Enhancing Endocrine Therapy for Hormone Receptor Positive Advanced Breast Cancer: Cotargeting Signaling Pathways

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1 JNCI J Natl Cancer Inst (2015) 107(10): djv212 doi: /jnci/djv212 First published online August 6, 2015 Review Enhancing Endocrine Therapy for Hormone Receptor Positive Advanced Breast Cancer: Cotargeting Signaling Pathways Stephen R. D. Johnston Affiliation of author: Department of Medicine, Royal Marsden NHS Foundation Trust, Fulham Road, Chelsea, London, UK. Correspondence to: Stephen R. D. Johnston, MA, FRCP, PhD, Professor of Breast Cancer Medicine, Department of Medicine, Royal Marsden NHS Foundation Trust, Fulham Road, Chelsea, London SW3 6JJ, UK ( stephen.johnston@rmh.nhs.uk). Abstract Overcoming primary or secondary endocrine resistance in breast cancer remains critical to further enhancing the benefit of existing therapies such as tamoxifen or an aromatase inhibitor (AI). Much progress has been made in understanding the molecular biology associated with secondary endocrine resistance. Cotargeting the estrogen receptor, together with various key intracellular proliferation and cell survival signaling pathways, has been explored as a strategy either to treat endocrine resistance once it develops in the second-line setting or to enhance first-line endocrine responsiveness by preventing secondary resistance from developing via blockade of specific pathways from the outset. While attempts to improve endocrine therapy by adding growth factor inhibitors have been disappointing, success resulting in new drug approvals has been seen in secondary endocrine resistance by treating patients with the mtor antagonist everolimus in combination with the AI exemestane and, more recently, in the first-line setting, by the addition of the CDK 4/6 inhibitor palbociclib to the AI letrozole. Numerous other therapeutics are being evaluated in combination with endocrine therapies based on supportive preclinical evidence, including inhibitors of PI3K, Akt, HDAC, Src, IGFR-1, and FGFR. Appropriate clinical trial design and patient selection based on prior therapy exposure, together with predictive biomarkers derived through real-time molecular profiling, are needed to enrich future trials and maximize any additional benefit that cotargeting may bring to current endocrine therapies for estrogen receptor positive breast cancer. Modern endocrine therapies are very effective as treatment for estrogen receptor positive (ER+) breast cancer. For patients with early-stage disease, adjuvant endocrine therapy given for five years after primary surgery substantially delays local and distant relapse and prolongs overall survival (OS) (1). Endocrine responsiveness, for the most part, is dependent on the presence of a functional estrogen receptor (ER), a protein that is detected in approximately 70% to 80% of primary breast cancers and regulates endocrine-dependent growth in these tumors. Hormonal manipulation is achieved either at a cellular level by using anti-estrogens, such as tamoxifen, to compete for ER in the breast tumor or systemically by lowering estrogen levels in premenopausal women with the use of luteinizing hormonereleasing hormone (LHRH) agonists and in postmenopausal women by aromatase inhibitors (AIs) that block estrogen biosynthesis in nonovarian tissues. All of these approaches have been extensively investigated within the context of large-scale international trials of adjuvant endocrine therapy over the last two decades. Likewise, endocrine therapy can be a very effective strategy for patients who either initially present with ER+ locally advanced/metastatic breast cancer (MBC) or relapse during or following completion of prior adjuvant endocrine therapy. However, not all patients with ER+ MBC respond to first-line endocrine treatment because of primary (or de novo ) resistance, while many others may initially respond but eventually progress with secondary (or acquired ) resistance (2). There has been substantial progress made in understanding the various Received: September 26, 2014; Revised: February 20, 2015; Accepted: July 8, 2015 The Author Published by Oxford University Press. All rights reserved. For Permissions, please journals.permissions@oup.com. 1 of 10

2 S. R. D. Johnston 2 of 10 biological mechanisms responsible for the development of endocrine resistance, which in recent years has led to new clinical therapeutic strategies aimed at enhancing the efficacy of current endocrine therapies either by reversing or by preventing endocrine resistance (3,4). Unfortunately, not all approaches that looked promising in experimental models have proved successful in the clinic. This article s the progress that has been made with various clinical strategies and examines some of the trial design issues and the efforts to identify predictive biomarkers that have made this area of clinical research challenging. Fundamental to all of these approaches has been an understanding of the molecular signaling pathways that account for endocrine resistance, together with preclinical evidence that manipulating specific signaling pathways using various targeted therapeutics can enhance or restore endocrine sensitivity in ER+ breast cancer. Potential Treatment or Reversal of Endocrine Resistance Historically, international guidelines state that at the time of progression on endocrine treatment alternative endocrine therapies are still considered appropriate for patients with ER+ MBC, in particular for those patients with low disease burden and evidence of a prior good response to first-line endocrine treatment (5,6). In postmenopausal ER+ MBC, there has been a clinical expectation that prior responsiveness to endocrine therapy in either the adjuvant or first-line metastatic setting is a strong predictor for further clinical benefit either from a non cross resistant steroidal aromatase inactivator, such as exemestane, or the selective estrogen receptor downregulator (SERD) fulvestrant. Recent trials (EFECT, SoFEA) in this setting have shown that both strategies are equally effective, although objective tumor response rates are low (<10%) and median time to progression (TTP) is relatively short, at three to four months only (7,8). As such, there is room for marked improvement in this setting, especially if the fundamental mechanism for acquired