Luminal breast cancer: from biology to treatment

Abstract

Oestrogen receptor (ER)-positive--or luminal--tumours represent around two-thirds of all breast cancers. Luminal breast cancer is a highly heterogeneous disease comprising different histologies, gene-expression profiles and mutational patterns, with very varied clinical courses and responses to systemic treatment. Despite adjuvant endocrine therapy and chemotherapy treatment for patients at high risk of relapse, both early and late relapses still occur, a fact that highlights the unmet medical needs of these patients. Ongoing research aims to identify those patients who can be spared adjuvant chemotherapy and who will benefit from extended adjuvant hormone therapy. This research also aims to explore the role of adjuvant bisphosphonates, to interrogate new agents for targeting minimal residual disease, and to address endocrine resistance. Data from next-generation sequencing studies have given us new insight into the biology of luminal breast cancer and, together with advances in preclinical models and the availability of newer targeted agents, have led to the testing of rationally chosen combination treatments in clinical trials. However, a major challenge will be to make sense of the large amount of patient genomic data that is becoming increasingly available. This analysis will be critical to our understanding how intertumour and intratumour heterogeneity can influence treatment response and resistance.

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Luminal breast cancer: from biology to treatment

Michail Ignatiadis and Christos Sotiriou

Abstract | Oestrogen receptor (ER)-positiveor luminaltumours represent around two-thirds of all breast cancers. Luminal breast cancer is a highly heterogeneous disease comprising different histologies, gene-expression profiles and mutational patterns, with very varied clinical courses and responses to systemic treatment. Despite adjuvant endocrine therapy and chemotherapy treatment for patients at high risk of relapse, both early and late relapses still occur, a fact that highlights the unmet medical needs of these patients. Ongoing research aims to identify those patients who can be spared adjuvant chemotherapy and who will benefit from extended adjuvant hormone therapy. This research also aims to explore the role of adjuvant bisphosphonates, to interrogate new agents for targeting minimal residual disease, and to address endocrine resistance. Data from next-generation sequencing studies have given us new insight into the biology of luminal breast cancer and, together with advances in preclinical models and the availability of newer targeted agents, have led to the testing of rationally chosen combination treatments in clinical trials. However, a major challenge will be to make sense of the large amount of patient genomic data that is becoming increasingly available. This analysis will be critical to our understanding how intertumour and intratumour heterogeneity can influence treatment response and resistance.

Ignatiadis, M. & Sotiriou, C. Nat. Rev. Clin. Oncol. 10, 494506 (2013); published online 23 July 2013; http://www.nature.com/doifinder/10.1038/nrclinonc.2013.124

Web End =doi:10.1038/nrclinonc.2013.124

Introduction

Breast cancer is the most frequently occurring cancer in women in the developed world, with oestrogen receptor (ER)-positive disease representing around two-thirds of all cases.1,2 It has long been established that these tumours are heterogeneous in terms of histology (they are mainly ductal, but also lobular, mixed ductal and lobular, cribriform, mucinous and tubular carcinomas), natural history, and response to treatment.3

Studies conducted over 10years ago using microarray technology showed that this tumour heterogeneity was also present at the gene-expression profiling level, and two main ER-positive breast cancer subtypes were identified.46 These subtypes are referred to as luminal A and luminalB and have been shown to have different gene-expression profiles, prognosis and treatment response.47

When compared to the luminal A subtype, luminal B tumours often have lower expression levels of ER or oestrogen-regulated genes, lower or no progesterone receptor (PR) expression, higher tumour grade, higher expression of proliferation-related genes, and activation of growth factor receptor signalling pathways, such as IGF-1R and PI3K/AKT/mTOR.4,7 Luminal B tumours are also considered to have lower sensitivity to endocrine treatment and higher sensitivity to chemotherapy than luminal A tumours.2,810

Several single-sample predictors1113 and subtype classification models (Box1)1416 have been developed to assign patients to molecular subtypes (including luminal A and B) with fair to substantial concordance among them.16,17 Moreover, several prognostic gene signa tures (Box1) that have been developed to improve breast cancer prognostication18,19 are able to discriminate between luminal A and luminal B tumours, and prognostic performance of these signatures is mainly driven by the inclusion of genes associated with proliferation.8,15

High concordance has been observed among these prognostic signatures and single-sample predictors.20 In

clinical practice, the use of prognostic gene signatures and single-sample predictors to define women with good prognosis luminal A tumours that can be spared adjuvant chemotherapy will rely not only on appropriate demonstration of analytical and clinical validity, but ultimately on demonstration of clinical utility.2123

Three prospective trialsTAILORx,24 RxPONDER25 and MINDACT26are evaluating the clinical utility of two such signatures. Since prospective validation requires major recourses and considerable time, another approach to accelerate patient access to prognostic gene signatures might be their approval after appropriate analytical and clinical validation and demonstration of clinical utility in retrospective analysis from at least two prospective studies.22 In such cases, the approval should be conditional; a close follow-up of the clinical outcome of patients treated using the prognostic signatures should be required (patient registries).

Department of Medical Oncology and Breast Cancer Translational Research Laboratory, Institut Jules Bordet, Universit Libre deBruxelles, 121Boulevard deWaterloo, 1000Brussels, Belgium (M. Ignatiadis, C. Sotiriou).

Correspondence to: C. Sotiriou mailto:[email protected]

Web End =christos.sotiriou@ mailto:[email protected]

Web End =bordet.be

Competing interestsC. Sotiriou is co-inventor of a gene-expression grade index patent and co-inventor of a gene module PIK3CA patent. M.Ignatiadis declares no competing interests.

