tumor growth but substantially reduced lung and liver metastases. Therefore, these data suggest that CDK 4/6 inhibitors may inhibit EMT and metastases independent of Rb.
Goel et al20 recently reported a novel mechanism by which CDK 4/6 inhibitors promote antitumor immunity through activation of tumor cell expression of endogenous retroviral elements and intracellular levels of double-stranded RNA, resulting in the production of type III interferons and tumor antigen presentation. Additionally, Goel et al20 showed that CDK 4/6 inhibitors suppress the proliferation of regulatory T cells and DNA methyltransferase 1, resulting in cytotoxic T cell–mediated clearance of tumor cells. Overall, these data suggest that CDK 4/6 inhibitors may contribute antitumor effects through modulation of the immune system.

Selection of Patients FOR CDK 4/6 Inhibitors

Although CDK 4/6 inhibitors have shown substantial efficacy in patients with HR+, HER2 metastatic breast cancer, the use of these medicines involve additional toxicity (from both side effect and financial standpoints). Therefore, selecting when and in whom to administer a CDK 4/6 inhibitor is a topic of considerable debate. Previously reported subgroup analyses of the randomized phase III registration trials concluded that all subgroups benefit from the addition of CDK 4/6 inhibitors.13,14,17 However, the absolute benefit of CDK 4/6 inhibitor therapy may depend on the clinical scenario. For example, a recent analysis of over 1,000 patients enrolled in the Monarch 2 and 3 studies evaluated a broad set of common clinical and pathologic variables associated with the prognosis of patients receiving endocrine monotherapy. In this analysis, patients with liver metastases, high-grade tumors, PR− tumors, or a short treatment-free interval had a poor prognosis. Conversely, patients with bone-only disease, excellent performance status, or a long treatment-free interval exhibited substantially better prognosis. Although abemaciclib conferred benefit regardless of baseline characteristics, the patients with the poorest prognosis (e.g., liver metastases) derived the largest absolute benefit from the addition of abemaciclib to endocrine therapy. In contrast, in the Monarch 3 study, little if any benefit was noted for the addition of abemaciclib to endocrine therapy in women with a prolonged treatment-free interval. Although these data are hypothesis generating, they suggest that there may exist a subset of patients with highly endocrine-sensitive breast cancer for whom endocrine monotherapy (e.g., an aromatase inhibitor or fulvestrant) may be the optimal first-line therapy followed by the addition of a CDK 4/6 inhibitor at progression. Additional clinical studies are necessary to determine the optimal strategy.

Mechanisms of Resistance TO CDK 4/6 Inhibitors

Similar to the paradigm of treating ER+ breast cancer, the mechanisms of resistance to CDK 4/6 inhibitors can be divided into de novo and acquired resistance. Currently, ER status is the only selection criteria used for women with metastatic breast cancer being considered for a CDK 4/6 inhibitor. Biologically plausible biomarkers of the cyclin D-CDK 4/6-Rb pathway (e.g., loss of Rb and consequent upregulation of p16INK4A and downregulation of cyclin D1) have not been consistently associated to the benefit of CDK 4/6 inhibitors in the randomized trials.21 For example, cyclin D1 amplification and/or loss of CDKN2A were not associated with palbociclib resistance in the PALOMA 1 study.12 Furthermore, Rb, cyclin D1, p16, and Ki-67 (all evaluated by immunohistochemistry) were not predictive of palbociclib benefit in the PALOMA 2 study.22 Although loss of RB1 function is relatively rare in newly diagnosed patients with ER+ breast cancer,23 the incidence of Rb loss, E2F amplification, and/or loss of CDKN1 and their association with acquired resistance to CDK 4/6 inhibitors are unknown.