endocrine resistance can be identified and targeted within these tumors. One of the key signaling pathways in ER+ breast cancer is the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mtor) pathway. There is good evidence that this pathway becomes activated during secondary endocrine resistance in ER+ breast cancer, accounting for cell survival despite the presence of continued endocrine blockade (9). Preclinical models of both ER+ hormone-sensitive and resistant breast cancer have examined the effects of combining mtor antagonists with endocrine therapy, with clear evidence for additive/synergistic effects (10,11). These laboratory data provided a strong rationale for combining endocrine therapy with mtor inhibition as a means of treating endocrine resistance. Two landmark clinical trials have investigated the addition of the mtor antagonist everolimus to endocrine therapy for postmenopausal women with ER+ MBC who have already received prior endocrine therapy. In both studies, patients had often developed endocrine resistance following initial response to endocrine therapy, and as such might be expected to have acquired either cell survival or adaptive signaling pathways such as PI3K/Akt/mTOR that might be susceptible to an appropriate therapeutic agent. Tamoxifen plus everolimus was investigated in patients with AI-resistant MBC in the phase TAMRAD study and showed that the combination therapy provided an improvement in TTP (8.6 vs 4.5 months), six-month clinical benefit rate (61% vs 42%), and median OS (not reached vs 33 months) compared with tamoxifen alone (Table 1 and Table 2) (12). Importantly, the trial design included stratification according to type of resistance to previous treatment with AIs and showed that the greatest clinical benefit for the combination arm occurred in patients with secondary resistance as opposed to those with primary resistance. These clinical data support a hypothesis that tumors that initially respond to AIs but then develop secondary resistance utilize the PI3K/Akt/mTOR cell survival pathway and that a combined mtor antagonist/endocrine therapy approach is an effective strategy for those patients with ER+ advanced disease that progresses during or after nonsteroidal AI therapy (26). This was subsequently confirmed in the Breast Cancer Trials of Everolimus-2 (BOLERO-2) study, a large randomized phase trial that assigned 724 postmenopausal patients with ER+ MBC in a 2:1 ratio to either exemestane alone or the combination of exemestane and everolimus (15,27). All patients had progressed on a nonsteroidal AI, and importantly 84% had demonstrated prior hormone-sensitive disease defined as at least 24 months of endocrine therapy before recurrence in the adjuvant setting, or a response or stabilization for at least 24 weeks of endocrine therapy for advanced disease (27). In BOLERO-2 there was a statistically significant and clinically relevant improvement in progression-free survival (PFS) for the combination (median 7.8 vs 3.2 months, hazard ratio [HR] = 0.45, log-rank P <.0001) (15). The clinical benefit was primarily because of better disease control, although there was a statistically significant improvement in tumor response rates from only 0.4% in the exemestanealone group to 9.5% in the everolimus plus exemestane group (P <.001) (27). Everolimus is associated with an increase in various toxicities such as stomatitis, fatigue, rash, and a low incidence of pneumonitis. A recent update of BOLERO-2 reported a non statistically significant difference in overall survival for the combination (OS, 31.0 vs 26.6 months, HR = 0.89, 95% confidence interval [CI] = 0.73 to 1.10, log-rank P =.14), which may be attributable to other therapies that patients with MBC subsequently receive during the course of their disease that make identification of statistically significant OS differences in these trials challenging (16). The statistically significant improvement in PFS seen with everolimus plus exemestane was sufficient for regulatory approval of this combination in the United States and the European Union in 2012 (and subsequently in several other countries worldwide), representing a new standard of care for the treatment of endocrine-resistant postmenopausal breast cancer compared with using endocrine therapy alone (28,29). Despite the encouraging results from TAMRAD and BOLERO-2, two key regulatory feedback loops may limit the effectiveness of mtor inhibitors at a molecular level. A negative feedback loop exists downstream in the PI3K/Akt/mTOR pathway, whereby the mtor-activated kinase S6K1 phosphorylates and destabilizes the IRS1 and IRS2 proteins in insulin-like growth factor (IGF) responsive cells (30), which in turn allows enhanced activation of insulin-like growth factor (IGFR)-1 dependent Akt activity (31,32). In addition, a positive regulatory loop exists involving the mtorc2 complex that can be activated more directly by growth factors and activated phosphorylated Akt (33,34). While these two mechanisms may limit the benefit of the current generation of mtor inhibitors in combination with endocrine therapy, several other drugs that target the PI3K/Akt pathway upstream of mtor are currently being tested in clinical trials in patients with advanced, endocrine-resistant ER+ breast cancer (Figure 1) (35). These include pan-pi3k inhibitors, isoform-specific PI3K inhibitors, dual PI3K/mTOR inhibitors, and Akt inhibitors (Table 3). For example, buparlisib (BKM120) is a potent oral pan-pi3k inhibitor

