Mechanisms of endocrine resistance.

Introduction

Two-thirds of breast cancers are associated with oestrogen receptor (ER) signalling, and hormonal manipulation was first shown to be a successful strategy against breast cancer over 100 years ago [1]. Currently available endocrine strategies include targeting the ER itself with the selective ER modulator tamoxifen or the ER downregulator fulvestrant, as well as suppressing the amount of available ligand for the receptor with gonadal suppression in premenopausal women or aromatase inhibitors (AIs) in postmenopausal women.

Among postmenopausal women with early ER-positive breast cancer, endocrine therapy has been shown to have a greater impact on reducing annual breast cancer death rate than adjuvant chemotherapy (31% versus 20%) [2]. Given their favourable side effect profile and their potential for significant anti-tumour activity, endocrine therapies represent an important tool in our armamentarium against breast cancer. Unfortunately, despite documented levels of ERs, up to 50% of patients with metastatic disease do not respond to first-line endocrine treatment (de novo resistance), while the remainder will eventually relapse (acquired resistance) [3]. Understanding the various biological mechanisms responsible for the development of endocrine resistance can identify new strategies to enhance the efficacy of hormone receptor-positive breast cancer treatment.

A number of theories, supported by preclinical data, have been proposed to explain endocrine resistance. These vary according to the sustained dependence on ER-mediated signalling and a significant number implicate growth factor-mediated mitogenic signalling (Table 1). Identifying which resistance mechanism(s) is (are) operational is of obvious clinical relevance in tailoring the most appropriate subsequent therapy (i.e. non-ER-targeted treatment, further endocrine manipulation, or a combination of both).

Normal ER function

The ER belongs to the steroid nuclear receptor family of ligand-dependent transcription factors (Figure 1A). The transcription of ER-regulated genes is regulated by two activating function (AF) domains. AF-2 is located at the C-terminus and requires binding of oestradiol (E2) to the ligand-binding domain (LBD) of the receptor, whereas AF-1 is located at the N-terminus and is hormone independent. E2 binding to the LBD induces a conformational change in the receptor, leading to the recruitment of nuclear co-activators (NCOAs) that increase ER interaction with DNA [4]. The activated ER binds as a dimer to oestrogen response elements in the promoters of target genes and activates transcription. In contrast, AF-1 can be phosphorylated by growth factor receptors or other downstream effectors and can act both independently or synergistically with AF-2 to increase the efficiency of ER transcriptional activity [5].

[FIGURE 1 OMITTED]

Tamoxifen acts as a competitive inhibitor. Upon binding to the LBD, it induces a conformational change that inactivates AF-2, but has no effect on AF-1 transcriptional activity [6]. Because ER activity in the breast is predominantly mediated via AF-2, tamoxifen has an overall antagonist effect in the breast, but can act as an agonist in other tissues primarily driven by AF-1, such as the uterus [7].

In addition, tamoxifen recruits co-repressors (NCORs) thereby further inhibiting transcription. Alterations in the relative contributions of AF-1/AF-2 to transcription, as well as the amounts of NCOAs/NCORs available, may shift the balance of tamoxifen activity towards agonism and have been implicated in endocrine resistance. In addition to its role as a classical transcription factor, ER can also enhance transcription without direct DNA binding by participating in protein-protein interactions with other transcription factors (non-classical genomic activity). For example, ER can essentially act as a co-activator and increase the activity of the Jun/Fos activator protein 1 (AP-1) transcription complex [8] (Figure 1B).

Finally, in addition to these nuclear transcriptional functions, non-genomic actions have more recently been ascribed to the ER. Oestrogen-bound ER has been shown to interact directly with, and phosphorylate, membrane-associated growth factor receptors [9,10] as well as downstream effectors such as the p85 subunit of phosphoinositide-3-kinase (PI3K) [11], resulting in further pro-survival and anti-apoptotic signalling (Figure 1C).