Combination Therapy Strategies

There is great rational for combining CDK 4/6 inhibitors with drugs that target growth factor signaling pathways upstream of cyclin D1, including drugs that target the PI3K/mTOR/AKT pathway.24 These data have resulted in several ongoing studies, in which CDK 4/6 inhibition is combined with drugs that target this pathway. Furthermore, there exists substantial rationale for the use of CDK 4/6 inhibitors in combination with HER2-directed therapy in patients with HER2-amplified breast cancers. In addition to the surprising single-agent activity of abemaciclib in ER+/HER2+ breast cancer,25 there are extensive preclinical data identifying cyclin D1 as a critical downstream target of HER-induced transformation26 and providing evidence of synergy when combining CDK 4/6 inhibitors with anti-HER2–based therapy. Based on these data, multiple clinical trials are ongoing to evaluate the combination of HER2-directed therapy and CDK 4/6 inhibitors in ER+/HER2+ breast cancer.
Finally, recent data suggest the FGFR kinase may also mediate resistance to CDK 4/6 inhibitors. Formisano et al27,28 not only identified FGFR1 as associated with resistance to AI-based therapy27 but additionally, identified FGFR1 amplification as a mechanism of resistance to the combination of ribociclib and fulvestrant in vitro.28 In this report, the addition of the FGFR tyrosine kinase inhibitor erdafitinib to fulvestrant/palbociclib resulted in marked regressions in vivo. Based on these data, a clinical trial is planned to combine erdafitinib with a CDK 4/6 inhibitor in ER+ FGFR–amplified breast cancer.


A myriad of molecular mechanisms have been postulated to be associated with resistance to various HER2-targeted therapies (Fig. 1). Indeed, a comprehensive outline of humanized monoclonal anti-HER2 antibody trastuzumab resistance biomarkers in HER2-overexpressing breast cancer has been cataloged in a scholarly review by Menyhárt et al.29 Among the types of biomarkers most well studied to date are (1) perturbation of HER family receptors or binding of therapeutic antibodies to HER2 (e.g., shedding of the HER2 extracellular domain,30 expression of the Δ16HER2 splice isoform expression,31 overexpression of MUC4/MUC1 resulting in steric hindrance to trastuzumab binding to the HER2 extracellular domain,32 and increased phosphorylation of HER333); (2) parallel receptor pathway activation (e.g., upregulation of IGF-1 receptor,34 erythropoietin receptor,35 AXL receptor,36 or MET receptor37); and (3) activation of downstream signaling events distal to HER2 receptor (e.g., hyperactivation of the PI3 kinase/Akt pathway by loss of PTEN or PIK3CA mutational activation,38 cyclin E amplification/overexpression,39 upregulation of miR-21,40 and expression of the ER41). Of these and other potential resistance pathways, not all have been confirmed to occur in human clinical/translational cohorts with annotated outcomes, and even those that have at least some clinical evidence from discovery cohorts lack validation cohorts and/or independent confirmation across multiple trials. Fewer still have prospective clinical/translational efforts of new therapeutic approaches to overcome resistance to HER2-targeted therapeutics (Table 1). Herein, we discuss three resistance mechanisms addressed by recent/ongoing interventional clinical trials: (1) the use of antibody-drug conjugate (ADC) ado-trastuzuman emtansine (T-DM1) to overcome resistance as a result of PIK3CA mutation, (2) the use of novel approaches to enhance antibody-dependent cell-mediated cytotoxicity (ADCC) of immune effector cells to address resistance caused by low-affinity activating Fcγ receptor (FcγR) polymorphisms, and (3) solutions to overcome anatomic resistance by the blood-brain barrier in HER2+ brain metastasis.
Figure 1. Selected Examples of Resistance to HER2-Targeted Therapies
IGF-1 receptor (far left) and MET (far right) are shown as two examples of parallel receptor pathway activation to bypass HER2 (downstream signaling in light blue). Truncated HER2 C-terminal fragments/isoforms resulting from proteolysis of p185HER2 (p95-HER2), alternative initiation of translation (p110-HER2), or alternative splice variation (del exon 16) lead to loss of antibody (or ADC) binding epitopes as well as hyperactivation of downstream pathways, resulting in HER2 MAb/ADC resistance (middle section). Glycosylated MUC4 or MUC1 (middle section) has been shown to sterically hinder binding of HER2 antibodies to HER2 receptor. PI3 kinase mutation, loss of PTEN, HER2 L755S mutation, and upregulation of miR-21 as well as upregulation of IRS4 result in downstream signal activation uncoupled from control at the receptor level (maroon in the middle). Upregulation of extracellular matrix and collagen II genes leads to activation of the integrin-β1/Src signaling pathway (gold in the middle right). Alteration of HER3 expression (far right) and/or NRG-1 overexpression can result in attenuated response to HER2 targeting agents. Decreased FOXO3a can result in transcriptional upregulation of the ER, leading to relative resistance to HER2-targeting agents (yellow in the bottom left); amplified cyclin E or loss of negative regulation by p27 drives the transition from G1 to S phase (light green in the bottom right). Finally, the TGFβ/SMAD3 axis and EMT can induce cell surface CD73 expression and consequent adenosine generation, leading to tumor immune escape (left in the top box).
Abbreviations: ECM, extracellular matrix; NK, natural killer; TKI, tyrosine kinase inhibitor; ER, estrogen receptor; ADC, antibody-drug conjugate.
Table 1. Postulated Mechanisms of Resistance to HER2-Targeted Therapies in Breast Cancer

