Tackling endocrine resistance in ER-positive HER2-negative advanced breast cancer: A tale of imprecision medicine

Accepted Manuscript Title: Tackling endocrine resistance in ER-positive HER2-negative advanced breast cancer: a tale of imprecision medicine Authors: Alexios Matikas, Theodoros Foukakis, Jonas Bergh PII: DOI: Reference:

S1040-8428(16)30339-0 http://dx.doi.org/doi:10.1016/j.critrevonc.2017.04.002 ONCH 2371

To appear in:

Critical Reviews in Oncology/Hematology

Received date: Revised date: Accepted date:

24-11-2016 28-3-2017 4-4-2017

Please cite this article as: Matikas Alexios, Foukakis Theodoros, Bergh Jonas.Tackling endocrine resistance in ER-positive HER2-negative advanced breast cancer: a tale of imprecision medicine.Critical Reviews in Oncology and Hematology http://dx.doi.org/10.1016/j.critrevonc.2017.04.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Tackling endocrine resistance in ER-positive HER2-negative advanced breast cancer: a tale of imprecision medicine Running head: Endocrine resistant advanced breast cancer

Alexios Matikas1, Theodoros Foukakis1, Jonas Bergh1 1

Department of Oncology-Pathology, Karolinska Institutet and University Hospital, Stockholm,

Sweden Corresponding Author: Dr. Alexios Matikas, Department of Oncology-Pathology, Karolinska Institutet, 17176, Stockholm, Sweden; e-mail: [email protected]; tel: +46767823322 Author contributions: AM and TF conceptualized and designed the review. All authors participated in drafting the manuscript. JB contributed critical revisions. Running head: Endocrine resistant advanced breast cancer

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Highlights     

Optimal treatment after endocrine failure in advanced breast cancer is not clearly defined Combinations of endocrine agents have met with mixed results PI3K/mTOR inhibition has modest efficacy; no biomarkers have been identified CDK4/6 inhibitors significantly prolong PFS, but not OS, in randomized trials Biomarkers are critical for the integration of novel combinations in clinical practice

ABSTRACT The selection of patients with advanced breast cancer as appropriate for endocrine manipulation according to hormone receptor status is a successful strategy. Unfortunately, the emergence of resistance is inevitable and subsequent treatment is not well defined. Numerous mechanisms have been implicated in the development of resistance; central among them is the activation of compensatory signaling pathways. Despite the rationale that supports combining agents targeting these pathways with hormonal therapies in an attempt to delay or even reverse endocrine resistance, most clinical trials have failed to demonstrate improved outcomes. Although the inhibition of the PI3K/mTOR pathway and of CDK 4/6 function has led to meaningful prolongations of progression free survival, no overall survival gains have been reported yet. Considering the associated toxicity and costs, genomic-driven trials are eagerly needed in order to refine management strategies and achieve a truly personalized approach for this patient subgroup. Keywords: breast cancer; CDK 4/6; endocrine; metastatic; mTOR; PI3K; resistance

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1. Introduction The endocrine manipulation of hormone receptor (ER) positive metastatic breast cancer (MBC) represents a highly successful example of precision medicine, where treatment selection is based on the presence of a predictive biomarker. For most patients clinical practice guidelines support initial treatment with endocrine therapies (Cardoso et al., 2017), such as gonadotropin-releasing hormone agonists (GnRH-A), selective ER modulators (tamoxifen), aromatase inhibitors (AI) or the ER antagonist fulvestrant which blocks ER dimerization and nuclear uptake (Possinger, 2004). Unfortunately, eventual disease progression is inevitable. Clinical resistance to endocrine treatment can be classified into primary (progression in under 6 months for advanced disease or after less than two years of adjuvant treatment) and acquired (progression in over 6 months for advanced disease or after more than two years of adjuvant treatment or less than 12 months after its discontinuation) (Cardoso et al., 2014). The evolution of the clonal architecture of ER-positive MBC under the pressure of endocrine treatment is promoted by several escape mechanisms which result in the expansion of resistant clones, clinically overt disease progression, need for cytotoxic chemotherapy which is associated with worse quality of life compared to endocrine treatment (Gupta et al., 2014) and, eventually, death. These mechanisms include the acquisition of ESR1 mutations, loss of ER expression, imbalances between positive and negative coregulators, overexpression of growth factors and enhanced crosstalk with compensatory pathways (Osborne and Schiff, 2011). Subsequent treatment after the failure of endocrine therapy is not clearly defined. In an attempt to delay the manifestation of resistance or even reverse it, agents targeting several cell processes have been combined with continuous endocrine treatment with variable levels of success. The results of these trials, presented in table 1, are reviewed with the aim to explore the reasons of previous