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Another approach to defining luminal A and B tumours is to use standard immunohistochemical markers, including ER, PR, HER2, and the proliferation marker Ki-67. Luminal A tumours are ER and/or PR positive, with a Ki-67 level of <14%.2 However, despite ongoing international efforts to improve Ki-67 testing, including recommendations on preanalytical, analytical issues, interpretation and scoring,27 a recent Ki-67 reproducibility study involving experienced pathologists showed significant interobserver variability.28

Worryingly, when a cutoff of 13.5% was used to define Ki-67 low versus Ki-67 high tumours, around one-third of the tumours were classified differently between two participating laboratories. This discrepancy demonstrates that Ki-67 cutoff points to distinguish luminalA and luminal B tumours cannot be freely transferred between laboratories, and that local recalibration against specific clinical end points is needed.28

The breast cancer classification system has been refined as a result of combining data on gene-

expression profiling and copy-number alterations from 2,000 patients.29 Moreover, in 2012, the genomic landscape of breast cancer tumours was extensively characterized using next-generation sequencing (NGS) in the context of large-scale collaborative initiatives, such asThe International Cancer Genome Consortium3032 and

TheCancer Genome Atlas (TCGA),33 as well as efforts from major institutions.34,35 These studies have provided new avenues to advance treatment in luminal tumours,36

but have also highlighted new challenges stemming from the tremendous intertumour and intratumour h eterogeneity that was revealed.

In this Review, we focus on ER-positive/HER2-negative breast cancer, and we address clinically relevant questions. These questions include, which patients can be spared the use of adjuvant chemotherapy? Which patients benefit from extended adjuvant hormone therapy that can target tumour dormancy? What is the role of adjuvant bisphosphonates in targeting crosstalk between tumour cells and the bone micro environment? Can we target minimal residual disease and tumour- initiating cells in these tumours? How we can reverse endocrine resistance? And, finally, what are the c hallenges for drug development in luminal tumours?

Sparing adjuvant chemotherapy

In 2012, an Early Breast Cancer Trialists Collaborative Group (EBCTCG) meta-analysis showed that treatment with adjuvant chemotherapy reduces 10-year breast cancer mortality by one-third.37 Interestingly, the observed reduction in breast cancer mortality did not differ according to age or available tumour characteristics, including ER status and histological grade.37 Of

note, differences in reductions in breast cancer mortality based on gene-expression profiling data have not been estimated, because these data were not included in the EBCTCG meta-analysis. In the meta-analysis, the absolute benefit from chemotherapy was associated with the absolute risk without chemotherapy. This risk is low for women with ER-positive/HER2-negative, low

prolifer ative, node-negative disease, and current guidelines suggest that women with this disease should be treated with endocrine therapy only.2

There are commercially available genomic predictors that are designed to aid oncologists in deciding whether to administer adjuvant chemotherapy. These predictors include oncotypeDX,18 MammaPrint,19 MapQuant Dx,38 PAM50 risk of recurrence (ROR),13,39 Breast Cancer IndexSM,40 and EndoPredict;41,42 of these, oncotype DX is the most extensively validated test.18,43 The clinical utility of oncotype DX and MammaPrint is being evaluated in two prospective trials (TAILORx24 and RxPONDER25) and one prospective trial (MINDACT26,44), respectively. Patients with predefined characteristics who were enrolled in these trials were randomly assigned to receive adjuvant chemo-therapy and endocrine therapy or endocrine therapy alone (Figure1). In the TAILORx trial,24 women with ER-positive, node-negative breast cancer and a low recurrence score based on oncotype DX are treated with

Key points

Results from trials using gene-expression assays will better define the women with oestrogen receptor-positive breast cancer and 03 positive axillary lymph nodes who do not need adjuvant chemotherapy

Extended adjuvant hormone therapy might be an important strategy to target tumour dormancy in luminal tumours

The intriguing hypothesis that bisphosphonates might decrease recurrence in women with a low-oestrogen environment is supported by subgroup analyses from several prospective trials

Detecting and targeting minimal residual disease is currently being explored asa way to improve outcome for women with these tumours

Targeting the PI3K/AKT/mTOR pathway is one of the most-promising approaches to reversing endocrine resistance; the intertumour and intratumour heterogeneity of luminal breast cancer has implications for designing newtherapies

The challenge will be to identify and target epistatic gene interactions in luminal tumours and to tailor therapy based on tumour evolution in space and time

Box 1 | Glossary

Driver mutations: Mutations observed in cancer genes that have an important role in oncogenesis or cancer progression by providing clonal advantage

Epistasis: When the effect of a mutation in one gene is modified by mutation(s) inone or several other genes

Exome sequencing: Sequencing of the coding regions (exons) of the genes inagenome

Oncogene addiction: The dependency of a tumour cell on the activity of anoncogene

Passenger mutations: Mutations that do not confer clonal advantage Prognostic gene signature: Classifier assigning a breast cancer case intogood or bad prognosis based on an algorithm that relies on the expression ofapredefined gene set Single-sample predictor: Classifier assigning a breast cancer case into a molecular subtype (basal-like, HER2-enriched, luminal A and luminal B) based on similarities in gene expression between this case and molecular subtype centroids Subtype classification model: Classifier assigning a breast cancer case into a molecular subtype (basal-like, HER2-enriched, luminal A and luminal B) based onGaussian distributions of gene expression of three gene sets associated withoestrogen receptor, HER2 and proliferation Whole-genome sequencing: Sequencing of the coding and non-coding regions ofthe genome

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Add chemotherapy? (MINDACT,26 TAILORx,24 RxPONDER25)

Target tumourhost interaction? Bisphosphonates, denosumab (NATAN,87 D-CARE89)