Overcoming Resistance as A Result of PIK3CA Mutation USING ADC T-DM1

One of the best characterized mutational events associated with resistance to both HER2-directed monoclonal antibodies (MAbs) and small molecule HER2 kinase inhibitors is somatic mutation of the PIK3CA gene—the most common molecular alteration in human breast cancer. We hypothesized that treatment with T-DM1 would be agnostic to the presence or absence of downstream activating PIK3CA mutations, because cytotoxicity of the derivative of maytansine 1 cytotoxic payload of T-DM1 is not dependent on activation status of the PI3 kinase signaling pathway. We had the opportunity to test this hypothesis by investigating whether the efficacy of T-DM1 was correlated with PIK3CA mutation in the phase III pivotal registrational EMILIA study—a randomized phase III study of T-DM1 versus lapatinib and capecitabine in 991 patients with metastatic HER2+ cancer who had prior treatment with trastuzumab and a taxane.86 Tumor tissue was collected (with additional consent) and subjected to PIK3CA DNA sequence analysis (259 patients) using the cobas PIK3CA mutation test (Roche Molecular Diagnostics) for exon 1: R88Q; exon 4: N345K; exon 7: C420R; exon 9: E542K, E545X, and Q546X; and exon 20: M1043I, H1047X, and G1049R. PFS and overall survival were analyzed using the Kaplan–Meier method and a Cox regression model.67 Moreover, T-DM1 was also tested on cell lines and in HER2+ breast cancer xenograft models containing defined activating PIK3CA mutations. PIK3CA mutation frequency (30.5%) was similar across both treatment arms and consistent with previously reported data.23,87,88 PIK3CA mutations were associated with shorter median PFS (mutant vs. wild type: 4.3 vs. 6.4 months) and overall survival (17.3 vs. 27.8 months) in patients treated with capecitabine plus lapatinib but not patients treated with T-DM1 (PFS, 10.9 vs. 9.8 months; overall survival, not yet reached in mutant or wild-type groups). Additionally, T-DM1 showed potent activity in cell lines and xenograft models with known activating PIK3CA mutations.67 We concluded that, despite the observation that other standard HER2-directed therapies are less effective in tumors with PIK3CA mutations, T-DM1 seems to be effective in both PIK3CA-mutated and PIK3CA wild-type tumors.

Humanized HER2 Monoclonal Antibody Resistance Mediated by Low-affinity Polymorphisms IN Activating FCΓRS

In the case of therapeutic immunoglobulin G1 isotype humanized MAbs (like trastuzumab and pertuzumab), it is not only perturbation of downstream receptor signaling that accounts for clinical efficacy in vivo but also, FcγR-dependent ADCC mediated by various immune effectors, such as macrophages and natural killer cells.89 ADCC occurs when the Fc portion of the tumor-bound antibody is recognized by FcγRs. In knockout mice deficient in activating FcγR genes, the antitumor effects of trastuzumab are significantly blunted.90 Consistent with these observations, engineered anti-HER2 antibodies with disabled Fc domains fail to induce tumor responses in vivo, despite retained HER2 binding and growth inhibition in vitro. Conversely, antitumor antibodies are 10-fold more effective in mice deficient in inhibitory FcγRs, and antitumor antibody potency is greatly increased by engineering Fc domains to bind activating FcγR with greater affinity/avidity than inhibitory Fc receptors.58 Moreover, studies in the metastatic and neoadjuvant settings suggest that single-nucleotide polymorphisms in activating and decoy FcγRs (FcγR3A and FcγR2A, respectively) may be associated with differential response to trastuzumab by modulating ADCC.91,92 This question was recently explored by Gavin et al93 from the National Surgical Adjuvant Breast and Bowel Project (NSABP) in their analysis of polymorphisms in FcγRs in early-stage breast cancer in the NSABP B-31 adjuvant trastuzumab trial.93 As expected, patients with genotype FcγR3A-158V/V or FcγR3A-158V/F received greater benefit from trastuzumab (HR 0.31; 95% CI, 0.22–0.43; p < .001) than patients who were homozygous for the low-affinity allele (HR 0.71; 95% CI, 0.51–1.01; p = .05), thus confirming prior published observations in metastatic disease to an adjuvant early-stage setting.91,92