The degree to which these individual functions of the ER contribute to malignant proliferation may vary from one patient to another and evolve over time thereby contributing to the development of endocrine resistance.

Implication of growth factor receptor and downstream mitogenic signalling in endocrine resistance

Membrane growth factor receptors such as the epidermal growth factor receptor (EGFR), the human epidermal growth factor receptor 2 (HER2) or the insulin growth factor 1 receptor (IGF1R) have been implicated in endocrine resistance. Upregulation of HER2 occurs in approximately 15-20% of breast cancers and has been associated with poor prognosis and de novo resistance to tamoxifen in the neoadjuvant setting [12]. Similarly, EGFR is overexpressed in a number of breast cancers and has also been associated with poor response to tamoxifen [12]. It is important to note that cell models of acquired resistance to both tamoxifen and oestrogen deprivation (ED) have shown that the development of resistance is associated with adaptative upregulation of growth factor signalling pathways [13,14].

Activation of these membrane receptors stimulates two major intracellular kinase signalling cascades--the ras/mitogenic-activated protein kinase (MAPK) pathway and the PI3K/Akt pathway. These pathways activate downstream effectors leading to a cascade of signals involved in malignant growth and survival, and can be involved in endocrine resistance by a number of mechanisms including downregulation of ER expression, a switch to ER-independent growth, or bi-directional crosstalk between ER and mitogenic signalling.

ER downregulation

ER and HER2 expression are frequently described as inversely related. ER-positive cell lines stably transfected with full-length HER2 demonstrate downregulation of ER, while quantitative measurements of ER levels in tumour samples show consistently lower levels of the receptor among patients with HER2-amplified breast cancer [15].

There is some evidence that hyperactive growth factor signalling can suppress ER expression, and may eventually lead to complete loss of ER [16]. Conversely, interruption of hyperactive MAPK or EGFR has been shown to re-induce ER expression in cell lines and xenograft models [17,18]. In fact, in a small study of 10 patients with ER-negative/HER2-positive breast cancer receiving trastuzumab, three patients acquired ER expression with treatment [19]. Studies with the dual EGFR/HER2 inhibitor lapatinib have shown that long-term treatment with this agent was associated with adaptive increase in ER signalling [20]. The degree to which complete loss of ERs secondary to mitogenic signalling may contribute to acquired resistance needs further confirmation as serial clinical samples from women who have relapsed on endocrine therapy suggest that this is a rare event (10-15%) [21,22]. However, this 'yin and yang' dynamic interaction between ER and growth factor signalling would support using ER- and growth factor-targeted therapies in combination, or in fact in sequence, as one may sensitise to the other.

Switch to ER-independent growth

Other mechanisms are likely to account for endocrine resistance in HER2-amplified breast cancer, as measurable levels of ERs are expressed in 50% of cases. Another possible mode of escape from the inhibitory effects of endocrine therapy is an adaptive switch to ER-independent growth, where the ER is still present but no longer driving proliferation. A cell model for acquired resistance to fulvestrant has shown that cells adapt to long-term treatment with the ER downregulator by pronounced upregulation of multiple growth-stimulatory pathways, resulting in oestrogen-independent autocrine-regulated proliferation [9]. This would imply pan-endocrine resistance and support the use of non-ER-targeted therapies.

Endocrine resistance is usually associated with a functional ER

Most in vitro and clinical observations would suggest that even following the development of endocrine resistance, ER signalling continues to play an important role in the proliferation of breast cancer. Biopsies of tumours from breast cancer patients who have relapsed while receiving treatment with an anti-oestrogen agent show a functional ER [23], while women who have become refractory to tamoxifen or non-steroidal AIs actually respond to further endocrine manipulation with the ER[alpha] downregulator fulvestrant [24,25], indicating that ER-mediated signalling remains functional. There is increasing evidence that crosstalk with growth factor and downstream mitogenic pathways can augment the genomic and non-genomic functions of ERs.