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failures and suggest possible solutions for implementation in future clinical and translational studies. 2. Combinations of endocrine agents Since the most commonly used agents in MBC inhibit ER signaling through distinct mechanisms, an intuitive approach would be to combine them in order to achieve maximal vertical blockade of the ER axis. An early attempt was the combination of GnRH-A with tamoxifen, with a metaanalysis supporting its superiority compared to GnRH-A alone (Klijn et al., 2001); its relative efficacy compared to tamoxifen is unclear. Nevertheless, this combination has become a standard option for premenopausal women with MBC. More data are available for postmenopausal patients. Based on preclinical findings (Macedo et al., 2008), the combination of anastrozole and low-dose fulvestrant (F-LD) was compared to the AI alone at the first line setting in two trials, with conflicting results (Bergh et al., 2012; Mehta et al., 2012). The prolongation of overall survival (OS) with the combination in the S0226 trial (47.7 versus 41.3 months, P=0.05) could be attributed to the large fraction of endocrine naïve patients enrolled (60%) compared to the FACT trial, while another difference was that 38.9% in the former trial were diagnosed with de novo metastatic disease. These observations, along with the fact that in the S0226 trial the prolongation of progression free survival (PFS) only concerned endocrine naïve patients [17.0 versus 12.6 months, Hazard Ratio (HR)=0.74, P=0.006] and not patients that had received prior tamoxifen (HR=0.89, P=0.37), supports the management of sensitive patients with polyendocrine therapy. Regarding second line treatment, the combination of F-LD with anastrozole was not superior to neither F-LD nor exemestane monotherapy in the SoFEA study (Johnston et al., 2013).

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Interestingly, the detection in the plasma of ESR1 mutations predicted benefit from fulvestrant versus exemestane (Fribbens et al., 2016). It should be noted that in most of these trials F-LD was used, which has been found to be inferior to high dose fulvestrant (500 mg monthly, F-HD) in terms of both PFS and OS in the CONFIRM trial (Di Leo et al., 2010). Importantly, an unplanned analysis of the FIRST study also revealed a prolongation of OS with F-HD compared to first line AI (Ellis et al., 2015) and the confirmatory phase III FALCON trial was stopped early due to the demonstration of PFS prolongation favoring F-HD (Ellis et al., 2016), implying that fulvestrant may be the optimal first line choice in ERpositive MBC. These results leave unresolved the issue of how a combination of F-HD with AI would compare to alternatives in ER-positive MBC. Presently, such a combination or F-HD monotherapy represent reasonable options as first line treatment for ER-positive MBC. 3. Inhibition of the PI3K/AKT/mTOR pathway 3.1 Biology and mechanisms of resistance The phosphoinositide 3-kinase/phosphatase and tensin homolog/protein kinase B/mechanistic target of rapamycin (PI3K/PTEN/AKT/mTOR) pathway is a central mediator of several vital cellular functions, such as growth, differentiation and proliferation. Its dysregulation and constitutive activation is common in tumorigenesis and has been implicated in the emergence of resistance to endocrine manipulation in BC (Miller et al., 2010). Consequently, inhibitory molecules of this pathway have been evaluated in clinical trials based on preclinical data that indicate that mTOR inhibitors act synergistically with AI when co-administered and may delay the development of resistance (Boulay et al., 2005).

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Resistance mechanisms to mTOR inhibition exhibit significant diversity, with a complex interplay of the genomic profile of the tumor such as the presence of V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations (Di Nicolantonio et al., 2010), the increased intrinsic kinase activity of mTOR (Rodrik-Outmezguine et al., 2016) and the activation of escape routes through bypass signaling [insulin growth factor receptor 1 (IGF-1R) and mitogen activated protein kinase (MAPK) being the most prominent pathways (O'Reilly et al., 2006)] contributing to the emergence of resistance. Efforts to circumvent it have already been explored in preliminary clinical trials (Ma et al., 2013). 3.2 Clinical trials The BOLERO-2 phase III trial lead to the regulatory approval of everolimus for patients with ERpositive MBC, resistant to AI (Baselga et al., 2012). Patients pretreated with a non-steroid AI were randomized to receive exemestane with or without everolimus. Locally assessed PFS, which was the primary endpoint, favored the combination group, 7.8 versus 3.2 months (HR=0.45, P<0.0001) (Yardley et al., 2013b). This effect was similar regardless of prior receipt of chemotherapy, absence of prior treatment for advanced disease (Beck et al., 2014), the presence of visceral metastases (Campone et al., 2013) and older age (Pritchard et al., 2013). However, there was no OS benefit (31.0 versus 26.6 months, P=0.1426) (Piccart et al., 2014). The most common grade 3/4 adverse events for the combination were stomatitis (8%), non-infectious pneumonitis (4%), new-onset diabetes (6%) and infections (6.6%); these events were rare in the control group. Furthermore, 62% of patients had dose reductions or interruptions and 26% discontinued due to toxicity in the experimental group; the respective rates for the control group were 12% and 5% (Baselga et al., 2012). The combination of everolimus with tamoxifen in AI resistant MBC was evaluated in the phase 2 TAMRAD trial, which was not designed to perform a formal statistical comparison. Time to progression (TTP) and OS significantly favored the combination compared 6

to tamoxifen monotherapy, with patients with acquired endocrine resistance benefiting the most. Due to the design of this trial the authors acknowledge that these findings should be considered to be hypothesis generating (Bachelot et al., 2012). Recently, the efficacy of another everolimusbased combination, with fulvestrant, was proven superior in terms of PFS compared to the endocrine agent alone. In the randomized phase II PrECOG 0102 trial, the combination statistically significantly prolonged PFS (10.4 versus 5.1 months, P=0.02), offering yet another option to patients progressing on an AI (Kornblum et al., 2016). Another mTOR inhibitor, temsirolimus, has been evaluated in the HORIZON phase III trial. Its combination with letrozole did not improve PFS compared to first line letrozole alone. Since temsirolimus was administered to patients sensitive to AIs at an oral intermittent schedule, inadequate blockade of mTOR may have contributed to these results (Wolff et al., 2013). In addition, the single arm BOLERO-4 study of first line everolimus and letrozole and, following disease progression, exemestane combinations demonstrated a 12-month PFS rate of 71.4% (Royce et al., 2016); the non-randomized nature of this trial does not allow for robust conclusions. Preclinical data have demonstrated that PI3K inhibitors can induce synthetic lethality when combined with endocrine therapy in ER-positive BC (Crowder et al., 2009). In the BELLE-2 trial, AI resistant patients with MBC received F-HD with or without the PI3K inhibitor buparlisib with the combination modestly prolonging PFS (Baselga et al., 2015). Similarly, in the BELLE-3 trial which enrolled heavily pretreated patients (35% had received chemotherapy for advanced disease, 69% had received two or more endocrine therapies, all were resistant to mTOR inhibitors), the combination of buparlisib and fulvestrant significantly but modestly prolonged PFS compared to fulvestrant alone (3.9 versus 1.8 months, HR=0.67, P<0.001), a difference which became apparent only in patients with visceral disease (HR=0.56, 95% CI 0.43 – 0.74) but not in those with non-