3 3 of 10 JNCI J Natl Cancer Inst, 2015, Vol. 107, No. 10 Table 1. Summary of randomized clinical studies evaluating progression-free survival/time to progression as primary endpoint with combination regimens in hormone receptor positive, advanced breast cancer, including endocrine therapy (ET) pretreated or ET-naive subsets (when available) ITT population ET-pretreated population ET-naive population Study Phase Treatment arms Patients, n Median, mo P Patients, n Median, mo P Patients, n Median, mo P Endocrine combination therapy SWOG 0226 (13) + FUL FACT* (14) + FUL NA NA SoFEA (8) + FUL FUL # Same as ITT population NA Endocrine therapy plus a targeted agent mtor inhibitor TAMRAD (12) BOLERO-2 (15,16) HORIZON (17) EGFR inhibitor NCT (18) NCT (19) EGFR/HER2 inhibitor EGF (HER2-negative population) (20) EGFR/HER2/HER3 inhibitor MINT (21) IGFR inhibitor NCT (22) HDAC inhibitor ENCORE 301 (23) Src inhibitor NCT (24) CDK 4/6 inhibitor TRIO-18 (25) EVE + TAM TAM EVE + + TEM GEF + TAM TAM GEF + + LAP AZD GAN + FUL/ FUL/ ENT + + DAS Palbociclib Same as ITT population NA (exploratory) <.0001 Same as ITT population NA NA NR NA NR NA Same as ITT population.44 Same as ITT population NA.055** Same as ITT population NA NR NA NA.0004 NA NA.27 NR NR.522 * 70% of patients in the + FUL arm and 66% in the arm had previously received endocrine therapy. = anastrozole; DAS = dasatinib; ENT = entinostat; ET = endocrine treatment; EVE = everolimus; = exemestane; FUL = fulvestrant; GAN = ganitumumab; GEF = gefitinib; HER = human epidermal growth factor receptor; LAP = lapatinib; = letrozole; NA = not applicable; NR = not reported; SWOG = Southwest Oncology Group; TAM = tamoxifen; TEM = temsirolimus. In HER2-negative patients, the two populations shown are either those patients relapsing in six months or less after adjuvant endocrine therapy (listed as ET pretreated in this table) or those untreated with prior endocrine therapy or relapsing more than six months after completion of adjuvant endocrine therapy (listed as ET-naive in this table). Interim analysis of AZD mg dose only. 40% of patients in the + DAS arm and 51% in the arm had previously received endocrine therapy. 32% of patients in the palbociclib + arm and 35% in the arm had previously received endocrine therapy. + FUL vs FUL. # FUL vs. ** One-sided. that, when given either continuously or intermittently in combination with letrozole in a phase I study, has been demonstrated to be safe, with evidence of antitumor efficacy as assessed by fluorodeoxyglucose positron emission tomography scans (36). A randomized phase study of buparlisib with fulvestrant in patients with hormone receptor positive/human epidermal

4 S. R. D. Johnston 4 of 10 Table 2. Summary of randomized clinical studies evaluating overall survival with combination regimens in hormone receptor positive, advanced breast cancer ITT population Phase Endocrine combination therapy SWOG 0226 (13) FACT (14) SoFEA (8) Patients, n Median, mo + FUL + FUL + FUL FUL EVE + TAM TAM EVE + + TEM NR NE NE.007 (exploratory) GAN + FUL/ FUL/ NE.28 ENT (exploratory) Palbociclib P * * + FUL vs FUL. = anastrozole; ENT = entinostat; EVE = everolimus; = exemestane; FUL = fulvestrant; GAN = ganitumumab; = letrozole; NE = not estimable; NR = not reached; SWOG = Southwest Oncology Group; TAM = tamoxifen; TEM = temsirolimus. FUL vs. Figure 1. Growth factor receptor and intracellular targets investigated in clinical trials in hormone receptor positive advanced breast cancer. Data from Zardavas D, Baselga J, Piccart M. Nat Rev Clin Oncol. 2013;10(35): CDK = cyclin-dependent kinase; EGFR = epidermal growth factor receptor; FGFR = fibroblast growth factor receptor; HDAC = histone deacetylase; IGFR = insulin-like growth factor receptor; MDM2 = murine double minute 2; Me = methylation; mtor = mammalian target of rapamycin; PI3K = phosphatidylinositol-3 kinase; STAT3 = signal transducer and activator of transcription 3. Endocrine therapy plus a targeted agent mtor inhibitor TAMRAD (12) BOLERO-2 (15,16) HORIZON (17) IGFR inhibitor NCT (22) HDAC inhibitor ENCORE 301 (23) CDK 4/6 inhibitor TRIO-18 (25) Treatment arms Study

5 5 of 10 JNCI J Natl Cancer Inst, 2015, Vol. 107, No. 10 Table 3. Summary of ongoing or planned clinical studies evaluating the efficacy of targeted combination regimens in postmenopausal hormone receptor positive, advanced breast cancer ClinicalTrials. gov identifier Study name Agent/comparator Setting Study design Estimated enrollment, N PI3K/Akt/mTOR inhibitors NCT MK-2206 (Akt inhibitor) + or or or FUL MBC, AI/fulvestrant naive or pretreated Phase I, non open label 31 NCT * XL147 (PI3K inhibitor) or XL765 (PI3K/mTOR dual inhibitor) + MBC, refractory to NSAI Phase I/, non open label 72 NCT FERGI NCT BELLE-2 NCT BELLE-3 IGF-1 inhibitors NCT NCT GDC-0941 (pan-pi3k inhibitor) or GDC-0980 (PI3K/mTOR dual inhibitor) + FUL or placebo + FUL BKM120 (pan-pi3k inhibitor) + FUL vs placebo + FUL BKM120 + FUL vs placebo + FUL MEDI-573 (dual IGF-1/2- neutralizing antibody) + AI vs AI BMS vs BMS MBC, refractory to AI AI treated MBC AI treated MBC, mtor inhibitor refractory MBC untreated for metastatic disease unless prior adjuvant AI and/ or tamoxifen ended 2 wk prior to 1st dose of MEDI-573 MBC refractory to an NSAI NCT MM vs alone MBC progressed after 1 st -line endocrine therapy or during (within 6 mo of completing) adjuvant NSAI and/or tamoxifen FGFR inhibitors NCT GLOW NCT Src kinase inhibitors ISRCTN ARISTACAT CDK 4/6 inhibitors NCT PALOMA-2 NCT PALOMA-3 AZD4547 (FGFR1, 2, and 3 selective inhibitor) + FUL vs FUL Dovitinib (FGFR, VEGFR, and PDGFR inhibitor) + FUL vs FUL AI + saracatinib (AZD0530) vs AI alone Palbociclib (PD ) + vs placebo + Palbociclib (PD ) + FUL vs placebo + FUL MBC with FGFR1 polysomy or gene amplification progressing after endocrine Tx MBC refractory to endocrine therapy MBC AI-sensitive/naive or refractory to NSAI (without PD for 24 mo in the [neo] adjuvant setting or 6 mo in the advanced setting) 1 st -line MBC (no prior Tx for MBC and >12 mo since completion of adjuvant endocrine Tx) 2 nd -line MBC (progressed within 12 mo of prior adjuvant endocrine Tx or 1 mo of prior endocrine Tx for MBC) Phase, Phase, Phase, Phase Ib/, open label Phase, open label Phase, Phase I//a, Phase, Phase, open label Phase, Phase, NCT MONALESSA-2 LEE011 + vs placebo + 1 st -line MBC (no prior Tx for MBC and >12 mo since completion of adjuvant endocrine Tx) Phase, 500 NCT MONARCH 2 LY FUL vs FUL 1 st -line MBC (no prior endocrine therapy) or 2 nd -line MBC (progression on either an antiestrogen or an aromatase inhibitor) Phase, 550 NCT MONARCH 3 LY or vs or 1 st -line MBC (no prior endocrine Tx for MBC and >12 mo since completion of adjuvant endocrine Tx) Phase, 450 * Study has been completed but no results reported thus far. Based on information available at AI = aromatase inhibitor; = anastrozole; CBR = clinical benefit rate; CDK = cyclin-dependent kinase; =exemestane; FGFR = fibroblast growth factor receptor; FUL = fulvestrant; IGF = insulin-like growth factor; = letrozole; MBC = metastatic breast cancer; NSAI = nonsteroidal aromatase inhibitor; PDGFR = platelet-derived growth factor receptor; Tx = therapy; VEGFR = vascular endothelial growth factor receptor.