Target minimal residual disease/tumour-initiating cells? Trastuzumab in HER2-negative breast cancer (NSABP-B47,101 Treat-CTC100)

Target secondary endocrine resistance? Endocrine therapy +/ everolimus (UNIRAD122)

Target tumour dormancy, late relapses? (LEAD,55 SALSA,57 DATA,58 SOLE,60

NSABP-B-42,56 MA17R59)

Target primary endocrine resistance? Endocrine therapy +/ everolimus (SWOG-S1207121)

Time from diagnosis

Figure 1 | Research questions and respective ongoing studies in patients with oestrogen receptor-positive early stage breast cancer. Example clinical trials in brackets.

endocrine therapy only, women with a high recurrence score with endocrine therapy and chemotherapy, and women with an inter mediate recurrence score are randomly assigned between endocrine therapy alone versus endocrine therapy plus chemotherapy. The RxPONDER trial interrogates whether women with ER-positive/ HER2-negative breast cancer, 13 infiltrated axillary nodes and recurrence score 25 can be spared adjuvant chemotherapy.25 The MINDACT trial aims to more-accurately assign risk categories using the molecular classifier MammaPrint compared to the computer tool Adjuvant! Online45 that summarizes risk of relapse based on standard clinicopathological characteristics, and thus reduce by 1020% the use of adjuvant chemotherapy in women with 03 positive nodes.26 Although the results of these trials are eagerly awaited, oncotype DX is already being broadly used in clinical practice in the USA. The use of these genomic predictors in Europe and other parts of the world is currently low, but we expect that this will rapidly increase in the future depending on the results of the above trials.

Women with ER-positive, HER2-negative, low- proliferative (that is, luminal A) tumours defined using genomic predictors such as oncotype DX have not only been shown to have a lower risk of recurrence when treated with hormone therapy, but also to derive less benefit from chemotherapy.18,43,46,47 Indeed, in retrospective analyses of prospective trials, benefit from adjuvant CMF (cyclophosphamide, methotrexate and 5-fluorouracil) in women with node- negative breast cancer46 and adjuvant CAF (cyclophosphamide, adriamycin, 5-fluorouracil) in women with node-positive disease47 was largely confined to women with ER-positive tumours who also had a high oncotype DX recurrence score. Recently, NGS studies have suggested mechanisms that could explain why luminal A tumours are not sensitive to chemotherapy.30,33,36 These tumours frequently exhibit abrogation of stress-induced apoptotic kinase JNK signalling, either through loss-of-function mutations in the MAP3K1 or MAP2K4 genes, or though activating mutations in the genes in the PI3K/AKT pathway, and this abrogation has been associated with reduced response to chemotherapy compared with patients with normal JNK signalling.48

Extended adjuvant hormone therapy

The EBCTCG meta-analysis included 10,645 patients with ER-positive disease and the data showed that 5years of adjuvant treatment with the ER antagonist tamoxifen reduced breast cancer mortality by one-third throughout the first 15years.49 These results led to 5years of adjuvant tamoxifen becoming the standard of care for premenopausal women. For postmenopausal women, a meta-analysis that assessed randomized trials of aroma tase inhibitors (such as letrozole, anastrozole and exemestane) versus tamoxifen as initial monotherapy for 5years showed that aromatase inhibitor treatment reduced recurrence (absolute benefit 3% at 5.8years of follow-up), but not breast cancer mortality, whereas 23years of tamoxifen followed by aromatase inhibitor treatment reduced both breast cancer recurrence and mortality (absolute benefit 3% and 0.7%, respectively, at 3.9years of follow-up) compared to tamoxifen alone.50

It is established that in ER-positive tumours, late recurrences can occur up to 20years or more after diagnosis despite adjuvant endocrine treatment. Several trials have demonstrated the value of extended use of aroma tase inhibitors (letrozole, anastrozole and exemestane) to decrease late relapses after 5years of adjuvant tamoxifen.5154 Ongoing trials are testing the value of extended adjuvant hormone therapy with aromatase inhibitors after an initial 5years of tamoxifen followed by aromatase inhibitor treatment in postmenopausal women (LEAD,55 NSABP-B42,56 SALSA57 and DATA58), or after 5years of treatment with an aromatase inhibitor (NSABP-B4256 and SALSA57), or after 10years of treatment, including tamoxifen followed by an aromatase inhibitor (MA17R59). The SOLE trial60 is addressing the question of continuous versus i ntermittent extended letrozole (Figure1).61

In the ATLAS trial, women with breast cancer who have completed 5years of tamoxifen treatment were randomly assigned to either continue with another 5years of tamoxifen or to stop tamoxifen.62 In the 6,846 women with ER-positive disease, the investigators observed a reduction in breast cancer recurrence and mortality for women taking the additional 5years of tamoxifen, mainly from year 10 after diagnosis (recurrence rate ratio 0.75, 95% CI 0.620.90 and breast cancer mortality rate

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ratio of 0.71, 95% CI 0.580.88 for 10years). Overall, 10years of tamoxifen treatment was associated with an increased risk of endometrial cancer and pulmonary embolism, but no increase in stroke, and a decreased incidence of ischaemic heart disease. Other smaller trials that assessed long-term treatment with tamoxifen had previously reported conflicting results.6365 If the results of the ATLAS trial are confirmed by ongoing trials (for example, ATTOM66), 10years of adjuvant tamoxifen will become an interesting option for premenopausal women.

Extended hormone therapy in ER-positive breast cancer effectively decreases late recurrences; thus, providing an example of how tumour dormancy can be targeted in the clinic.61 However, we do not yet have the optimal tools to select the patients who will benefit from extended adjuvant hormone therapy.