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visceral metastatic sites (HR=0.96, 95% CI 0.61 – 1.50) (Leo et al., 2016). In contrast, pictilisib was not found to be effective in the similarly designed FERGI trial, possibly because of the significant toxicity and subsequent dose reductions (Krop et al., 2016). A matter of concern regarding these first generation, non-selective PI3K inhibitors are the significant adverse events associated with their use. Serious (grade 3 or higher) toxicity was reported in 77.3% receiving buparlisib and 61% of those receiving pictilisib in the BELLE-2 and FERGI (part 1) trials, respectively. The most commonly observed adverse events are elevated liver function enzymes, rash and hyperglycemia. Psychiatric disorders such as anxiety and depression have also been noted in patients receiving buparlisib. Taken together, these data demonstrate the modest efficacy of PI3K/AKT/mTOR inhibition. No trial of PI3K/mTOR inhibition has demonstrated an OS advantage associated with these agents, presumably due to the lack of statistical power, to the availability of multiple effective post-trial therapeutic agents, to imbalances in post-trial treatments (Piccart et al., 2014), or to the paradoxical activation of AKT because of a negative feedback loop which may induce resistance to subsequent treatments (Wan et al., 2007). The development and ongoing evaluation of selective PI3K inhibitors, such as alpelisib, which inhibit the α-subunit of the PI3K domain may shed some light on whether more selective inhibition could lead to improved outcomes with reduced toxicity. 3.3 Exploring putative biomarkers Although several lines of evidence indicate that the inhibition of the PI3K pathway benefits endocrine resistant patients, better patient selection is needed. Case reports describe exceptional responses to mTOR inhibitors in various neoplasms that were attributed to the presence of specific molecular aberrations, such as TSC1 and TSC2 (tuberous sclerosis 1 and 2) mutations or PTEN

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loss (Iyer et al., 2012; Marsh et al., 2008; Wagle et al., 2014). With these few exceptions, the available literature is fraught with contradicting reports. Activation of the PI3K pathway does not seem to predict benefit from these agents, as seen in data from the TAMRAD study, where patients with increased levels of late effectors of mTOR such as phosphorylated 4E-binding protein 1 (p4EBP1) but low phosphorylated AKT, indicating noncanonical mTOR activation, benefited the most from the addition of everolimus (Treilleux et al., 2015). Moreover, in the BOLERO-2 trial genotyping using next generation sequencing (NGS) in archival specimens from 227 patients revealed that the presence of all wild type or at most one mutated gene among the PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha), CCND1 (cyclin D1) and FGFR1/2 (fibroblast growth factor receptor 1/2) genes predicted a greater treatment effect by everolimus (HR=0.27) (Hortobagyi et al., 2013). Recently, patients enrolled in BOLERO-2 harboring ESR1 mutations, as assessed by plasma circulating tumor DNA (ctDNA), were shown to have worse PFS compared to wild type ones when treated with the combination, although the addition of everolimus did improve outcomes in mutated patients compared to exemestane alone (Chandarlapaty et al., 2016). Confusingly, contradictory data were reported from the FERGI and BELLE-2 trials. In the former, the presence of PIK3CA mutations did not predict the efficacy of pictilisib (HR=1.07) (Krop et al., 2016). In the latter, patients with activated PI3K pathways at archival samples (defined as the presence of PIK3CA mutations or PTEN loss) benefited from the addition of buparlisib (PFS 6.8 versus 4.0 months, HR=0.76, P=0.014). Intriguingly, the detection of PIK3CA mutations in ctDNA was associated with a PFS prolongation (7.0 versus 3.2, P<0.001) while wild type patients did not benefit at all (Baselga et al., 2015), underscoring the feasibility of liquid biopsies in this setting. Importantly, these results were corroborated by the findings from the aforementioned BELLE-3 trial in an