6 S. R. D. Johnston 6 of 10 growth factor receptor 2 (HER2) negative locally advanced/mbc who have progressed after prior AI therapy is ongoing (BELLE 2; NCT ). Likewise, FERGI (NCT ) is a multicenter, international, ed, placebo-controlled phase trial in patients with advanced or metastatic breast cancer who have previously received treatment with an AI. Patients were randomly assigned to receive fulvestrant with or without the pan-pi3k inhibitor pictilisib (GDC-0941). Preliminary results from part I of FERGI showed no overall difference in PFS for the combination and, in particular, no difference in efficacy in relation to PIK3CA mutation status (wild-type vs mutant) (37). Results from further studies with more selective isoform-specific PI3K inhibitors are awaited. A key question remains as to whether the combination of an mtor or PI3K inhibitor with endocrine therapy will only be effective in endocrine-resistant breast cancer or if this is a new option for endocrine-sensitive MBC in the first-line setting that could delay or prevent endocrine resistance from developing and enhance the benefit of first-line AI therapy. A large firstline phase study (HORIZON) recently reported the efficacy of the oral mtor antagonist temsirolimus (30 mg orally for 5 days every 2 weeks) in combination with letrozole vs letrozole plus placebo in 1112 patients with AI-naive ER+ advanced breast cancer (Tables 1 and 2) (17). In contrast to BOLERO-2, the population in this larger study was mainly totally endocrine therapy naive (approximately 60%) and had received no prior AI therapy for locally advanced/metastatic disease. In HORIZON, there was neither improvement in PFS overall (median 8.9 months [temsirolimus plus letrozole] vs 9.0 months [letrozole], HR = 0.90, P =.25) nor improvement in the 40% patient subset that had received prior adjuvant endocrine therapy. These data suggest that as first-line therapy the combination of an mtor inhibitor with an AI may not be any better than an AI alone. One explanation for this could be that the PI3K/Akt/mTOR pathway is not operative in all endocrine-sensitive ER+ breast cancer cells, but instead only becomes substantially switched on during the development of secondary endocrine resistance. As discussed further below, identifying biomarkers that might predict benefit from the addition of mtor inhibitor therapy has become an important research priority. Potential Prevention of Endocrine Resistance Any strategy that can prevent secondary (acquired) endocrine resistance in ER+ MBC will improve the clinical benefit of firstline endocrine therapy, which in turn ultimately could be translated into the adjuvant setting as a cure for more women with ER+ primary breast cancer. Secondary endocrine resistance develops as a consequence of a series of complex adaptive changes occurring in breast cancer cells during the selective pressure of long-term endocrine treatment (9). In preclinical models, activation of various intracellular signaling pathways leads to endocrine resistance, and targeting a number of these pathways has been a valid strategy to prevent/delay resistance developing over time to endocrine therapy. There is evidence for increased cross-talk between various peptide growth factor receptor signaling pathways and ER at the time of relapse on endocrine therapy, with ER often becoming activated and super-sensitized by a number of different intracellular kinases, including mitogen-activated protein kinases (MAPKs) that are activated by peptide growth factor receptors (including epidermal growth factor receptors [ie, EGFR, HER2, and HER3], the insulin-like growth factor receptor [IGFR], or the fibroblast growth factor receptor [FGFR]) (38 40). Experimental data provided a strong clinical rationale that combining these various established growth factor therapies with endocrine therapy can prevent or delay endocrine resistance (41,42). Most clinical trials testing this strategy have been conducted in the first-line advanced breast cancer setting with the expectation that combined endocrine and targeted growth factor receptor therapy might delay the TTP on endocrine therapy alone by blocking a key resistance mechanism from the outset. Two phase randomized trials assessed whether cotargeting EGFR using gefitinib in combination with either tamoxifen or anastrozole could enhance the benefit of endocrine therapy in the firstline setting (18,19). In endocrine therapy naive patients in the gefitinib-plus-tamoxifen trial, there was a numerical increase in PFS from 8.9 to 12.1 months, whereas patients who had been pre-exposed to AIs did not gain any benefit from the combination (Table 1) (18). A second randomized phase trial of gefitinib and anastrozole vs anastrozole alone in 93 patients reported a statistically significant prolongation of PFS from a median of 8.4 months with anastrozole to 14.7 months with the combination (HR = 0.55, 95% CI = 0.32 to 0.94) (Table 1) (19). A subsequent combined analysis of both clinical trials suggested that the benefit for the combination was seen exclusively in those patients who were endocrine therapy naive, including no prior endocrine therapy in the adjuvant setting (43). On the basis of these results, a prospective randomized placebo-controlled multi-center phase first-line study (MINT) was conducted in postmenopausal, endocrine therapy naive, ER+ MBC utilizing a novel tyrosine kinase inhibitor, AZD8931, a potent inhibitor of EGFR, HER2, and HER3 (21). There was no statistical improvement in PFS after the addition of either 20 or 40 mg AZD8931 (Table 1), with greater toxicity seen in the experimental arms (21). Thus, there are no conclusive clinical data to support a strategy of cotargeting EGFR as first-line therapy in ER+ MBC. In contrast, HER2 has been a more attractive target in hormone receptor positive breast cancer given the evidence that HER2 expression in breast cancer models is associated with primary resistance to endocrine therapy. Three randomized trials in MBC (20,44,45) have confirmed that this approach can treat this form of endocrine resistance in tumors with known co-expression of ER and HER2. However, the more intriguing question is whether blockade of the HER2 pathway in ER+ but HER2-nonamplified tumors could be an effective strategy to enhance the benefit of endocrine therapy by