Fc-engineered HER2 Monoclonal Antibody Margetuximab Elicits Potent ADCC Reactions, Even with Low-affinity Activating Fc Receptors

It may be theoretically possible to overcome such deficits in antibody Fc domain binding to low-affinity FcγRs by engineering the Fc domain to achieve higher binding affinity to activating FcγRs (and lower binding affinity to decoy receptors). This theoretical ideal has been achieved in the case of chimeric anti-HER2 antibody margetuximab (MGAH-22), which binds with elevated affinity to both lower- and higher-affinity forms of FcγR3A.58 In phase I, first-in-human dose-finding studies of margetuximab, tumor reductions were observed in over one-half (18 of 23; 78%) of response-evaluable patients with breast cancer, including durable (greater than 30 weeks) responders.94 Ex vivo analyses of patient peripheral blood mononuclear cell samples confirmed the ability of margetuximab to support enhanced ADCC compared with trastuzumab. In addition, margetuximab was very well tolerated, with mostly grades 1 and 2 toxicities consisting of pyrexia, nausea, anemia, diarrhea, and fatigue—similar to infusion-related reactions observed with other HER2-directed therapeutic monoclonal antibodies and thus far, with no apparent increase in cardiac adverse events.94 Clinical development of margetuximab has proceeded to an ongoing phase III pivotal trial comparing margetuximab directly with trastuzumab in combination with salvage chemotherapy in pretreated HER2+ metastatic breast cancer (SOPHIA trial; NCT02492711).

Augmenting ADCC USING Agonist Antibodies Directed Against CD137

Another means to augment ADCC is by activation of the costimulatory receptor CD137 (4-1BB) on natural killer cells using agonist antibodies (for example, utomilumab [the proposed nonproprietary name for PF-05082566]).95 CD137 activation occurs both in vitro and in the peripheral blood of women with HER2-overexpressing breast cancer after trastuzumab treatment.95 Stimulation of trastuzumab-activated human natural killer cells with agonistic MAb specific for CD137 euthanized breast cancer cells (including an intrinsically trastuzumab-resistant cell line) more efficiently both in vitro and in vivo in xenotransplant models of human breast cancer. The dual antibody strategy combining a tumor-targeting antibody with a second antibody that activates the host innate immune system may improve the therapeutic effects of antibodies against breast cancer and other HER2-expressing tumors.95 An investigator-initiated phase IB/II clinical trial of agonist CD137 antibody utomilumab in combination with trastuzumab (or T-DM1) is currently underway (NCT03364348).

Overcoming Anatomic Resistance as A Result of the Blood-brain Barrier: the Central Nervous System Niche as A Sanctuary Site