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everolimus-resistant population. The presence of PIK3CA mutations in archival tissue and/or in newly obtained ctDNA predicted benefit from buparlisib and fulvestrant (HR=0.39, P<0.001 and HR=0.46, P<0.001 for PIK3CA mutated patients in tissue and ctDNA based analysis, respectively). The superiority of the combination compared to fulvestrant monotherapy was marginal in patients with wild type ctDNA (HR=0.73, P=0.026) while no benefit was observed in the wild type tumor by tissue testing subgroup (HR=0.83, P=0.117). These results indicate that a new option, PI3K inhibitors, could become useful for a subset of patients (those with visceral disease and PIK3CA mutations) experiencing disease progression under everolimus, a population currently lacking any approved or prospectively evaluated non-cytotoxic therapies. 4. CDK4/6 inhibitors 4.1 Rationale The dysregulation of cell-cycle control and the subsequent uninhibited proliferation is a hallmark of cancer (Hanahan and Weinberg, 2011). Central in the complex network of cell-cycle regulators are cyclin dependent kinases (CDKs). Specifically, CDK4 and CDK6 control the transition from the G1 to the S phase of the cell cycle, although this over-simplified view has been challenged because of the overlapping actions of the CDKs (O'Leary et al., 2016). Aberrations of CDK4/6 function and cell-cycle control are commonly observed in luminal BC and include CDK4 and CDK6 amplification, p16INK4A loss, CCND1 amplification, Cyclin D overexpression, epigenetic alterations and microRNA silencing (Musgrove et al., 2011). In addition, an interplay between ER signaling and CDKs has been described, manifested through the ER-regulated activation of the CCND1 promoter and, conversely, through the facilitation of ER transcriptional activity by Cyclin D1 (Musgrove et al., 2011). Importantly, CDK4/6 inhibition has been shown to reverse endocrine resistance in ER-positive MBC (Finn et al., 2009; Wardell et al., 2015). These observations have led to the clinical development of CDK4/6 inhibitors. 10

Although several biomarkers have been postulated to predict resistance to CDK4/6 inhibitors, such as Rb (Retinoblastoma protein) loss, CCNE1 (Cyclin E1) amplification and E2F amplification, none has been clinically validated (O'Leary et al., 2016). These alterations bypass CDK4/6 dependency and activate alternative S phase entry (Herrera-Abreu et al., 2016). Intriguingly, these mechanisms of resistance could be prevented, but not reversed, by the simultaneous blockade of the PI3K pathway, a notion that is explored later in this review (Herrera-Abreu et al., 2016; Miller et al., 2011). 4.2 Palbociclib The best studied CDK4/6 inhibitor is palbociclib. Based on its encouraging preliminary activity (Hamilton and Infante, 2016), palbociclib was approved following the results of the phase II PALOMA-1/TRIO-18 study, where its addition to first line letrozole roughly doubled PFS but did not prolong OS. In addition, CCND1 amplification and p16 loss were not predictive of outcomes (Finn et al., 2015). Corroborating these findings are the results of the confirmatory phase III PALOMA-2 trial which was recently published in full form. Again, the combination significantly improved PFS (24.8 versus 14.5 months, HR=0.58, P<0.001) in all clinical and demographic subgroups; the final OS analysis is pending (Finn, R.S. et al., 2016). An attempt to identify putative predictive biomarkers by the use of immunohistochemical stains for ER, Ki67, Rb, Cyclin D1 and p16 was not successful, since the addition of palbociclib was found to improve PFS regardless of patient subgroup (Finn, R. et al., 2016). In AI resistant patients, adding palbociclib to second line F-HD was found to be effective in the PALOMA-3 trial (PFS 9.5 versus 4.6 months, P<0.0001). Importantly, 21% of enrolled patients were premenopausal so this trial offers evidence for the use of the combination in this population. While the benefit from the combination was consistent in all patient subgroups, the detection of

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PIK3CA mutations in the plasma was not found to have predictive value (Cristofanilli et al., 2016) and neither did the presence of ESR1 mutations (Fribbens et al., 2016). These results highlight the significant activity of palbociclib in both endocrine naïve and pretreated patients. Its use is associated with distinct but manageable toxicity: in the PALOMA-3 trial, grade 3/4 neutropenia occurred in 65% of treated patients and in 34% there were dose reductions. However, due to its cytostatic rather than cytotoxic effect on marrow progenitors and lack of mucosal damage, febrile neutropenia was rare (1%). Other adverse events of clinical interest were thrombocytopenia, fatigue, alopecia and arthralgia, while only 4% discontinued treatment due to adverse events (versus 2% in the control group) (Cristofanilli et al., 2016). 4.3 Abemaciclib and Ribociclib Abemaciclib and ribociclib are CDK4/6 inhibitors under evaluation in ongoing trials (table 2). The former was administered as monotherapy in the MONARCH phase II trial in heavily pretreated patients (median 3 lines, 90% had visceral disease). The response rate was 19.7% and median PFS and OS were 6 and 17.7 months, respectively (Dickler, M. et al., 2016). These results underscore the activity of CDK4/6 inhibitor monotherapy and raise the question whether a sequential endocrine and CDK4/6 inhibition approach would be comparable to their co-administration. Interestingly, the dosing schedule (continuous with abemaciclib versus intermittent with palbociclib and ribociclib) and safety profile (gastrointestinal versus hematopoietic adverse events respectively) seem to be unique for this agent. The clinical implications of this observation, if any, are as yet unclear (Patnaik et al., 2016). Ribociclib is also being evaluated in several trials; among them is the MONALEESA-7 trial which only enrolls premenopausal patients. Recently, the results of the MONALEESA-2 trial were reported: the addition of ribociclib to first line letrozole extended PFS rates at 18 months from 12