Large tumour size and positive lymph-node status are associated with increased late relapse rates in postmenopausal women who have received 5years of tamoxifen.67

Moreover, it has been suggested that women who were premenopausal at diagnosis,68 whose tumours measured >2 cm,53 and who had node-positive53,54 and ER-positive/ PR-positive disease52,69 derived more benefit from extended aromatase inhibitor treatment after 5years of tamoxifen. It should be noted that in the ATLAS trial, there was no significant heterogeneity in risk reduction after 10years of tamoxifen according to patient character istics, including tumour size and nodal status.62

Nevertheless, currently, most postmenopausal women at high risk of relapse are treated upfront with an aroma-tase inhibitor, and women with less-poor prognosis are treated with 23years of tamoxifen followed by 23years of aromatase inhibitor treatment.

Recently, data from the ABCSG-8 and the TransATAC studies showed that classic clinicopathological characteristics and three gene signatures (Breast Cancer IndexSM,

EndoPredict and PAM50-ROR) are independent predictors of late recurrence, with most information provided by the clinicopathological characteristics.7073 On the one hand, using clinicopathological characteristics and these signatures, the investigators were able to predict which women with low risk of late relapse could be spared the toxicity of extended hormone therapy.7073 On the other hand, positive lymph-node status is already used to select women who might benefit from extended hormone therapy. We believe that validation of these signatures in completed or ongoing trials of extended adjuvant hormone therapy, as well as a better understanding of the biology of dormant disseminated tumour cells,7476

will help us better tailor extended adjuvant endocrine treatment in the future.

Adjuvant bisphosphonates

Preclinical studies have suggested that bisphosphonates have antitumour effects, mainly by interrupting a vicious cycle of growth factors and cytokines between occult tumour cells and osteoclasts in the bone marrow micro-environment (Figure2).7779 Moreover, the bisphospho-nate zoledronic acid reduced the incidence of bone marrow disseminated tumour cells in women receiving

chemotherapy.80,81 Subgroup analyses from several trials raise the hypothesis that bisphosphonates might decrease recurrences in women with early stage breast cancer in the presence of a low-oestrogen environment (either premenopausal women undergoing chemical ovarian ablation or postmenopausal women).79

The AZURE trial showed that the addition of 5years of zoledronic acid to standard systemic treatment did not improve disease-free survival (DFS) or overall survival in the overall population, but improved DFS (hazard ratio [HR] = 0.75, 95% CI 0.590.96, P = 0.02) and overall survival (HR = 0.74, 95% CI 0.550.98, P = 0.04) in women who were postmenopausal at least 5years before study entry.82 The ABCSG-12 trial showed that, in premenopausal women, zoledronic acid every 6months for 3years improved DFS (HR = 0.64, 95% CI 0.460.91, P = 0.01) when added to hormone therapy.83 In an updated analysis there was an overall survival benefit from the addition of zoledronic acid, mainly in women over 40years of age.84

In the ZO-FAST study, immediate use of zoledronic acid resulted in increased DFS compared to delayed zoledronic acid treatment in postmenopausal women with early stage breast cancer who were receiving letrozole (HR = 0.66, P = 0.03),85 and in the NSABP B34 trial, the addition of the oral bisphosphonate clodronate improved the recurrence-free interval only in patients who were over 50years old (HR = 0.75, 95% CI 0.570.99).86

A formal meta-analysis of available or ongoing trials (such as, NATAN87) is warranted to investigate interactions between benefit from bisphosphonates and factors such as age and menopausal status. Although

Tumour cells

RANKL antibody

PTHrP

Bone-derived growth factors

(TGF-~, FGFs, IGFs, PDGF, BMPs), Ca2+

OPG RANKL

Osteoclast precursor

Osteoblast

Bisphosphonates

PTHrP IL-8 IL-11

Osteoclast

Bone matrix

Bone resorption

Figure 2 | Vicious cycle of occult tumour cells and osteoclasts in breast cancer.7779 Bone marrow disseminated tumour cells that have escaped dormancy produce factors (for example, PTHrP) that promote the formation and activation of osteoclasts. Osteoclast activation results in bone resorption and thus release of factors by bone matrix, such as TGF-, which stimulate tumour cell proliferation.

Bisphosphonates and denosumab can interrupt this vicious cycle. Abbreviations: BMPs, bone morphogenetic proteins; FGFs, fibroblast growth factors; IGFs, insulin-like growth factors; IL, interleukin; OPG, osteoprotegerin; PDGF, platelet derived growth factor; PTHrP, parathyroid hormone-related peptide; RANKL, receptor activator of nuclear factor-B ligand; TGF-, transforming growth factor-.

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Table 1 | Studies addressing endocrine resistance in ER-positive tumours*

Trial Population (n)

Treatment Median PFS (months)

Median OS (months)

Optimize endocrine therapy administration

CONFIRM102 ER+ (736) Fulvestrant 500 mg (loading-dose regimen) vs250 mg

6.5vs 5.5,P = 0.006

25.1vs 22.8, P = 0.91

Combine different endocrine therapy

SWOG-S0226103 ER+ (694) Anastrozole + fulvestrant (250 mg + loading-dose regimen) vs anastrozole

15vs 13.5,P = 0.007

47.7vs 41.3, P = 0.05

FACT104 ER+ (514) Anastrozole + fulvestrant (250 mg + loading dose regimen) vs anastrozole

10.8vs 10.2,P = 0.91

37.8vs38.2, P = 1.0

Combine endocrine therapy with anti-HER2 agents

EGF30008108 ER+/HER2+ (219)