A final novel form of resistance is that of anatomic resistance. Consider, for example, the central nervous system (CNS) microenvironment, which consists of a unique vascular endothelium (the so-called blood-brain barrier), pericytes, astrocytes, and glial cells, all of which may contribute in concert to pathogenesis of the CNS metastatic niche.96 HER2+ breast cancer has a strong predilection for metastasis to the CNS, with as many as one-half of all patients with HER2+ metastatic breast cancer experiencing brain metastasis during their treatment course. CNS metastases can develop during efficacious treatment of extracranial metastasis with HER2 monoclonal antibodies, suggesting that the CNS is a sanctuary site for HER2+ cancer cells. Indeed, it is argued that the macromolecular size of monoclonal antibodies (150 kd) is a severe handicap for their diffusion in therapeutic concentrations across the blood-brain barrier. This model assumption affords the novel hypothesis that diffusion of macromolecular antibody therapeutics, such as trastuzumab, may be achieved through mass action (compare with the Le Chatelier Principle) simply by increasing the concentration of trastzumab in the circulation (recall that trastuzumab had no defined maximum tolerable dose during dose escalation, and therefore, the dose can arguably be increased with impunity). To test this hypothesis, a trial of high-dose (6 mg/kg weekly) trastuzumab (in combination with standard dose pertuzumab for control of extracranial metastasis) for HER2+ CNS metastasis is currently ongoing (NCT02536339).61 Consistent with this hypothesis, previous publications have documented diffusion of positron-emitter 89Zr-conjugated trastuzumab with localization to HER2+ CNS in humans.97 Moreover, multiple groups have documented clinical responses to ADC T-DM1 (with molecular weight slightly higher that of trastuzumab) in HER2+ CNS metastasis.98,99
Meanwhile, small molecule HER2 tyrosine kinase inhibitors continue to be developed as treatment of HER2+ CNS metastasis. Historically, modest results have been obtained from the combination of lapatinib with capecitabine. Newer encouraging data have recently been presented for neratinib in combination with capecitabine62 and highly HER2-selective tyrosine kinase inhibitor tucatinib (ONT-380)-based regimens.63 If pathogenic factors within the CNS metastatic niche can be identified (such as chemotactic factors, adhesion, and transendothelial tumor cell extravasation factors as well as peptide growth factors), there may be unique opportunities for exploiting novel treatment approaches or perhaps more importantly, opportunities for prophylaxis against HER2+ brain metastasis altogether.96

Triple Negative Breast CANCER

TNBCs account for 10% to 20% of primary breast cancers and are so named, because they express low levels of ER, PR, and HER2. Early efforts to develop targeted therapy strategies resulted in lackluster clinical response rates in TNBC, which is now presumed to be related, in part, to molecular heterogeneity within the “catchall” diagnosis of TNBC. Recent advances in molecular characterization have shown subtypes of TNBC, each with distinct targetable molecular aberrations.100,101 Although it is anticipated that ongoing clinical trials will lead to additional targeted therapies for TNBC, it is important to note that the only currently FDA-approved targeted therapy is olaparib for the treatment of BRCA-associated TNBCs.

BRCA-associated Triple Negative Breast CANCER

BRCA1 and BRCA2 are required for homologous recombination repair of DNA strand breaks, leading to a defect in DNA repair in cancers harboring these mutations.102 As such, these tumors are more sensitive to chemotherapy-inducing DNA breaks, such as those induced by platinum-based therapies. The TNT trial randomized 376 patients with advanced TNBC to receive either carboplatin or docetaxel for six to eight cycles or until disease progression.103 There was no significant difference in objective response rate (ORR) or median PFS between the two arms (p = .44); however, in patients with BRCA1/2-associated breast cancers (43 patients), both ORR and PFS were significantly improved with carboplatin compared with docetaxel (p = .03). Notably, a diagnostic assay to measure tumor deficiencies in homologous recombination failed to predict benefit for carboplatin in non-BRCA–associated TNBC.
PARP enzymes catalyze the formation of chains of poly(adenosine 5′-diphosphate)-ribose units, which recruit the necessary DNA repair proteins.104 PARP inhibition leads to accumulation of single-strand DNA breaks, which lead to double-strand breaks at replication forks. In the absence of PARP, tumors that lack the ability to repair double-strand DNA breaks through homologous recombination must use less efficient mechanisms, such as nonhomologous end joining, which lead to further genomic instability and cell death.105 Early-phase trials of PARP inhibitors showed single-agent responses in BRCA-associated tumors, including in patients with TNBC.106 The OlympiAD trial was an open label, randomized phase III trial that compared single-agent olaparib with physician’s choice of standard chemotherapy (capecitabine, eribulin, or vinorelbine) for the treatment of BRCA-associated breast cancers (302 patients), one-half of which were TNBC.107 Median PFS was significantly longer in the olaparib group (7.0 vs. 4.2 months; HR 0.58; p < .001), and the ORR was higher (59.9% vs. 28.8%). Similar results were seen in the EMBRACA trial when the PARP inhibitor talazoparib was compared with physician’s choice of chemotherapy.108 However, it is important to note that neither of these trials contained a DNA-damaging agent in the “physician’s choice” arm and that both excluded patients with a history of disease progression while receiving platinum-based therapy for metastatic disease.