42.2% to 63.0% (HR=0.56; P=3.29×10-6), a result that is similar to the one reported in the PALOMA-2 trial, while the adverse event profile was within what is expected from CDK4/6 inhibitors (Hortobagyi et al., 2016), therefore representing another option for this patient population. In summary, CDK4/6 inhibitors are an effective addition to the therapeutic armamentarium. Translational studies from the PALOMA-2 and PALOMA-3 studies and the results of the multiple ongoing trials are eagerly awaited, in hopes of determining which patient subgroup benefits the most from these agents. 5. Other combinations of targeted agents and endocrine therapy 5.1 Bevacizumab Bevacizumab is a monoclonal antibody (MoAb) that targets the vascular endothelial growth factor A (VEGF-A). Based on the premise that the efficacy of endocrine therapy depends on VEGF levels (Manders et al., 2003), endocrine-bevacizumab combinations were evaluated in two phase III trials, with only the recent CALGB 40503 study reporting a significant prolongation in PFS (20.2 versus 15.6 months, P=0.016) but no benefit in OS (47.2 versus 43.9 months, P=0.188) (Dickler, M.N. et al., 2016; Martin et al., 2015). These results once again challenge the validity of PFS as a surrogate marker for OS, at least in the first line setting. 5.2 Dasatinib Proto-oncogene tyrosine-protein kinase Src (Src) has been implicated as a promoter of acquired resistance in ER-driven BC and of the development of bone metastases (Vallabhaneni et al., 2011; Zhang et al., 2009). Dasatinib, an oral multikinase inhibitor that targets Src, has been evaluated in MBC in several small trials. Although inactive as monotherapy (Schott et al., 2016), the combination with letrozole was found to double PFS in AI naïve patients (Paul et al., 2013). However, in AI resistant patients the addition of dasatinib to either fulvestrant or exemestane did 13

not improve outcomes (Llombart et al., 2011; Wright et al., 2011). Predictive biomarkers are needed before the use of dasatinib is further explored for this population. 5.3 Ganitumab Enhanced signaling through the IGF-1R cascade is thought to mediate resistance to endocrine treatment (Pollak, 2008). Thus, its inhibition with the MoAb ganitumab was evaluated in a phase II trial of endocrine resistant patients, in combination with either fulvestrant or exemestane. PFS was not significantly improved while, unexpectedly, OS favored the monotherapy group (Robertson et al., 2013). The reasons that explain this deleterious effect are unclear and further development of this agent for this indication has been abandoned. 5.4 Gefitinib and other EGFR inhibitors Gefitinib is an oral inhibitor of the epidermal growth factor receptor (EGFR) which has been approved for use in EGFR mutated lung cancer. Since resistance to endocrine therapy has been shown to be mediated by the EGFR / human epidermal growth factor receptor 2 (HER2) axis (Arpino et al., 2004) and gefitinib has been shown in vitro to reverse resistance to tamoxifen (Zhang et al., 2015), it was combined with either tamoxifen (Osborne et al., 2011) or anastrozole (Cristofanilli et al., 2010) in relatively endocrine sensitive patients, with the latter study reporting a significant prolongation of PFS. Two other agents that target the same axis have been evaluated for this indication. Despite encouraging preclinical data (Morrison et al., 2014), the results of early clinical trials regarding the pan-EGFR inhibitor AZD8931 (Johnston et al., 2014) and the antiHER3 antibody MM-121 (Higgins et al., 2013) do not suggest any activity in endocrine sensitive patients. In summary, activation of EGFR signaling seems to be common during the development of endocrine resistance, but not critical for the survival of the cancer cell. Thus, further exploration of the role of EGFR inhibitors is not warranted.

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5.5 Entinostat Epigenetic alterations such as methylation and histone acetylation are commonly described in hormone refractory BC and entinostat, a histone deacetylase (HDAC) inhibitor, has been shown to restore hormone sensitivity (Sabnis et al., 2011). In the phase II ENCORE 301 study, the addition of entinostat to exemestane in endocrine resistant patients marginally improved PFS (HR=0.73, P=0.055), especially in those with primary resistance to AI (HR=0.47). Interestingly, the exploratory survival analysis revealed a 9-month prolongation of OS (P=0.036) (Yardley et al., 2013a). These encouraging results are awaiting confirmation from an ongoing phase III trial. 5.6 AS1402 MUC-1 is a modulator of ER activity and it was hypothesized that targeting it with the MoAb AS1402 would enhance letrozole efficacy. Unfortunately, a phase II trial of the combination was discontinued early due to lack of efficacy (Ibrahim et al., 2011).

6. Overcoming current challenges When administered to patients with ER-positive MBC, combinations of mTOR or CDK4/6 inhibitors with endocrine treatment have been consistently shown to improve PFS, at least in postmenopausal patients. In contrast, multiple other agents have been tested and failed to improve outcomes (table 3). Taking into account the diversity of the molecular mechanisms at play, this is hardly a paradigm of precision medicine: a one size fits all approach, not dissimilar to cytotoxic chemotherapy and far from its successful implementation in HER2-positive BC or other neoplasms. Important questions remain unanswered. The lack of an OS benefit could be attributed to the disease itself which is amenable to multiple interventions, leading to the need for unrealistically large samples in clinical trials in order to detect an OS advantage (Broglio and Berry, 2009). Therefore, although PFS has only been shown to be an adequate surrogate for OS at 15