Letrozole + lapatinib vsletrozole

8.2vs 3, P = 0.019

33.3vs 32.3, P = 0.113

TANDEM109 ER+/HER2+ (207)

Anastrozole + trastuzumab vs anastrozole

4.8vs2.4, P = 0.0016

28.5vs 23.9, P = 0.325

Combine endocrine therapy with CDK inhibitor

NCT01740427148 ER+/

HER2(165)

Letrozole + PD 0332991vs letrozole

26.2vs 7.5,P <0.001

Combine endocrine therapy with agents targeting the PI3K/AKT/mTOR pathway

BOLERO II117 ER+/

HER2(724)

Exemestane + everolimus vs exemestane

6.9vs2.8, P <0.001

TamRAD/ GENICO118

ER+/ HER2(111)

Tamoxifen + everolimus vs tamoxifen

8.6vs4.5, P = 0.002

32.9vs not reached,P = 0.007

HORIZON119 ER+

(1,112)

Letrozole + temsirolimus vs letrozole

8.9vs 9, P = 0.25

*These studies were selected as the major published studies addressing endocrine resistance according to the authors opinion. Abbreviations: AI, aromatase inhibitors; ER, oestrogen receptor; NR, not reported; OS, overall survival; PFS, progression-free survival.

the use of bisphosphonates is not currently approved for reducing the risk of relapse in patients with early stage breast cancer, we believe that oncologists will increasingly consider these agents in postmenopausal women with significant bone loss when treated with aromatase inhibitors.88 Finally, results from ongoing trials in the early disease setting using newer agents targeting osteoclast formation and survival, such as the RANK-ligand inhibitor denosumab (for example, the D-CARE trial89)

are awaited (Figure1).

MRD and tumour initiating cells

Another approach to improve outcome in patients with ER-positive/HER2-negative breast cancer might be to target minimal residual disease (MRD). The presence of circulating tumour cells in the blood of a patient after surgery and chemotherapy are considered to be surrogates of treatment-resistant MRD, and technological advances have enabled the standardized detection of these cells.9092 Recent studies have suggested that circu lating tumour cell detection using the CellSearch system has adverse prognostic value in patients with early stage breast cancer.9395 Moreover, both clinical9698 and pre clinical data99 suggest that benefit from adjuvant treatment with

the anti-HER2 antibody trastuzumab might not be confined to HER2-amplified tumours only. Using cell lines, mouse models and human tumours, investigators from Max Wichas laboratory showed that HER2 expression drives self-renewal of the tumour-initiating cells in luminal breast cancer. Since treatment-resistant tumour initiating cells might be responsible for tumour relapses and given the important role of HER2 for the self-renewal of tumour-initiating cells in luminal breast cancer, targeting HER2 might decrease tumourrelapses.99 Moreover, trastuzumab blockedtumour growth when administered to mice immediately after luminal tumour inoculation, whereas it was not effective in established luminal breast cancer mouse xenografts.99 Based on these pre-clinical and clinical data, the European Organisation for Research and Treatment of Cancer launched the TREAT-CTC phaseII trial.100 This trial is being run under the Breast International Group umbrella and will investigate whether trastuzumab can eliminate chemotherapy- resistant circulating tumour cells in women with HER2 non-amplified breast cancer and whether trastuzumab can improve clinical outcome in these women. The NSABP is also running a phaseIII trial in the USA addressing a similar question.101

Strategies to overcome endocrine resistance

In metastatic luminal breast cancer, denovo or acquired resistance to endocrine therapy eventually develops, and several approaches have been used to reverse it (Table1 and Figures1 and 3).

Optimize endocrine treatment administration

One approach to the problem of endocrine therapy resistance is to optimize the schedule and dose of endocrine therapies. This method was assessed in postmenopausal patients after disease progression on prior endocrine therapy in the CONFIRM trial that led to the approval of the fulvestrant 500 mg loading-dose regimen by the FDA in 2010.102 Another approach is to combine endocrine therapies. The SWOG S0226 trial demonstrated that anastrozole plus fulvestrant was superior to anastrozole alone in terms of progression-free survival (PFS) and overall survival.103 However, the FACT and the SOFEA trials failed to show improved outcome when assessing the same combination,104,105 and combining endocrine treatment (anastrozole and tamoxifen) was not shown to be effective in early stage disease.106 Beyond optimizing endocrine treatment administration, it is much more important to identify and target crosstalk between ER and other signalling pathways (Figure3).

Crosstalk between ER and HER2

Crosstalk between ER and growth factor receptors, such as HER2, has been implicated in endocrine resistance.107

In a randomized, placebo controlled phaseIII trial, the addition of lapatinib to letrozole resulted in prolonged PFS in patients with ER-positive/HER2-positive metastatic breast cancer (HR = 0.71, 95% CI 0.530.96, P = 0.019), but not in ER-positive/HER2-negative metastatic breast cancer.108 In addition, the TANDEM

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PI3K/AKT/mTOR PIK3CA mutation 40%