delaying treatment resistance. In the large EGF30008 study, this hypothesis was prospectively evaluated in patients with ER+ HER2 tumors (20). However, just as in the MINT study that targeted EGFR and HER2/ HER3, combination treatment did not result in an improvement in PFS (median PFS = 14.7 vs 15.0 months). This implies that specific cotargeting of HER2 together with ER from the outset does not delay resistance in ER+ endocrine-sensitive breast cancer. Indeed, this result is consistent with experimental models that demonstrated the failure of trastuzumab and letrozole combined together from the outset to delay endocrine resistance in hormone receptor positive xenografts, in contrast to combined therapy that was very effective once resistance to letrozole (and presumably HER2 expression) had developed (41). While targeting growth factor receptors has been a largely disappointing strategy in the clinic for preventing endocrine resistance in the first-line setting, a more successful approach has been to inhibit the cell cycle in luminal ER+ breast cancer cells. Palbociclib is a novel oral selective inhibitor of cyclindependent kinase 4/6 (CDK 4/6) that prevents cellular DNA synthesis by blocking cell cycle progression from G1 to S phase

7 7 of 10 JNCI J Natl Cancer Inst, 2015, Vol. 107, No. 10 (46,47). Recently, it was reported that the combination of palbociclib and letrozole statistically significantly improved PFS in a randomized phase study (TRIO-18, PALOMA-1) in patients with advanced ER+ breast cancer (median PFS = 20.2 months for palbociclib plus letrozole vs 10.2 months for letrozole) (Table 1) (25). The combination appeared to be well tolerated, with uncomplicated neutropenia the most frequent drug-related toxicity. On the strength of these data, accelerated new drug approval was granted in February 2015 by the US Food and Drug Administration (FDA), conditional upon results from the multicenter,, phase study of palbociclib plus letrozole vs placebo plus letrozole in postmenopausal women with ER+, HER2 MBC (PALOMA-2, NCT ). Similar randomized phase studies are underway in the first-line setting using letrozole in combination with the CDK 4/6 inhibitor ribociclib (LEE011; MONALEESA-2, NCT ) and letrozole in combination with the CDK 4/6 inhibitor abemaciclib (LY ; MONARCH 3, NCT ) (Table 3). Benefit for this approach may also exist in endocrine resistant disease. Results from a phase study of palbociclib with fulvestrant recently showed a significant improvement in PFS compared with fulvestrant in endocrine pre-treated ER + metastatic breast cancer (PALOMA-3, NCT ) (median PFS 9.2 months for palbociclib + fulvestrant vs 3.8 months for fulvestrant, HR = 0.42, P < ) (48). Other Potential Strategies to Cotarget Endocrine and Signaling Pathways The reasons for emergence of resistance during prolonged endocrine therapy remain complex, and it is unlikely that any single mechanism is dominant. While the EGFR/HER2 growth factor receptor pathway and the cell survival PI3K/Akt/mTOR pathway have been studied extensively, numerous other signaling pathways may also be implicated in causing endocrine resistance. The role of the insulin-like growth factor (IGF) system in endocrine-resistant breast cancer has also been investigated. To date, the only study to report results involved AMG-479 (ganitumab), which is a monoclonal antibody against IGFR-1. In a randomized phase study in the second-line setting, AMG- 479 in combination with either exemestane or fulvestrant failed to improve PFS or OS compared with exemestane or fulvestrant alone (NCT ) (22) (Tables 1 and 2). Other monoclonal neutralizing antibodies against the IGF-I/ ligands (MEDI-573) and tyrosine kinase inhibitors against IGFR-1 (BMS ) are being investigated in combination with endocrine therapies in the first- and second-line settings (Table 3). The fibroblast growth factor receptor-1 gene (FGFR1) is amplified in up to 20% of ER-positive breast cancers, and amplification or overexpression of FGFR1 may be a major contributor to poor prognosis and endocrine therapy resistance in luminal-type B breast cancers (49). AZD4547 is a potent selective inhibitor of FGFR-1, -2, and -3 receptor tyrosine kinases (based on enzyme and cellular phosphorylation endpoints) and has a markedly lower potency for inhibition of IGFR-1 and kinase insert domain receptor (KDR) (50). Likewise, dovitinib is a tyrosine kinase inhibitor that targets FGFR in addition to vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR). Randomized phase studies are ongoing with both FGFR inhibitors in combination with fulvestrant in the second-line setting (Table 3). Another possible approach to reverse hormone resistance is the use of histone deacetylase inhibitors (HDACi) to resensitize breast cancer cells to hormone manipulation. It has been shown that in some breast cancers expression of ER can be repressed or lost by epigenetic modifications such as methylation and histone deacetylation, and this could be a mechanism contributing to endocrine resistance. Entinostat is an HDACi that has been shown to increase expression of ER and the enzyme aromatase in a dose-dependent manner in vitro and in vivo and sensitized breast cancer cells to estrogen and subsequent inhibition by the AI letrozole (51). In a randomized phase trial (ENCORE 301, NCT ), entinostat in combination with exemestane was compared with exemestane plus placebo in patients who had received prior hormonal therapy (nonsteroidal AI), and the results showed prolongation of median PFS (4.3 vs 2.3 months) and extension of OS benefit (28.1 vs 19.8 months) for the combination (Tables 1 and 2) (23). A confirmatory phase intergroup study (E2112) in 600 patients with ER+ MBC that has progressed on prior nonsteroidal AI therapy is now underway. The ER-Src kinase axis plays an important role in promoting endocrine resistance by proto-oncogenes such as HER2 and PELP1, and blocking this axis can prevent the development of hormonal independence in vivo (52). Dasatinib is a potent, broad spectrum ATP-competitive inhibitor of Src tyrosine kinase. However, the addition of dasatinib to either fulvestrant or exemestane did not improve PFS, clinical benefit rate, or OS in two separate randomized phase studies (53,54). In contrast, a randomized phase first-line study (NCT ) suggested in an