Immune Modulation FOR Treatment of TNBC

Immunotherapy is a rapidly evolving strategy for the treatment of TNBC. Tumor-infiltrating lymphocytes have been recognized as a positive prognostic biomarker by analysis of data from multiple adjuvant therapy trials in unselected breast cancer.109,110 Additionally, the presence of tumor-infiltrating lymphocytes has been favorably associated with higher rates of pathologic complete response to neoadjuvant chemotherapy in TNBC.109
Mittendorf et al111 found that PD-L1 expression occurred in 20% of TNBC tumors, suggesting that targeting PD-1 or PD-L1 may have therapeutic benefit in TNBC. Currently, the most mature studies evaluating immunotherapy in TNBC involve drugs targeting the PD-1/PD-L1 axis. The KEYNOTE-012 trial was a multicenter, nonrandomized phase Ib trial of single-agent pembrolizumab (anti–PD-1) in PD-L1+ (greater than or equal to 1%) TNBC.112 The drug was well tolerated, with toxicities similar to those reported in other solid tumor types (low-grade arthralgia, fatigue, myalgia, and nausea). In the 27 patients evaluated for response, the overall response rate (complete response or partial response) was 18.5%, with the median duration of response not reached at the time of publication (range of 15–47+ weeks). Importantly, some responders continued treatment of over 1 year. The follow-up phase II trial (KEYNOTE-086 trial) enrolled 170 patients with previously treated TNBC and showed an overall response rate of 5%, regardless of PD-L1 expression. Median PFS and overall survival were 2.0 and 8.9 months, respectively.113 Similar response rates have been reported with avelumab (anti–PD-L1 antibody). The Javelin study determined the overall response rate to single-agent avelumab to be 9% in patients (58 patients) with advanced TNBC.114 These single-agent results suggest that combination strategies as well as novel predictors of response must be pursued to further improve clinical outcomes.
Tumor cell death induced by cytotoxic chemotherapy or radiation also has the potential to expose the immune system to higher levels of tumor antigens; thus, inhibiting PD-L1/PD-1 signaling in combination with these therapeutic modalities may theoretically result in deeper and more durable responses. Ongoing studies will determine if combining immunotherapy with chemotherapy or radiation therapy improves clinical outcomes in patients with metastatic TNBC. Preliminary safety data from the study GP28328 indicate that atezolizumab (anti–PD-L1) can be safely combined with chemotherapy, including nab-paclitaxel and carboplatin.115
At least two groups have identified subtypes of TNBC with enhanced expression of genes involved in immune signaling. Lehmann et al100 initially described the immunomodulatory subtype; however, when tumor cells were separated from stroma using laser capture microdissection, this subtype was no longer identified, suggesting that this signature likely identifies immune infiltrate within stroma.116 Burstein et al101 described a basal-like immune-activated subtype that overexpressed CTLA-4 in addition to other immune-related genes. As PD-1/PD-L1 inhibitors advance as a therapeutic strategy for TNBC, it will be interesting to determine if either of these subtypes is associated with enhanced response to immunotherapy.

Pi3k/akt Pathway

The LOTUS trial investigated the benefit of administering the oral Akt inhibitor ipatasertib in combination with paclitaxel as first-line therapy in patients (124 patients) with metastatic TNBC in a placebo-controlled, double-blind phase II trial. Median PFS was 6.2 months with ipatasertib versus 4.9 months with placebo for the entire cohort (p = .037); however, the difference in PFS was much more profound in a subgroup of tumors (42 patients) with PI3K/AKT1/PTEN-altered tumors (9.0 vs. 4.9 months; p = .041).
Gene expression signatures have also identified breast cancer subsets enriched in EMT features. Initially, a breast cancer subset enriched in EMT was identified and named “claudin low,” because this group of tumors showed low gene expression of the tight junction proteins claudin 3, 4, and 7.117-119 Most claudin-low tumors are TNBC.117 Lehmann et al100 and Burstein et al101 also independently identified distinct subtypes of TNBC that contained gene expression profiles enriched in EMT, which they termed mesenchymal (Lehmann et al100 and Burstein et al101) and mesenchymal stem-like (Lehmann et al100). Importantly, on microdissection, the mesenchymal stem–like subtype was no longer identified in a majority of tumors tested, suggesting that this subtype call was also strongly weighted by stromal gene expression.116
Mesenchymal TNBCs carry a high rate of molecular aberrations that activate the PI3K/Akt/mTOR axis, suggesting that this subgroup may be responsive to therapeutic regimens targeting this pathway.100,117,120,121 In support of this concept, metaplastic breast cancers account for 10%–57% of TNBCs characterized as claudin low and can be clinically identified by light microscopy because of an admixture of epithelial and mesenchymal components within the tumor. These tumors are also associated with a high rate of PI3K mutations and/or activation of the PI3K pathway.121,122 Patients with metastatic, metaplastic breast cancer (52 patients) were treated in a clinical trial with liposomal doxorubicin, bevacizumab, and the mTOR inhibitor temsirolimus or everolimus (DAT or DAE).122 The ORR was 21% for DAT/DAE, and the clinical benefit rate was 40% (complete response of four, partial response of seven, and standard deviation greater than or equal to 6 months of 10). Notably, in the 43 patients who had tissue available for genomic analyses, there was a 74% incidence of activating PI3K/Akt/mTOR molecular aberrations, and this was associated with a significant improvement in ORR (31% vs. 0%; p = .04). Other therapeutic strategies with the potential to target EMT include dual PI3K/mTOR inhibitors, c-MET inhibitors, NOTCH pathway inhibitors, and TGFβ-targeted agents.