PTEN mutation/loss 18% INPP4B loss 12% AKT1 mutation 3%

Pan-PI3K inhibitors Isoform-speci[f_i]c PI3K inhibitors

Dual PI3KmTOR inhibitors AKT inhibitors mTOR inhibitors

CDK4/611q13 ampli[f_i]cation 37%

CCND1 ampli[f_i]cation 40% CDK4 ampli[f_i]cation 19% CDKN1B, CDKN2A,

CDKN2B loss 11% RB1 mutation 1%

CDK4/6 inhibitors

p53MDM2 TP53 mutation 22%

MDM2 gain 22%

MDM2 inhibitors

FGFR1 8p1112 ampli[f_i]cation 10%

FGFR1 ampli[f_i]cation 10%

Tyrosine kinase inhibitors FGFR monoclonal antibodies FGF-trap

HDACMLL3 mutation 7%

Hypermethylated Luminal B subtype 8%

HDAC inhibitors

RTK

PI3K

AKT

mTORC1

PTEN

INPP4B

4EBP1

Transcription

Cyclin D1

CDK4/6

FGFR1

PI3K

AKT

RAS

MEK

MDM2

p53

mTORC2

S6K

E2F mTOR MAPK

No transcription

Figure 3 | Genomic and epigenomic landscape, pathways and drugs to reverse endocrine resistance in oestrogen receptor-positive breast cancer. Data on genomic and epigenomic landscape are derived from next-generation sequencing studies3035 and from a study combining gene-expression profiling and copy number aberration data.29 Pink denotes gene or protein activation and blue denotes gene or protein suppression in breast cancer. Abbreviations: HDAC, histone deacetylase; RTK, receptor tyrosine kinase.

phaseIII trial showed improved PFS when trastuzumab was added to anastrozole in patients with ER-positive/ HER2-positive metastatic breast cancer (HR = 0.63, 95% CI 0.470.84, P = 0.0016).109

ER and PI3K/AKT/mTOR crosstalk

Crosstalk between the ER and the PI3K/AKT/mTOR signalling pathways has been associated with endocrine resistance.107 The mTORC1 complex integrates signals from growth factors (for example, insulin-like growth factor), the presence of nutrients (amino acids), energy signals through the AMP-activated kinase, and various stress signals (such as hypoxia and DNA damage) to promote cell growth and division by increasing mRNA translation and inhibiting autophagy.110 Genes from the PI3K/AKT/mTOR pathway are the most frequently mutated in luminal breast cancer; PI3K mutations are the most prevalent mutations and are identified in around 40% of cases.30,33,35,111

Interestingly, the majority of PI3K mutations in ER-positive tumours have been identified in the alpha catalytic subunit (PIK3CA).36 Moreover, preclinical data have demonstrated a synthetic lethal interaction when PIK3CA inhibition is combined with oestrogen deprivation in breast cancer cell lines,112 suggesting that this is a very promising approach to be tested in the clinic. However, gene-expression profiling and reverse-phase protein array data have shown that PIK3CA mutations in non-metastatic, ER-positive breast cancer are not associated with AKT/S6K pathway activation,33,113,114

as observed in cases that have PTEN loss in triple- negative breast cancer. Therefore, whether the approach of combined PI3K inhibition and endocrine treatment is effective in early stage breast cancer remains to be d emonstrated in future clinical studies.

Currently, there are numerous types of agents t argeting the PI3K/AKT/mTOR pathway, including the following: pan-PI3K inhibitors (such as, BKM-120, GDC-0941 and XL147); isoform-specific PI3K inhibitors (for example, the PI3K inhibitors BYL719, GDC-0032 and INK111; the PI3K inhibitors GSK2636771, TGX-221 and KIN-193; and the PI3K inhibitor CAL-101); dual PI3KmTOR inhibitors (such as, BEZ235, XL765, GDC-0890 and GSK1059615); AKT inhibitors (including ATP-competitive inhibitors such as GSK690693 and GDC-0068 or allosteric inhibitors such as MK-2206); and mTOR inhibitors (including allosteric inhibitors such as the rapalogues sirolimus, temsirolimus, ridaforolimus and everolimus or mTOR catalytic inhibitors such as the INK128, AZD8055, AZD2014 and OSI-027).115,116

The most promising results for targeting the crosstalk between ER and the PI3K/AKT/mTOR pathway have come from combining everolimus with endocrine therapy in patients with metastatic breast cancer in the BOLEROII phaseIII clinical trial.117 This trial randomly assigned 724 postmenopausal women with ER-positive/HER2-negative breast cancer that was resistant to anastrozole or letrozole to either exemestane plus everolimus or exemestane plus placebo in a 2:1 ratio. Of note, 84% of the women participating in the study had previously been sensitive to endocrine therapy. Everolimus improved median PFS when added to exemestane (HR = 0.36; 95% CI 0.270.47; P <0.001). However, the increased efficacy came at the cost of increased toxicity, namely higher incidence of grade 3 or 4 stomatitis, hyperglycaemia, fatigue, anaemia and pneumonitis. Based on this study, the FDA and European Medicine Agency (EMA) approved everolimus in combination with exemestane for the treatment of women with ER-positive/HER2-negative breast cancer with recurrence or progression after receiving letrozole or anastrozole.

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In another open-label phaseII study (Genico/ TamRAD), 111 patients with ER-positive/HER2-negative breast cancer that was resistant to aromatase inhibitors were randomly assigned to receive tamoxifen plus everolimus or tamoxifen alone.118 In the intent-to-treat population, time to progression was higher in the combination arm (HR = 0.54, 95% CI 0.360.81). In an exploratory subgroup analysis, benefit was mostly confined to patients with secondary endocrine resistance.

In contrast to the above studies, HORIZON, a placebo-controlled trial of letrozole plus temsirolimus in 1,112 postmenopausal women with aromatase inhibitor-naive advanced-stage or metastatic breast cancer showed no difference in PFS from the addition of temsirolimus to letrozole.119 The differences in results between HORIZON and the other studies can be attributed either to differences in patient populations (BOLERO II and TamRAD included women with aromatase inhibitor-resistant disease whereas HORIZON included women with aromatase inhibitor-naive disease) or to differences in the rapalogue used (molecule, dose, and schedule).