exploratory analysis that the addition of dasatinib to letrozole may improve PFS (median 20.1 vs 9.9 months, HR = 0.69) (Table 1) (24). Results from further similar studies are awaited, including with the Src-inhibitor saracatinib. Lessons for Clinical Trial Design Role of Biomarker Selection As highlighted above, it is clear that improving endocrine therapy through cotargeting of signaling pathways is not straightforward (55). While there have been successes with BOLERO-2 in second-line therapy and, more recently, with PALOMA-1 in first-line therapy, why has cotargeting endocrine therapy with various other therapeutics proved so challenging? Understanding which are the dominant signaling pathways in different clinical scenarios (ie, first-line vs second-line setting, prior exposure to tamoxifen vs AIs) is likely to be important. One important mistake made in some large phase first-line trials has been to include a mixed heterogenous population of ER+ breast cancer, including patients with de novo stage IV disease together with those who relapse during (or indeed sometimes after) completion of prior adjuvant endocrine therapy. Thus, tumor endocrine sensitivity and/or resistance may vary enormously, with de novo untreated stage IV patients potentially having a long PFS (15 months) with first-line AI therapy, whereas those relapsing during the first few years of adjuvant tamoxifen may have developed resistance mechanisms that mean benefit from AI therapy may only be six to eight months at best. By including both populations in a first-line study without clear clinical inclusion or exclusion criteria or correct stratification built around prior therapy exposure, these trial designs are likely to lead to inaccurate statistical consideration for the PFS benefit being evaluated in the so-called first-line population and risk diluting the clinical benefit that may be observed for any given cotargeting combination. Although most of the reported randomized trials have reported efficacy for patient subgroups that were either pretreated with endocrine therapy or were endocrine therapy naive, some trials with mixed populations have only reported efficacy for all patients, making it difficult to ascertain which groups of patients benefit from therapy (Tables 1 and 2). Another important consideration in a first-line cotargeting approach is that targeted blockade of any given signaling pathway in endocrine-sensitive breast cancer may be totally ineffective if the pathway is neither functional nor overexpressed in ER+ cells, and instead may simply allow other intracellular signaling

8 S. R. D. Johnston 8 of 10 resistance mechanisms to evolve during first-line AI therapy. Indeed, there is some concern that blockade of one pathway could even accelerate other compensatory pathways and shorten the PFS benefit observed with AI therapy (as seen in the MINT study) (21). Thus, identification of key signaling pathways from real-time biopsies of ER+ MBC rather than archival primary tissue samples has become a research priority. It is possible that some of these ER+ tumors are already inherently primed to respond to a given combination (eg, because of either a PIK3CA mutation or to amplification of FGFR1 or HER2). In future, all clinical studies in MBC should make a greater effort to enrich their trial population with the most appropriate patients, with a relevant real-time tumor biopsy taken in the metastatic setting to allow molecular profiling that may identify which targeted therapy to utilize. For example, in part of the recently recruited FERGI trial, only patients with proven PIK3CA mutations were randomly assigned to fulvestrant plus pictilisib or fulvestrant alone (37). Likewise, prospective stratification based on biomarker expression is being implemented in the BELLE-2 study of fulvestrant with or without the PI3K inhibitor buparlisib (BKM120) (56). Several academic centers have now set up real-time molecular profiling using metastatic biopsies to guide clinical trial selection for various targeted therapeutics in development. While some of these studies (ie, SAFIR-01) have demonstrated the many challenges and relatively low yield ( 12%) in deriving any actionable mutations and/or targets beyond PIK3CA mutation and CCND1 amplification (57), this remains a priority if precision medicine with cotargeted therapies is to be successful in ER+ MBC. In the absence of the ability to identify the molecular target in samples of metastatic disease because only the original archival primary tumor is available, understanding the inherent biology in the primary ER+ tumor that could account for subsequent relapse because of secondary endocrine resistance will become crucial. Genomic profiling using, for example, PAM-50 or other related platforms, together with next-generation sequencing to identify relevant genomic mutations, may help identify those ER+ breast cancers more likely to develop secondary resistance to endocrine therapy, or indeed the pathways that these tumors could utilize as escape mechanisms. It is unclear whether separate escape pathways and different rewiring of signaling pathways are utilized by breast cancer cells during exposure to either an anti-estrogen such as tamoxifen, or to estrogen deprivation by AI therapy, and further preclinical studies are needed to investigate this. Recent studies have started to demonstrate some of the common oncogenic pathways that intrinsic subtypes of breast cancer utilize at relapse, such as activation of growth factor signaling in luminal-type B ER+ breast cancer that accounts for the poorer prognosis of this subtype (58). As such, selection of patients by molecular profiling of their tumor at the start of endocrine therapy may help to determine the best combination strategy to use at the outset and then again at relapse, and in which sequence. This could be a much more successful approach than treating heterogeneous populations of unselected breast cancer patients with any given combination strategy that will only benefit a small group of unidentified patients. However, the major challenge will remain how predictive molecular profiling will be for HR + breast cancer cells that appear to have inherent plasticity in the various survival signals that cells use during specific therapeutic approaches. While overexpression of any given