Synergistic interaction between ER and mitogenic signalling resulting in enhanced genomic and non-genomic functions of ER

Increase in classical transcription via AF-1

Growth factor-mediated activation of MAPK or Akt can potentiate E2-mediated ER classical transcriptional activity by directly phosphorylating AF-1 [26]. Of note, both MAPK and Akt have been shown to phosphorylate ER within AF-1 (at serine 118 and serine 167, respectively) in the absence of E2, thereby contributing to ligand-independent ER transactivation [27,28]. Other downstream effectors of peptide growth factor receptors have also been shown to activate ERs, such as protein kinase A and the cyclin E-cdk-2 complex [29].

An increase in AF-1 transcription may predict for differential sensitivity to endocrine agents. Tamoxifen may efficiently block AF-2-mediated transcription; however, in tumours that exhibit high levels of AF-1 activity driven by mitogenic signalling, tamoxifen may act as an agonist [7]. This is supported by the high rate of primary tamoxifen resistance observed in neoadjuvant trials of HER2-amplified breast cancer [12]. There are some data to suggest that E2 deprivation using an AI, or ER downregulation using fulvestrant, may therefore be a more effective anti-cancer strategy in EGFR-positive/HER2-positive breast cancer [12,30,31].

Potentiation of genomic activity by altering the balance of co-regulators

ER transcription is tightly regulated by the balance of NCOAs/NCORs. Mitogenic signalling can alter the expression of some of these co-regulators thereby enhancing classical and non-classical ER transcription. The co-activator NCOA3, also known as amplified in breast cancer-1 (AIB1) is overexpressed in 50% of breast cancers and amplified in 5% [32]. Among untreated patients with ER-positive breast cancer it is associated with improved survival. In contrast, AIB1 has been associated with poor outcome on tamoxifen in HER2-amplified breast cancer because HER2 activates AIB1 and enhances the agonist effects of tamoxifen [33,34]. Similarly, decreased levels of NCORs have been shown to enhance tamoxifen agonism by shifting the balance towards ER transcriptional activity [35].

Cyclin D1 is overexpressed in 50% and amplified in 25% of breast cancers. As a transducer of both ER and growth factor-mediated cell cycle progression, cyclin D1 emerged as a potentially useful target in endocrine-resistant breast cancer. In addition, more recent data suggested that cyclin D1 could interact directly with ERs via recruitment of members of the steroid receptor co-activators (Src) family of NCOAs in the absence of endogenous ligand [36]. However, there are conflicting clinical data regarding the causal relationship between cyclin D1 and endocrine responsiveness [37,38].

In addition to binding directly to DNA and increasing transcription of ER-dependent genes (classical), ligand-bound ER can also complex with other transcriptional factors, such as Jun/Fos via AP-1 (non-classical genomic activity). Acquired tamoxifen resistance in tumour samples has been associated with increased AP-1 activity presumably via tamoxifen-liganded ER agonist activity [39]. The stress-activated kinase p38 MAPK is a major upstream modulator of AP-1 transcriptional activity and may potentiate the agonist effects of tamoxifen at AP-1 [40].

There are data to suggest that mitogenic signalling can alter the ratio of NCOAs/NCORs and result in a hypersensitive response of ERs to E2, or an agonist effect of tamoxifen-bound ERs. Whether profiling tumours by measuring the levels of various transcription co-regulators may offer useful predictive information regarding endocrine responsiveness has not been clearly established.

Enhanced non-genomic functions of ER

Conversely, as detailed in Figure 1, in addition to its effects on transcription, oestrogen-bound ER has also been shown to result in non-genomic effects via rapid activation of EGFR [9], IGF1R [10], HER2 [41] or the cleavage of membrane-bound growth factor receptor ligands such as epidermal growth factor or transforming growth factor [alpha] [13]. This bi-directional interaction between ERs and growth factor pathways creates a self-reinforcing synergistic loop that potentiates pro-survival signals and may allow breast cancer to escape normal endocrine responsiveness. Furthermore, the extranuclear functions of ERs appear to require a ligand, and both E2 and tamoxifen can act as agonists [9].