It is likely that the consistent benefit of everolimus when added to endocrine therapy in aromatase inhibitor- resistant disease is due to the higher prevalence of PI3K/ AKT/mTOR pathway activation in this setting than in aromatase inhibitor-naive disease. Interestingly, however, everolimus was also effective in previously untreated patients with non-metastatic breast cancer, showing markedly increased anti-proliferative response at day 15 when added to letrozole compared to letrozole alone.120

These results suggest that benefit from targeting mTOR might not be confined only to women with aromatase inhibitor-resistant disease.120 Two placebo-controlled phaseIII trials in patients with early stage breast cancer are now evaluating the role of everolimus (Figure1) in reversing primary (randomization before starting endocrine treatment121) or secondary endocrine resistance (randomization if relapse free after 2 to 3years of e ndocrine treatment122).

With the exception of everolimus, the pan-PI3K inhibitor BKM120 is the most advanced agent in development targeting the PI3K pathway with the aim of reversing endocrine resistance. Two ongoing phaseIII trials are evaluating BKM120 in combination with fulvestrant in women with ER-positive/HER2-negative breast cancer that is either refractory to aromatase inhibitors (BELLE2 trial123) or who have disease progression on or after treatment with mTOR inhibitors and endocrine therapy (BELLE 3 trial124). Another phaseII trial is evaluating the combination of fulvestrant with either the pan-PI3K inhibitor GDC-0941 or the dual PI3KmTOR inhibitorGDC-0980.125

Several phaseI and IIIII trials are evaluating endocrine therapy with different agents targeting the PI3K/ AKT/mTOR pathway, such as the pan-PI3K inhibitor XL147;126 the dual PI3KmTOR inhibitors XL765126 and BEZ235;127 the isoform specific PI3K inhibitors BYL719128,129 and GDC-0032;130 the allosteric

AKT inhibitor MK-2206;131 and the catalytic mTOR inhibitorAZD2014.132

Currently, there are no validated biomarkers predicting response or resistance to PI3K/AKT/mTOR pathway inhibitors. Initial efforts to correlate pathway activation with antitumour effect when pathway inhibitors were used gave inconclusive results, often because mutations were analysed only in a few genes (mainly PIK3CA and PTEN).133,134 Rapalogues are the most clinically studied agents; therefore, considerable effort has been invested in identifying biomarkers for these drugs. Mutations in the PI3K/AKT/mTOR axis (PIK3CA or AKT mutations, PTEN loss) or mutations in the LKB1/AMPK/mTORC1 axis (LKB1, TSC1 mutations) have been shown to predict sensitivity to everolimus in various tumour types.110,135,136

Two studies have shown that mTOR activation defined using either a PIK3CA mutation-related gene signature137 or expression of 4EBP1, a downstream target of mTORC1,138 is associated with sensitivity to everolimus and endocrine therapy in breast cancer. Feedback loops acting though AKT activation have been suggested to promote resistance to everolimus.139,140 To overcome

these feedback loops, rapalogues have been combined with either IGF1 inhibitors (phaseI study of ridaforolimus plus dalotuzumab141 or MEK inhibitors).142 Another approach is to use second-generation inhibitors that target the kinase domain of mTOR (dualkinase inhibitors of mTOR and PI3K or mTOR-selective kinaseinhibitors).142

Several questions have not been answered concerning the use of agents targeting the PI3K/AKT/mTOR axis in luminal breast cancer. These questions include, which class of agent and drug combinations should be priori-tized in a given genetic context? For example, for patients with metastatic ER-positive disease with PIK3CA mutations, should we prioritize a combination of hormone therapy with a PI3K inhibitor, or a pan-PI3K inhibitor, or a dual PI3KmTOR inhibitor? Do these agents differ in terms of activity in this setting? Does the activity of an agent targeting the PI3K/AKT/mTOR pathway depend on effective pathway inhibition? If so, how can we optimally assess this inhibition? What are the mecha nisms of resistance to these agents, and what are the optimal combinations to overcome them? Recent unbiased efforts using NGS, such as the those employed in the BOLEROII trial143 or in the phaseI BYL719 trial,144 hold promise to uncover the genetic landscape leading to response or resistance to these agents.

CDK4 and CDK6

The cyclin D1 complex (cyclin D1 bound to CDK4 or CDK6) and the cyclin ECDK2 complex phosphory-late the retinoblastoma (Rb) protein, preventing it from inactivating the E2F transcription factor, thus leading to cell cycle progression from G1 to S phase.145,146 In luminal tumours, inhibition of the Rb protein is mediated through CCND1 (the gene coding for cyclin D1) or CDK4 amplification or overexpression, or loss of the endogenous CDK inhibitors.33,36 The TCGA

showed amplification of CCND1 in 29% of patients with luminal A tumours and in 58% of patients with luminal B tumours.33 Moreover, the METABRIC Group

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identified a high-risk ER-positive 11q1314 cis-acting subgroup (IntClust 2) with particularly poor outcome and who exhibited alterations in cell-cycle-related genes, i ncluding amplification of CCND1.29

From these data, one could hypothesize that inhibitors of CDK4 and CDK6 would be more active in luminalB tumours or tumours with CCND1 amplification. Preclinical studies have shown that PD0332991, a highly selective oral inhibitor of CDK4 and CDK6, was active in luminal-like breast cancer cell lines.147 In

linewith these preclinical results, a study of 165 patients with ER-positive breast cancer showed that the addition of PD0332991 to letrozole resulted in improved PFS when compared to letrozole alone (HR = 0.37, 95% CI0.210.63, P <0.001).148 However, an exploratory analysis showed that, beyond ER-positive status, CCND1 amplifi cation or loss of the cyclin-dependent kinase inhibitor2A (p16) did not seem to help to better select patients for treatment benefit, highlighting the challenges we face in biomarker research.