oncogene or molecular target might identify the best group of ER+ patients to cotarget with any given novel therapeutic, the inherent complexity of breast cancer means that it is not that straightforward. Despite initial expectations that a predictive biomarker for everolimus might be identified in BOLERO-2, retrospective analysis of 309 archival tissue samples demonstrated no predictive biomarker for everolimus efficacy in the four most frequently altered genes and pathways, including PIK3CA mutations upstream of mtor (59). In contrast, Hortobagyi et al. reported lack of benefit from everolimus in the 24% of biomarker-assessable patients who had multiple genomic alterations in PIK3CA, CCND1, or FGFR1/2 pathways combined (59). This suggests that development of other co-existing mutations and molecular alterations within a complex web of interrelated signaling networks will mitigate against response to inhibition of any given key target. As such, translational studies continue to remain crucial for optimizing clinical benefit from any new targeted therapy, whether these studies utilize the original primary tumor or the relapsed metastatic sample, where the molecular profile may have changed. Future Directions There are now a multitude of targeted therapeutics in various stages of clinical development for ER+ breast cancer that target a signaling pathway potentially involved in the pathogenesis of endocrine resistance. Substantial improvements in PFS have been seen for both second- and first-line therapies in ER+ MBC. Everolimus combined with exemestane is approved to treat secondary endocrine resistance in postmenopausal hormone receptor positive advanced breast cancer following prior therapy with a nonsteroidal AI, while palbociclib combined with letrozole is a potential new first-line treatment option that doubled PFS compared with letrozole alone. These two developments alone represent substantial progress, prolonging the clinical benefit of endocrine therapy for women with advanced ER+ breast cancer, providing greater disease control and delaying the time when cytotoxic chemotherapy is required. These are clinically meaningful benefits for thousands of patients. However, the ultimate goal is to apply these developments to patients with early-stage breast cancer who may remain at risk of relapse because of failure of their adjuvant endocrine therapy. As such, the clinical benefit of the targeted combination strategies with proven efficacy in HR + advanced breast cancer is being evaluated in, controlled clinical studies in the adjuvant setting. These studies are being conducted either in those patients with higher risk HR + disease based on established clinical features such as nodal involvement (everolimus: UNIRAD [NCT ] and SWOG S1207 [NCT ]) or those with residual disease following neo-adjuvant systemic chemotherapy (palbociclib: PENELOPE-B [NCT ]). If study results are positive, these adjuvant trials could result in these combination strategies starting to cure more patients with early-stage HR + breast cancer. However, it is probably naive to believe that targeting a solitary signaling pathway will reverse all endocrine resistance mechanisms. As indicated above, blockade of any given pathway may just promote or even enhance development of other compensatory escape mechanisms that will drive resistance. Therefore, the emphasis is now shifting towards targeting networks and pathways with combinations of key signaling inhibitors, either in parallel (so-called combined horizontal blockade) or in series (combined vertical blockade). For example, strategies are being explored to examine triplet vertical combinations of endocrine therapy combined with at least two signaling inhibitors, especially CDK 4/6 inhibitors and PI3K pathway inhibitors. Preclinical studies suggest a mechanistic role for CDK 4 in estrogen-independent cell growth and for PI3K signaling in endocrine resistance, providing a strong rationale for the combination of

9 9 of 10 JNCI J Natl Cancer Inst, 2015, Vol. 107, No. 10 a PI3K inhibitor and CDK 4/6 inhibitor in the setting of endocrine resistance (60). At least two phase Ib/ studies using this approach are underway using either endocrine therapy (letrozole) combined with the CDK 4/6 inhibitor LEE001 and the isoform-specific PI3K inhibitor BYL219 (NCT ), or endocrine therapy (fulvestrant) combined with either BYL219 or the pan-isoform inhibitor BKM120 (buparlisib; NCT ). While it is hoped that some of these new approaches could potentially produce a further quantum leap in response to endocrine therapy in ER+ advanced breast cancer, ensuring that clinical trial design utilizes the most appropriate population will remain crucially important, whether it be defined by clinical criteria such as prior endocrine therapy exposure and/or responsiveness, or via a relevant biomarker from a real-time biopsy. This approach will ensure that there is rapid and efficient transition from early proof-of-concept studies into pivotal phase efficacy studies, thus maximizing the likelihood of success in preventing or reversing endocrine resistance. Funding I have not received any support for this work. I acknowledge research funding support to the National Institute for Health Research Biomedical Research Centre at The Royal Marsden National Health Service Foundation Trust. Notes Financial support for medical editorial assistance was provided by Novartis Pharmaceuticals. I thank Ann Marie Fitzmaurice (ProEd Communications, Inc.) for editorial assistance. References 1. Early Breast Cancer Trialists Collaborative Group. Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival: an overview of the randomized trials. Lancet. 2005;365(9472): Ring A, Dowsett M. Mechanisms of tamoxifen resistance. Endocr Relat Cancer. 2004;11(4): Ali S, Coombes RC. Endocrine-responsive breast cancer and strategies for combating resistance. Nat Rev Cancer. 2002;2(2): Osborne CK, Schiff R. Estrogen-receptor biology: continuing progress and therapeutic implications. J Clin Oncol. 2005;23(8): Network NCC. NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines). Breast Cancer. Version NCCN Guidelines for Patients available at Cardoso F, Costa A, Norton L, et al. ESO-ESMO 2nd international consensus guidelines for advanced breast cancer (ABC2)dagger. Ann Oncol. 