It is important to note that this crosstalk does not seem to be operational in hormone-sensitive ER-positive MCF7 cells; its relevance appears to be limited to HER2-amplified cell lines or those with acquired endocrine resistance [42]. Unfortunately, while supported by extensive in vitro models, the clinical relevance of the non-genomic actions of the ERs remains controversial as membrane and/or cytoplasmic ERs have yet to be conclusively demonstrated in clinical samples.

Clinical implications

Growth factor signalling has been extensively implicated in endocrine resistance. In some cases the interaction between ER and mitogenic pathways can be described as a dynamic inverse relationship, where inhibition of one results in a compensatory increase in the other. This is supported by pre-clinical and clinical data which show that growth factor inhibition may increase ER expression or function and re-sensitise breast cancer cells to endocrine therapy, and which would support combination, or in fact sequential treatment.

Alternatively, growth factor signalling can interact synergistically with the ER and augment both genomic and non-genomic functions of the receptor. This would provide a strong rationale for simultaneous blockade of both ERs and mitogenic pathways. Given this body of evidence, a number of trials have been completed exploring the benefit of various inhibitors of growth factor receptors or downstream effectors [e.g. mammalian target of rapamycin (mTOR)] in modulating endocrine resistance (for reviews see [43,44]). As shown in Table 2, despite strong pre-clinical evidence, some of these studies have yielded disappointing results, which may be in part attributable to a poor selection of patients. It is unlikely that patients will respond to a combination of specific inhibitors unless the intended target is a significant driver of endocrine-resistant growth. Conversely, while significant overexpression of HER2 is a known requirement for benefit from trastuzumab, further studies are needed to determine whether more moderate receptor expression or activation may be relevant in the setting of endocrine resistance. Most ongoing studies are therefore including parallel biological studies in an effort to define the subset of patients most likely to benefit from specific combinations (Table 3).

Other mechanisms implicated in endocrine resistance

Loss of ER expression due to promoter Hypermethylation

ER expression is an obligate, albeit insufficient, requirement for sensitivity to endocrine therapy, and loss of ERs with progressive disease due to clonal selection could account for acquired endocrine resistance. ER silencing as a result of promoter hypermethylation has been documented in a proportion of breast cancers [45]. It is important to note that this process has been shown to be reversible. Demethylating agents or histone deacetylase (HDAC) inhibitors can re-activate expression of a functional ER in cell lines with ER silencing due to promoter methylation [46,47]. These observations are provocative and have obvious clinical implications for a proportion of patients with ER-negative tumours who may benefit from endocrine therapy if ER expression could be reactivated using a demethylating agent. A trial of tamoxifen in combination with an HDAC inhibitor in patients who have relapsed after endocrine therapy is ongoing to investigate whether the HDAC inhibitor may restore endocrine sensitivity by enhancing ER expression.

Adaptive increase in ER and E2 hypersensitivity 

The biological mechanisms contributing resistance to ED using AIs or gonadal suppression are less well defined than those underpinning tamoxifen resistance and may in fact be quite distinct. Long-term ED can be associated with adaptive aberrant mitogenic signalling as previously described. However, as ED strategies do not involve direct interaction with ERs, none of the paradoxical agonist activities of tamoxifen would be expected. In addition, whereas acquired tamoxifen resistance has been frequently associated with relative decrease in ER expression, adaptation to ED may lead to upregulation of ERs. In vitro models of long-term ED have shown that part of the adaptive process involves an increase in ER expression and E2 hypersensitivity to very low levels of residual oestrogen [14]. This is supported by a recent clinical trial showing that women who have relapsed on an AI respond to more potent endocrine manipulation using the irreversible AI exemestane, or ER downregulation with fulvestrant [25]. If part of the adaptive process to AIs involves E2 hypersensitivity, then there may be a rationale for continued ED in combination with a novel agent, rather than switching to it. The current Study of Faslodex, Exemestane and Arimidex (SOFEA) trial will address this question. In this trial, women who have relapsed on a non-steroidal AI (anastrozole or letrozole) are randomly assigned to the irreversible steroidal AI exemestane, fulvestrant or fulvestrant in combination with anastrozole.