the second line and beyond for MBC (Adunlin et al., 2015), it is commonly used as the primary endpoint of trials in ER-positive disease. On the other hand, the lack of a survival benefit could be a result of ineffective suppression of resistance, either due to the use of non-specific agents or because non-critical pathways are targeted, providing tumor cells with escape routes as demonstrated by the multiple failed trials of once promising agents. The latter hypothesis merits particular mention. Targeting a pathway that is critical for the survival and proliferation of breast cancer cells such as HER2 has been associated with consistent improvements in OS, both in the first (Slamon et al., 2001; Swain et al., 2015) and subsequent line settings (Krop et al., 2014; Verma et al., 2012). Since trials reporting on the efficacy of CDK4/6 inhibitors have failed to demonstrate a prolongation of OS, longer follow-up will hopefully illustrate whether this is caused by the inherent insignificance of the target, masked by the currently short follow-up or by a rebound effect in proliferation after the discontinuation of CDK4/6 inhibition which could lead to shortened postprogression survival. Currently, it is not known which combinations should be administered and in which sequence. Since CDK4/6 and mTOR inhibitors cause clinically relevant toxicity and their use is associated with substantial costs but not all patients benefit, refining management algorithms by selecting appropriate candidates for each agent with the help of predictive biomarkers could lead to a more compartmentalized and efficient approach. In the absence of such biomarkers, horizontal blockade of multiple compensatory signaling pathways represents a promising avenue. Preclinical data demonstrate that inhibition of CDK4 can overcome resistance to PI3K inhibitors (Vora et al., 2014) and that the triple combination with AI is feasible (O'Brien et al., 2014), forming the basis for several ongoing phase I trials. The lack of clinical data regarding these combinations dictates the consecutive use of targeted agents, which has important implications: the efficacy of CDK4/6

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inhibition after exposure to PI3K/mTOR inhibitors is currently unknown, and vice versa. Taking into account the significant activity of first line palbociclib or ribociclib combined with letrozole, CDK4/6 inhibitors are likely to become the first line standard of care. How well everolimus or novel PI3K inhibitors perform at the post-CDK4/6 inhibitor setting is unknown, with no clinical data available yet. Moreover, whether the demonstration of PFS prolongation in AI-resistant patients for novel PI3K/endocrine combinations will be enough for them to receive approval by regulatory authorities remains to be seen. Confusingly, the recently reported positive results from the BELLE-3 trial offer yet another pathway in the evidence-based management algorithm, since patients progressing on AI monotherapy and everolimus/exemestane combination have now yet another possible treatment option based on a randomized trial, fulvestrant and buparlisib. Since the conduct of trials directly comparing CDK4/6 and PI3K/mTOR inhibitors seems unlikely, retrospective data derived from the real-world usage of targeted agents may offer some insight regarding their relative efficacy. In addition, although performing cross-trial comparisons is inherently hazardous, the safety profile of PI3K/mTOR and CDK4/6 inhibitors as reported in randomized trials is different, with the latter being better tolerated and leading to fewer treatment discontinuations due to toxicity. Taking into account the lack of OS benefit from all available agents and the lack of head-to-head comparisons, such an observation could affect treatment selection. Another important disadvantage of the previously mentioned trials is that the clonal evolution of the disease is not taken into account since in most trials patient enrollment was based on archival tissue. Discordance between the primary tumor and metastatic sites regarding ER status has been consistently illustrated (Amir et al., 2012), in addition to discrepancies in other relevant molecular aberrations such as PIK3CA and PTEN status (Gonzalez-Angulo et al., 2011). The merits of

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metastatic biopsies were prospectively evaluated in the SAFIR-01 trial, where they were shown to be both feasible and clinically relevant, leading to the detection of targetable alterations in 46% of patients while only 1% experienced grade 3 or worse adverse events (Andre et al., 2014). In addition, the BELLE-2 and BELLE-3 trials provided evidence that ctDNA based testing could lead to patient selection for treatment with PI3K inhibitors (Baselga et al., 2015; Leo et al., 2016). Since liquid biopsy facilitates serial testing, it accurately captures the molecular evolution of the underlying malignancy throughout the disease trajectory and could select appropriate patients for targeted agents. Biomarker based trials such as the MATCH-R trial where the genomic and proteomic landscape of resistance and disease progression of various oncogene driven tumors is being prospectively evaluated may be the key to addressing these currently unresolved issues. Trials based on upfront patient stratification according to genomic alterations or on treatment personalization are two complementary approaches (Arnedos et al., 2015). The comprehensive characterization of patients progressing under endocrine treatment and subsequent enrollment in such trials represents a rational, genuinely personalized and targeted approach and a major step forward from contemporary practice. 7. Conclusions Deciphering the complexity of endocrine resistance has led to the evaluation of an abundance of targeted agents that could potentially overcome it and to the approval of two novel drug classes with clinically meaningful efficacy, mTOR and CDK4/6 inhibitors. While this represented an important milestone, several matters remain unresolved. No biomarkers have been identified, the comparative efficacy of available combinations is unknown and whether it is best to reverse established resistance or attempt to suppress its emergence is as yet unclear. As a result, although

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these agents are by definition targeted, their use exhibits all the hallmarks of imprecision medicine. Importantly, the increasing costs of novel agents represent a considerable strain on healthcare systems since the financial toxicity associated with the use of these agents cannot be ignored (Diaby et al., 2014; Matter-Walstra et al., 2016). Hopefully, the widespread screening of patients for targetable alterations with high throughput techniques such as NGS and whole exome sequencing (Van Allen et al., 2014) and their enrollment in rationally designed clinical trials will offer the opportunity to truly personalize the management of ER-positive MBC. Acknowledgement: Dr Matikas is supported by fellowships from the European and Hellenic Societies of Medical Oncology Conflict-of-interest statement: The authors report no relevant financial conflicts of interest

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Table 1: Randomized trials of combinations in the treatment of hormone receptor positive metastatic breast cancer Reference

N

Patient Characteristics

Compari son

PFS (months)

OS (months)