2014;25(10): Chia S, Gradishar W, Mauriac L, et al. Double-blind, randomized placebo controlled trial of fulvestrant compared with exemestane after prior nonsteroidal aromatase inhibitor therapy in postmenopausal women with hormone receptor-positive, advanced breast cancer: results from EFECT. J Clin Oncol. 2008;26(10): Johnston SR, Kilburn LS, Ellis P, et al. Fulvestrant plus anastrozole or placebo versus exemestane alone after progression on non-steroidal aromatase inhibitors in postmenopausal patients with hormone-receptor-positive locally advanced or metastatic breast cancer (SoFEA): a composite, multicenter, phase 3 randomized trial. Lancet Oncol. 2013;14(10): Miller TW, Balko JM, Arteaga CL. Phosphatidylinositol 3-kinase and antiestrogen resistance in breast cancer. J Clin Oncol. 2011;29(33): degraffenried LA, Friedrichs WE, Russell DH, et al. Inhibition of mtor activity restores tamoxifen response in breast cancer cells with aberrant Akt Activity. Clin Cancer Res. 2004;10(23): Boulay A, Rudloff J, Ye J, et al. Dual inhibition of mtor and estrogen receptor signaling in vitro induces cell death in models of breast cancer. Clin Cancer Res. 2005;11(14): Bachelot T, Bourgier C, Cropet C, et al. Randomized phase trial of everolimus in combination with tamoxifen in patients with hormone receptor-positive, human epidermal growth factor receptor 2-negative metastatic breast cancer with prior exposure to aromatase inhibitors: a GINECO study. J Clin Oncol. 2012;30(22): Mehta RS, Barlow WE, Albain KS, et al. Combination anastrozole and fulvestrant in metastatic breast cancer. N Engl J Med. 2012;367(5): Bergh J, Jonsson PE, Lidbrink EK, et al. FACT: an open-label randomized phase study of fulvestrant and anastrozole in combination compared with anastrozole alone as first-line therapy for patients with receptor-positive postmenopausal breast cancer. J Clin Oncol. 2012;30(16): Yardley DA, Noguchi S, Pritchard KI, et al. Everolimus plus exemestane in postmenopausal patients with HR(+) breast cancer: BOLERO-2 final progression-free survival analysis. Adv Ther. 2013;30(10): Piccart M, Hortobagyi GN, Campone M, et al. Everolimus plus exemestane for hormone-receptor-positive, human epidermal growth factor receptor- 2-negative advanced breast cancer: overall survival results from BOLERO-2. Ann Oncol. 2014;25(12): Wolff AC, Lazar AA, Bondarenko I, et al. Randomized phase placebo-controlled trial of letrozole plus oral temsirolimus as first-line endocrine therapy in postmenopausal women with locally advanced or metastatic breast cancer. J Clin Oncol. 2013;31(2): Osborne CK, Neven P, Dirix LY, et al. Gefitinib or placebo in combination with tamoxifen in patients with hormone receptor-positive metastatic breast cancer: a randomized phase study. Clin Cancer Res. 2011;17(5): Cristofanilli M, Valero V, Mangalik A, et al. Phase, randomized trial to compare anastrozole combined with gefitinib or placebo in postmenopausal women with hormone receptor-positive metastatic breast cancer. Clin Cancer Res. 2010;16(6): Johnston S, Pippen J Jr, Pivot X, et al. Lapatinib combined with letrozole versus letrozole and placebo as first-line therapy for postmenopausal hormone receptor-positive metastatic breast cancer. J Clin Oncol. 2009;27(33): Johnston SRD, Basik M, Hegg R, et al. Phase randomized study of the EGFR, HER2, HER3 signaling inhibitor AZD8931 in combination with anastrozole (A) in women with endocrine therapy (ET) naive advanced breast cancer (MINT). J Clin Oncol. 2013;31(15 Suppl):abstract Robertson JF, Ferrero JM, Bourgeois H, et al. Ganitumab with either exemestane or fulvestrant for postmenopausal women with advanced, hormonereceptor-positive breast cancer: a controlled,, phase 2 trial. Lancet Oncol. 2013;14(3): Yardley DA, Ismail-Khan RR, Melichar B, et al. Randomized phase, doubleblind, placebo-controlled study of exemestane with or without entinostat in postmenopausal women with locally recurrent or metastatic estrogen receptor-positive breast cancer progressing on treatment with a nonsteroidal aromatase inhibitor. J Clin Oncol. 2013;31(17): Paul D, Vukelja SJ, Holmes FA, et al. Letrozole plus dasatinib improves progression-free survival (PFS) in hormone receptor-positive, HER2-negative postmenopausal metastatic breast cancer (MBC) patients receiving firstline aromatase inhibitor (AI) therapy. Cancer Res. 2013;73(24 Suppl):Abstract S Finn RS, Crown JP, Lang I, et al. The cyclin-dependent kinase 4/6 inhibitor palbociclib in combination with letrozole versus letrozole alone as first-line treatment of estrogen receptor-positive, HER2-negative, advanced breast cancer (PALOMA-1/TRIO-18): a randomized phase 2 study. Lancet Oncol. 2015;16(1): Rugo HS, Keck S. Reversing hormone resistance: have we found the golden key? J Clin Oncol. 2012;30(22): Baselga J, Campone M, Piccart M, et al. Everolimus in postmenopausal hormone-receptor-positive advanced breast cancer. N Engl J Med. 2012;366(6): US Food and Drug Administration. Afinitor product insert. July lbl.pdf. Accessed February 18, European Medicines Agency. Afinitor product information. August 3, Information/human/001038/WC pdf. Accessed February 18, Sabatini DM. mtor and cancer: insights into a complex relationship. Nat Rev Cancer. 2006;6(9): O Reilly KE, Rojo F, She QB, et al. mtor inhibition induces upstream receptor tyrosine kinase signaling and activates Akt. Cancer Res. 2006;66(3): Sun SY, Rosenberg LM, Wang X, et al. Activation of Akt and eif4e survival pathways by rapamycin-mediated mammalian target of rapamycin inhibition. Cancer Res. 2005;65(16): Sarbassov DD, Guertin DA, Ali SM, Sabatini DM. Phosphorylation and regulation of Akt/PKB by the rictor-mtor complex. Science. 2005;307(5712): Jacinto E, Loewith R, Schmidt A, et al. Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive. Nat Cell Biol. 2004;6(11): Zardavas D, Baselga J, Piccart M. Emerging targeted agents in metastatic breast cancer. Nat Rev Clin Oncol. 2013;10(4): Mayer IA, Abramson VG, Balko JM, et al. SU2C phase Ib study of pan-pi3k inhibitor BKM120 with letrozole in ER+/HER2- metastatic breast cancer (MBC). J Clin Oncol. 2012;30(15 Suppl):Abstract Krop I, Johnston S, Mayer IA, et al. The FERGI phase study of the PI3K inhibitor pictilisib (GDC-0941) plus fulvestrant vs fulvestrant plus placebo in patients with ER+, aromatase inhibitor (AI)-resistant advanced or meta-