Androgen Receptor+/Luminal Androgen Receptor Subtype

The luminal androgen receptor subtype was the most differential among the TNBC subtypes identified by Lehmann et al100 and accounted for approximately 11% of TNBC. This subtype was later confirmed by Burstein et al,101 who kept the name when describing their identified subtype.100,101 The luminal androgen receptor subtype is so named because of high messenger RNA expression of androgen receptor, which is noted to be ninefold greater than other subtypes.100 Additionally, androgen receptor expression by immunohistochemistry was significantly higher in the luminal androgen receptor compared with other TNBC subtypes.100 In a single-arm phase II trial of bicalutamide in patients with androgen receptor+, ER/PR− metastatic breast cancer, the clinical benefit rate at 24 weeks, defined as the percentage of patients who have complete response, partial response, or stable disease (standard deviation for 24 weeks), was 19%, and the median PFS was 12 weeks.123 A phase II study of enzalutamide in advanced androgen receptor+ TNBC showed a clinical benefit rate at 24 weeks of 29% and a median PFS of 14 weeks. In this trial, tumors that showed an androgen-related gene signature profile seemed to derive greater clinical benefit from enzalutamide, suggesting that the use of gene expression profiling may help to identify patients with TNBC who would most likely benefit from androgen receptor blockade.124

Antibodies AND Antibody-drug Conjugates

Reports of overexpression and/or enhanced EGFR signaling in TNBC125-127 led to strategies using the monoclonal anti-EGFR antibody cetuximab as a single agent or in combination with chemotherapy. Cetuximab in combination with carboplatin induced higher ORR (16%) compared with single-agent cetuximab (6%)128; however, the combination of cetuximab with cisplatin did not significantly improve ORR compared with cisplatin alone in metastatic TNBC.129 Although targeted therapy with monoclonal antibodies alone has not improved outcomes in TNBC, there is a rising interest in ADCs as a therapeutic strategy.130 ADCs allow for the select delivery of moderate to ultrapotent cytotoxic drugs by targeting tumor-associated antigens. ADC binding to these antigens induces internalization of the drug into the tumor cell and subsequent release of the “payload” cytotoxic. Promising targets for ADCs currently under development for the treatment of TNBC include trophoblast cell surface antigen, Ephrin A4, folate receptor alpha, and low-level expression of HER2.130