ER mutations

A single activating mutation in the ER gene has been identified and shown to result in constitutive ER transactivation [48]. However, screening of clinical samples has revealed that mutations occur in fewer than 1% of breast cancers and most are silent [3,49]. In addition, a significant number of naturally occurring mRNA splice variants encoding truncated ER have been identified; however, these have not been shown to modulate endocrine responsiveness in the clinical setting [49].

Altered drug metabolism and cellular clearance

Measurements of intratumoral levels of tamoxifen have shown that women with acquired resistance to tamoxifen have lower levels than sensitive controls [50]. Whether low intracellular levels of tamoxifen are attributable to decreased influx of the drug or increased efflux via a membrane pump such as p-glycoprotein has not been established. However, this mechanism is likely to be a minor contributor to tamoxifen resistance as clinical samples have consistently demonstrated that tamoxifen saturates ERs with greater than 99.9% occupancy [51]. The cytochrome P450 2D6 enzyme is required to convert tamoxifen to its more potent metabolite, endoxifen. CYP2D6 variants have been identified and bi-allelic polymorphism results in decreased levels of circulating endoxifen, fewer hot flushes and an increased risk of relapse among women treated with tamoxifen [52]. Similarly, CYP2D6 inhibitors, such as the selective serotonin reuptake inhibitor antidepressants, which are frequently used to treat postmenopausal hot flushes, also decrease endoxifen levels leading some to suggest that these agents should be avoided in women treated with tamoxifen [52,53]. Whether women with homozygous CYP2D6 variants would benefit from higher doses of tamoxifen or an alternative endocrine therapy has not been investigated.

Alterations in aromatase expression or function

In postmenopausal women, the only source of E2 is from the aromatisation of adrenal androgens. While peripheral conversion in adipose tissue contributes to measurable levels of circulating E2, local production via tumoural aromatase activity results in 10- to 20-fold higher E2 concentrations in the tumour than in plasma [54]. Variations in tumour aromatase levels could therefore contribute to responsiveness to AIs. A small study suggested that the level of intratumoral aromatase activity could predict for response to the first-generation AI aminoglutethemide [55]. However, more recent studies have shown no correlation between aromatase mRNA levels and response to AIs [56]. A number of single-nucleotide polymorphisms (SNPs) have been identified in the aromatase gene (CYP19); however, most do not translate into a clinically significant variation in circulating E2 levels [57]. One SNP has been shown in vitro to reduce affinity of the aromatase enzyme for exemestane [58]; however, there is no clinical evidence to date that genetic variations in CYP19 lead to resistance to aromatase inhibition in vivo.

Conclusion

A number of theories involving ER mutations or drug metabolism and clearance have been proposed to contribute to endocrine responsiveness. However, there is an increasing body of evidence implicating growth factor receptor and downstream kinases in both de novo and acquired endocrine resistance. The nature of the interaction between ER and mitogenic signalling probably varies over time and from one patient to another. In some models, activated growth factor-mediated signalling suppresses expression of ERs and function, raising the intriguing possibility that growth factor-targeted therapy may, over time, restore endocrine responsiveness. In other cases, ERs and growth factor signalling may interact synergistically providing the basis for combination strategies. Importantly, a significant proportion of endocrine-resistant breast cancer still depends on a functional ER. Acquired resistance can be attributed to tamoxifen agonism due to AF-1-driven transcription, or an increase in non-genomic ER activity. Degrading the receptor with fulvestrant might provide an effective strategy in this setting, while ED would be expected to abrogate ligand-dependent non-genomic activity. Comparatively little is known about the mechanisms underpinning resistance to ED; however, as more postmenopausal women are receiving first-line adjuvant treatment with AIs, this question has become increasingly relevant. Both growth factor signalling and E2 hypersensitivity have been shown to contribute and would suggest that continued ED with an AI might be a superior approach to growth factor targeting alone. A huge number of trials (Table 3) are currently exploring the benefit of various targeted agents in combination or in sequence with endocrine therapy and include biological analyses that may shed further light on the clinically relevant mechanisms of endocrine resistance.