Combinations of endocrine agents Mehta et al

694

Bergh et al

514

1st line, postmenopausal, 50% visceral mets, prior adjuvant tamoxifen 40% 1st line, 52% aged 18-65, 50% visceral mets, prior adjuvant endocrine 68% 2nd line after AI, 19% AI for MBC, 59% visceral mets, HER2: 7% (+), 37% unknown

AN ± F- 15.0 vs 13.5 47.7 vs 41.3 LD (P=0.007) (P=0.05)

2nd line after AI, postmenopausal, 56% visceral mets, 54% 3+ endocrine prior therapies 2nd line after AI, postmenopausal, 66% AI for advanced disease, 53% visceral mets 2nd line after AI, postmenopausal

EXE EVE

± 7.8 vs 3.2 31.0 vs 26.6 (P<0.0001) (P=0.1426)

TAM EVE

± 8.6 vs 4.5 (P=0.002) (TTP) ± 10.4 vs 5.1 (P=0.02) ± 8.9 vs 9.0 (P=0.25)

AN ± F- 10.8 vs 10.2 LD (P=0.91) (TTP) Johnson et al 723 AN + F- 4.4 vs 4.8 LD vs F- vs 3.4 (NS) LD vs EXE PI3K or mTOR inhibitors in combination with endocrine therapy Baselga et al, 724 Yardley et al, Piccart et al Bachelot et al 111

Kornblum et al

F-HD EVE st Wolff et al 110 1 line, 40% prior non-AI adjuvant, LET 6 postmenopausal, HER2: 20% (+), TEM 36% unknown Royce et al 202 1st line, 54% endocrine naïve EVE/LET → EVE/EX E Baselga et al 114 2nd line after AI, postmenopausal, F-HD ± 7 61% visceral mets BUP Leo et al Disease progression on F-HD ± 432 everolimus/AI, 73% visceral mets, BUP 35% prior chemotherapy, 69% 2+ endocrine prior therapies Krop et al 168 2nd line after AI, postmenopausal, F-HD ± 55% visceral mets, 24% 3+ prior PIC endocrine therapies CDK4/6 inhibitors in combination with endocrine therapy Finn et al

131

165

1st line, postmenopausal, 46% LET visceral mets, 49% de novo PAL metastatic

28

37.8 vs 38.2 (P=1.00) 20.2 vs 19.4 vs 21.6 (NS)

Not reached vs 32.9 (P=0.007) NR NR

Not reached NR (12-month PFS 71.4%) 6.9 vs 5.0 NR (P<0.001) 3.9 vs 1.8 NR (P<0.001)

6.6 vs 5.1 NR (P=0.096)

± 20.2 vs 10.2 37.5 vs 33.3 (P=0.0004) (P=0.42)

Finn et al

666

Cristofanilli et al

521

Hortobagyi et al

668

1st line, postmenopausal

LET PAL nd 2 line after AI, 21% F-HD premenopausal, 59% visceral mets, PAL 14% 3+ prior endocrine therapies 1st line, 48.2% endocrine naïve, LET 59% visceral mets RIB

± 24.8 vs 14.5 NR (P<0.001) ± 9.5 vs 4.6 NR (P<0.0001) ± Not reached NR vs 14.7 (P=3.29X1 0-6)

Other targeted agents in combination with endocrine therapy Martin et al

374

Dickler et al

343

Paul et al

120

Wright et al

99

Llombart et al

157

Robertson et al

156

Osborne et al

289

Cristofanilli et al

93

Johnston et al

359

Higgins et al

115

Yeardley et al

130

Ibrahim et al

109

1st line, 52% prior adjuvant LET or Fendocrine, 48% visceral mets LD ± BEV 1st line, 48% prior adjuvant LET ± endocrine, 25% bone only disease BEV 1st line AI (9% and 5% had prior 1st LET ± line TAM and 11% and 6% prior 1st DAS line chemotherapy in each arm) 2nd line after AI F-HD ± DAS nd 2 line after AI EXE ± DAS

19.3 vs 14.4 52.1 vs 51.8 (P=0.126) (P=0.518)

2nd line after endocrine (54% as metastatic) postmenopausal, HER2: 5% (+) 1st line, 35% visceral mets, HER2: 15% (+) 1st line, postmenopausal, no prior AI, 1st line, postmenopausal, 23% locally advanced disease

23.3 vs non estimable (P=0.025) 10.9 vs 8,8 NR (P=0.314) 14.7 vs 8.4 NR (HR=0.55) 10.9 vs 1.8 NR vs 14.0 (NS)

6.0 vs 5.3 (NS) 18 vs 16 weeks (P=0.148) or 3.9 vs 5.7 ± (P=0.44)

F-LD EXE GAN TAM ± GEF ANA ± GEF ANA ± AZD8931 20 or 40 mg 2nd line after endocrine, 25% bone EXE ± only disease MM-121 2nd line after AI, postmenopausal, 60% visceral mets, 19% 2+ prior endocrine therapies 1st line, postmenopausal, 74% visceral mets

20.2 vs 15.6 47.2 vs 43.9 (P=0.016) (P=0.188) 22 vs 11 NR (P=0.05)

EXE ENT

17.0 vs 21.7 (NS) NR

15.9 vs 10.9 NR weeks (P=0.249) ± 4.3 vs 2.3 28.1 vs 19.8 (P=0.055) (P=0.036)