In summary, the addition of CDK 4/6 inhibitors to endocrine therapy has markedly changed the landscape of ER+ metastatic breast cancer over a short period of time. Although the available drugs show remarkable similarities in terms of PFS benefit across the FDA registration trials, differences in both toxicity and response rates have been noted, and long-term follow-up of the ongoing clinical trials will be needed to identify the optimal strategy. This includes selection of both the best initial CDK 4/6 inhibitor/endocrine therapy combination and the optimal sequence (combination vs. sequential) for patients with highly endocrine–sensitive tumors. Furthermore, additional studies to elucidate the mechanisms of resistance and biomarkers that define response/resistance to this class of drugs are needed.
HER2+ breast cancers have myriad potential resistance pathways from which to choose. Lack of real-time monitoring for emergence of resistance pathways remains a critical unmet need. To this end, new HER2-targeting therapeutic strategies must be developed to exploit our better understanding of resistance pathways. As an example, it has been shown that ADC T-DM1 can overcome resistance to PI3 kinase pathway activation via PIK3CA mutants. It is hoped that augmenting the immunologic mechanism of action of HER2 therapeutic monoclonal antibodies via ADCC (Fc domain antibody engineering of HER2 antibodies or activation of CD137 with agonist antibodies) may also be able to bypass other vertical or horizontal HER2 intrinsic cellular resistance mechanisms. Finally, overcoming anatomic resistance (e.g., blood-brain barrier) may be possible with high-dose trastuzumab, potent HER2-directed ADCs, or improved tyrosine kinase inhibitors with greater CNS penetration and HER2 specificity (e.g., tucatinib). It is encouraging that a number of these approaches are the focus of intense ongoing clinical investigations.
Finally, the limited success previously seen with targeted therapy in TNBC is likely the result of the molecular heterogeneity of the disease, which leads to a dilution of drug effect in unselected patients. The approval of olaparib in BRCA-associated cancers (many of which are TNBCs) shows that appropriate selection of patients for targeted therapy can be beneficial in TNBC. Through modern molecular characterization, subtypes of TNBC have emerged, and targeted strategies are being developed based on their unique features. However, subtyping by gene expression is influenced by bioinformatic methods and can have problems with reproducibility in individual patients. It is critical that these barriers be overcome to better identify patients for targeted therapy strategies.

Authors' Disclosures of Potential Conflicts of Interest

The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO's conflict of interest policy, please refer to

Matthew P. Goetz

Consulting or Advisory Role: bioTheranostics, Eisai, Genomic Health, Lilly, Novartis
Research Funding: Lilly, Pfizer
Patents, Royalties, Other Intellectual Property: #Methods and Materials for Assessing Chemotherapy Responsiveness and Treating Cancer, #METHODS AND MATERIALS FOR USING BUTYRYLCHOLINESTERASES TO TREAT CANCER

Stacy L. Moulder

Honoraria: Novartis
Research Funding: Bayer (Inst), Genentech (Inst), Novartis (Inst), Oncothyreon (Inst), Pfizer (Inst), Takeda (Inst)
Travel, Accommodations, Expenses: Novartis

Mark D. Pegram

Employment: Loxo (I)
Consulting or Advisory Role: Genentech, Novartis, Pfizer
Travel, Accommodations, Expenses: DAVA Pharmaceuticals, Genentech, Novartis, Pfizer
Other Relationship: Genentech, Novartis, Pfizer

Clinton Yam

No relationship to disclose

Yu Zong

No relationship to disclose


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Published In

American Society of Clinical Oncology Educational Book
Pages: 65 - 77
PubMed: 30231328


Published online: May 23, 2018


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Mark D. Pegram, MD
From the Stanford Comprehensive Cancer, Stanford, CA; The University of Texas MD Anderson Cancer Center, Houston, TX; Mayo Clinic Cancer Center, Rochester, MN.
Yu Zong, MD
From the Stanford Comprehensive Cancer, Stanford, CA; The University of Texas MD Anderson Cancer Center, Houston, TX; Mayo Clinic Cancer Center, Rochester, MN.
Clinton Yam, MD
From the Stanford Comprehensive Cancer, Stanford, CA; The University of Texas MD Anderson Cancer Center, Houston, TX; Mayo Clinic Cancer Center, Rochester, MN.
Matthew P. Goetz, MD
From the Stanford Comprehensive Cancer, Stanford, CA; The University of Texas MD Anderson Cancer Center, Houston, TX; Mayo Clinic Cancer Center, Rochester, MN.
Stacy L. Moulder, MD [email protected]
From the Stanford Comprehensive Cancer, Stanford, CA; The University of Texas MD Anderson Cancer Center, Houston, TX; Mayo Clinic Cancer Center, Rochester, MN.


Corresponding author: Stacy L. Moulder, MD, Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd., Unit 1354, Houston, TX 77030; email: [email protected].

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Mark D. Pegram, Yu Zong, Clinton Yam, Matthew P. Goetz, Stacy L. Moulder
American Society of Clinical Oncology Educational Book 2018 :38, 65-77

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