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[61.] Polychronis A, Sinnett HD, Hadjiminas D et al. Preoperative gefinitib versus gefinitib and anastrozole in post menopausal patients with oestrogen-receptor positive and epidermal-growth-factor-receptor-positive primary breast cancer; a double- blind placebo-controlled phase II randomised trial. Lancet Oncol, 2005, 6, 383-391.

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Alexandra Leary and Stephen Johnston

Royal Marsden Hospital, London, UK

Correspondence to: Alexandra Leary, Department of Medicine, Royal Marsden Hospital, Fulham Road, London, SW3 6JJ, UK (email: [e-mail])

Table 1: Postulated mechanisms of endocrine resistance and potential
avenues for therapy

Biological mechanism                Therapeutic options / comments

Growth factor driven

Downregulation of ER expression     Sequential growth factor--ER-
  and/or function                     targeted therapy
Switch to ER-independent            Non-ER-targeted therapy
  proliferation
Enhanced genomic and non-genomic    Dual ER and growth factor
  functions of ER                     targeting

Other

ER silencing due to promoter        Demethylating agents or HDAC
  hypermethylation                    inhibitors
E2 hypersensitivity                 Continued ED plus ER downregulator
                                      or growth factor inhibitor
ER mutations                        Rare
Altered tamoxifen metabolism and    Higher tamoxifen doses in selected
  cellular clearance                  patients?
Aromatase expression and function   Relevance unknown

ER, oestrogen receptor; E2, oestradiol; HDAC, histone deacetylase; ED,
oestrogen deprivation.

Table 2: Combinations of endocrine therapies with inhibitors of
membrane receptors or downstream effectors: selected completed trials
(data from [59]).

Clinical                                    Clinical
setting          Phase     Intervention     endpoints        Reference

Combinations with EGFR inhibitor gefitinib (GEF)

MBC              II        ANA + GEF        PR=0 SD=0        [59]
Endocrine                                   ORR=61% vs       [60]
  resistant                                   48%, P=0.067
Neoadjuvant      II RCT    ANA vs ANA +     ORR=50% vs 54%
  PBC                        GEF x 16                        [61]
                             weeks
Preoperative     II RT     GEF vs GEF +
  PBC                        ANA
EGFR+ only                 x 4-6

Combinations with trastuzumab (TRAS)

HER2+ MBC        II        TRAS + LET       PR=26% SD=26%    [62]
HER2 MBC (all    III RCT   ANA vs ANA +     PFS=2.4 mo vs    [63]
  pts were IHC               TRAS             4.8 mo,
  3+ or FISH+)                                P=0.0016
                                            OS=23.9 mo vs
                                              28.5 mo,
                                              P=0.325

mTor inhibitors: everolimus (EVE) or temsirolimus (TEM)

MBC              Ib n=9    LET+EVE 1, 5     No grade 3/4     [64]
stable or                    or 10 mg         toxicities
  slowly                     daily            at 5 mg
  progressing                                 (6 pts) or
  on letrozole                                10 mg (3
                                              pts)
MBC              II n=92   Let vs LET+TEM   ORR=45% vs 33%   [65]
                             10 mg daily      vs 40%
                             vs LET+TEM       PFS=11.6 mo
                             30 mg            vs 11.5 mo
                             intermittent     vs 13.2 mo
MBC              III RCT   LET vs           ORR=24% vs 24%   [66]
                 n=992       letrozole+       SD=19% vs
                             TEM              16% PFS=9.2
                             intermittent     mo vs 9.2 mo