LET ± HR=0.947 AS1402

NR

PFS: progression free survival; OS: overall survival; TTP: time to progression; NS: non-significant; NR: not reported; HR: hazard ratio PI3K: Phosphoinositide 3-kinase; mTOR: mechanistic target of rapamycin; CDK: cyclin dependent kinase; AI: aromatase inhibitor; mets: metastases; AN: anastrozole; LET: letrozole; EXE: exemestane; F-LD: fulvestrant 250 mg; F-HD: fulvestrant 500 mg; TAM: tamoxifen; EVE: everolimus; TEM: temsirolimus; BUP: buparlisib; PAL: palbociclib; RIB: ribociclib; BEV: bevacizumab; DAS: dasatinib; GAN: ganitumab; GEF: gefiitnib; ENT: entinostat

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Table 2: Selected ongoing trials of combinations in ER-positive HER2-negative advanced breast cancer Agent

Pha se

Clinical Setting

Clinicaltrials.gov Identifier

PI3K/mTOR inhibitors Everolimu s Everolimu s Everolimu s Everolimu s Everolimu s Everolimu s Buparlisib

2 3

EVE + endocrine therapy beyond progression on NCT02269670 EVE/EXE Maintenance AI ± EVE after 1st line chemotherapy NCT02511639

2

GnRH/LET ± EVE after progression on TAM

3

2

EVE/EXE→FUL vs FUL→EVE/EXE after progression NCT02404051 on AI GnRH/LET ± EVE after progression on TAM, NCT02313051 premenopausal patients EVE/EXE vs EVE vs capecitabine, 2nd line after NCT01783444 progression on AI BUP/TAM after prior endocrine exposure NCT02404844

Alpelisib

3

FUL ± ALP after progression on AI

NCT02437318

Taselisib

3

FUL ± TAS after progression on AI

NCT02077933

AZD2014

2

EVE/FUL vs AZD2014/FUL vs FUL after progression on NCT02216786 AI

2 2

NCT02344550

CDK4/6 inhibitors Palbociclib

3

PAL/FUL or PAL/EXE vs capecitabine, 1st line

NCT02028507

Palbociclib

3

LET ± PAL 1st line Asian population

NCT02297438

Palbociclib

2

FUL ± PAL 1st line endocrine sensitive disease

NCT02690480

Palbociclib

2

PAL/FUL after progression on PAL/AI, single arm

NCT02738866

Palbociclib

2

PAL/FUL 1st line, 70+ years old

NCT02760030

Palbociclib

2

PAL/LET vs PAL/FUL 1st line

NCT02491983

Palbociclib

2

FUL ± PAL after progression on AI in molecularly NCT02536742 characterized patients

30

Palbociclib

2

PAL/TAM 1st line single arm, premenopausal are eligible

NCT02668666

Palbociclib

2

PAL/FUL after progression on PAL/AI

NCT02738866

Ribociclib

3

FUL ± RIB 1st or 2nd line

NCT02422615

Ribociclib

3

Ribociclib

2

GnRH/AI or GnRH/TAM ± RIB 1st line, exclusively NCT02278120 premenopausal FUL ± RIB after progression on CDK4/6i/AI NCT02632045

Abemacicl 3 FUL ± ABE after progression on AI ib Abemacicl 3 AI ± ABE 1st line ib Abemacicl 3 AI ± ABE vs FUL ± ABE, 1st line ib Abemacicl 2 TAM ± ABE, pretreated patients ib Combinations of PI3K/mTOR and CDK4/6 inhibitors

NCT02107703

Everolimu 1/2 s, ribocilib Buparlisib, 1/2 ribocilib, BYL719 Ribociclib, 1/2 BYL719 Palbociclib 1/2 , AZD2014 Other agents

EVE/RIB/EXE after progression on RIB

NCT02732119

RIB/FUL vs RIB/FUL/BUP vs RIB/BYL719

NCT02088684

RIB/BYL719/LET vs RIB/LET vs BYL719/LET

NCT01872260

PAL/FUL ± AZD2014

NCT02599714

Vandetani b Bortezomi b Erlotinib

2

FUL ± VAN after progression on AI

NCT02530411

2

FUL ± BOR after progression on AI

NCT01142401

2

FUL ± ERL after progression on AI

NCT00570258

Cabozantin 2 ib Entinostat 3

NCT02246621 NCT02763566 NCT02747004

FUL ± CAB after progression on 1st line, bone metastases NCT01441947 required EXE ± ENT after progression on AI NCT02115282

EVE: everolimus; EXE: exemestane; AI: aromatase inhibitor; LET: letrozole; GnRH: gonadotropin releasing hormone agonist; TAM: tamoxifen; FUL: fulvestrant; BUP: buparlisib; ALP: alpelisib; TEL: telalisib; PAL: palbociclib; RIB: ribociclib; ABE: abemaciclib; CDK4/6i: CDK4/6 inhibitor; VAN: vandetanib; BOR: bortezomib; ERL: erlotinib; CAB: cabozantinib; ENT: entinostat

31

Table 3. Possible explanations of the failure of targeted agent – endocrine combinations to improve overall survival in ER-positive, HER2-negative breast cancer Long natural history results in need for unrealistically large trials, powered adequately to demonstrate benefit Currently available agents may target non-critical pathways Inappropriate patient selection in trials based on archival primary disease tissue Significant toxicities lead to decreased doses and inadequate pathway blockade No known biomarkers that could select appropriate candidates Use of non-specific agents that inadequately inhibit critical pathways Post-progression disease may be more aggressive and resistant to subsequent treatments Imbalances in post-trial treatments

32