EGFR, epidermal growth factor receptor; MBC, metastatic breast cancer;
PBC, primary breast cancer; RCT, randomised controlled trial; RT,
randomised trial; PR, partial response; SD, stable disease; ORR,
objective response rate (PR+CR); ANA, anastrozole; LET, letrozole;
mTOR, mammalian target of rapamycin; PFS, progression-free survival;
OS, overall survival; vs, versus; +, positive; mo, months; pts,
patients; IHC, immunohistochemistry; FISH, fluorescence in situ
hybridisation.

Table 3. Endocrine combinations: selected ongoing or recently closed
trials.

                                                               NCT
Clinical        Trial                       Biological         protocol
setting         phase     Intervention      endpoints          number

Endocrine combinations

MBC             III       FUL vs EXEM vs    Serum hormone      00253422
Previously                FUL+ANA           levels
treated
with AI

MBC             III       ANA vs ANA +      Not available      00256698
1st line                  FUL

With EGFR inhibitors: gefitinib (GEF) or erlotinib (ERL)

Neoadjuvant     II        GEF x 2 wks       Tumour biomarker   00206492
PBC                       followed by       analysis at
(HER2+ only)              GEF+TAM           weeks 1, 2 and
                                            6, and surgery

Neoadjuvant     II        ANA + FUL + GEF   Tumour biomarker   00206414
PBC                                         analysis pre-
                                            and
                                            post-treatment

MBC             II RCT    TAM [+ or -]      Correlate RR to    00069290
                          GEF               HER2/A1B1 status
                                            and other
                                            biomarker
                                            studies

MBC             II RCT    ANA [+ or -]      biomarker study    00077025
                          GEF

MBC             II RCT    ANA [+ or -]      None specified     00066378
                          GEF

MBC             II        FUL + GEF         None specified     00234403

MBC             II RT     ANA + GEF vs      Identify           00057941
                          FUL + GEF         biologic
                                            predictors

MBC             II        LET + ERL         Correlation of     00179296
                                            EGFR, HER2 and
                                            pERSer118 to
                                            benefit

MBC             II RT     GEF [+ or -]      Correlate early    00080743
TAM resistant             TAM               changes on PET
                                            and in plasma
                                            DNA to response

With trastuzumab (TRAS)

HER2+ MBC       II        TRAS              Correlate          00238290
AI-resistant              monotherapy       benefit with
                          until PD          HER2 ECD at
                          followed by       baseline and
                          TRAS + LET        after treatment

HER2+ MBC       III RCT   ANA [+ or -]      None specified     00022672
                          TRAS

HER2+ MBC       IV        LET + TRAS        None specified     00171847
TRAS-naive

With lapatinib (LAP)

MBC             II        LAP + TAM         None specified     00118157
TAM resistant

MBC             III RCT   LET [+ or -]      Biomarker and      00073528
                          LAP               genetic variant
                                            analysis

With mTOR inhibitors: everolimus (EVE) and temsirolimus (TEM)

Neoadjuvant     II RCT    LET [+ or -]      None specified     00107016
PBC                       EVE

MBC             II RCT    LET [+ or -]      None specified     00061971
                          TEM

Combination with HDAC inhibitor SAHA

MBC             II        TAM+SAHA          Pre-and post       00365599
                                            treatment ER
                                            expression and
                                            histone
                                            acetylation

PBC, primary breast cancer; MBC, metastatic breast cancer; RCT,
randomised controlled trial; RT, randomised trial; RR, response rate;
ECD, extracellular domain: ANA, anastrozole; FUL, fulvestrant; EXEM,
exemestane; LET, letrozole; TAM, tamoxifen; SAHA, suberoylanalide
hydroxamic acid; PET, positron emission tomography.