Taking these results into consideration, a placebo- controlled phaseIII clinical trial is currently evaluating the role of adding PD0332991 to letrozole as first-line treatment in ER-positive/HER2-negative metastatic breast cancer.149 In addition to PD0332991, two other dual CDK4 and CDK6 inhibitors are currently under going testing in phaseI trials (LY2835219150 and LEE011151). Although we believe that these agents should mainly be tested in luminal B tumours, selection based on CCND1 amplification or overexpression is currently not justified.

MDM2p53 interaction

The TCGA showed that up to two-thirds of luminalB tumours may have defective p53 pathways, either through TP53 mutations or MDM2 amplification.33

Defects in the p53 pathway have been linked to endocrine resistance in NGS studies of neoadjuvant aromatase inhibitors.35 Targeting the MDM2p53 interaction using MDM2 inhibitors has been shown to increase apoptosis in cancer cells and might be a potentially powerful approach to reversing endocrine resistance in tumours with MDM2 amplification.36

FGFR pathway aberrations

FGFR signalling through FGF ligand dependent or indepen dent activation has been implicated in onco-genesis, angiogenesis and treatment resistance in various tumour types.152,153 FGFR1 amplification has been found in up to 10% of breast cancer tumours, and was associated with poor prognosis in ER-positive breast cancer.154

FGFR1 amplification has been associated with FGFR1 mRNA overexpression and high proliferative tumours (up to 27% of luminal B tumours).155 Moreover, pre-clinical data suggest that FGFR1 amplification drives anchorage-independent proliferation and resistance to endocrinetherapy.155

Approaches to targeting FGFR in various tumour types include tyrosine kinase inhibitors (TKIs), monoclonal FGFR antibodies, and FGF-trapping molecules, with TKIs being more clinically advanced. In a phaseII

study, dovitinib, a non-selective FGFR TKI, showed activity in the subgroup of patients with ER-positive/HER2-negative/FGFR1 amplified breast cancer.156 Based on these results, a randomized phaseII trial of fulvestrant with or without dovitinib in postmenopausal ER-positive/ HER2-negative breast cancer is ongoing with molecular screening for FGFR1, FGFR2 and FGFR3 amplification incorporated into its design.157 Another non-selective FGFR TKI, lucitanib, has shown significant activity in FGFR1 amplified breast cancer.158 Selective FGFR TKIs are at an earlier phase of development. We believe that further progress in breast cancer should consider toxicity issues and patient selection based on FGFR amplification.

Histone deacetylases

Several lines of evidence have suggested that histone deacetylase inhibitors combined with endocrine treatment might be a promising approach to reversing endocrine resistance in a subset of luminal tumours. First, several sequencing studies have shown that the MLL3 gene was mutated in about 8% of luminal tumours30,33,35

and that this gene belongs to a family that encodes histone methyltransferases regulating, among other aspects, ER-expression. Second, the TCGA has shown that a subset of luminal B tumours exhibits a hyper-methylation phenotype and a low frequency of PIK3CA, MAP3K1 and MAP2K4 mutations.33 Third, a phaseII trial has indicated that the histone deacetylase inhibitor entinostat improves PFS and overall survival when added to exemestane treatment in women with ER-positive metastatic breast cancer who have progressive disease on non-steroidal aromatase inhibitors.159

Challenges for drug development

Putting mutations in context

Successful drug development has been achieved by target ing oncogene addiction, that is, by targeting single oncogenes that were essential for tumour cell survival. Examples include HER2 amplification in HER2-positive breast cancer160 and the BCRABL kinase in chronic myeloid leukaemia.161 As the majority of newly identified cancer genes in ER-positive breast cancer have loss-of-function mutations,30,33 that traditionally were considered difficult to target, understanding the interaction between cancer genes (epistasis) in tumour evolution will be critical for the design of new therapeutic approaches in luminal tumours.162,163 An example is the synthetic lethal interaction between BRCA1 or BRCA2 mutations and poly(ADP-ribose) polymerase (PARP) inhibition in triple-negative breast cancer.164

Moreover, several mutations have been shown to have a different impact on clinical outcome, depending on the disease context. For example, PIK3CA mutations have been associated with poor prognosis in patients with metastatic HER2-positive breast cancer receiving trastuzumab or trastuzumab and pertuzumab-based treatment,165,166 but with good prognosis in patients with non-metastatic ER-positive breast cancer treated with adjuvant endocrine therapy (Table2).111,114 In patients

with early stage breast cancer who participated in the

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Table 2 | PIK3CA mutations and prognosis in breast cancer

Study Disease Treatment PIK3CA mutation(n)/ total (n), %

Clinical outcome

Cleopatra166 Metastatic HER2-positive

DTP vs DTPl 176/557, 32% PIK3CA wild type better PFS (HR = 0.63, 95%CI 0.490.80, P <0.0001)

FINHER2168 Early stage D-FEC vs N-FEC tamoxifen 687/174, 25% RFS (HR = 0.85, 95% CI 0.571.28, P = 0.44)

Team111 Early stage, ER-positive

1,702/4,272, 40% PIK3CA mutant better DRFI (HR = 0.76, 95%CI 0.630.91, P = 0.003)

Abbreviations: D-FEC, docetaxel, 5-fluoruracil, epirubicin and cyclophosphamide; DRFI, distant relapse-free interval; DTP, docetaxel, trastuzumab and pertuzumab; DTPl, docetaxel, trastuzumab and placebo; ER, oestrogen receptor; HR, hazard ratio; N-FEC, navelbine, 5-fluoruracil, epirubicin and cyclophosphamide; PFS, progression-free survival; RFS, recurrence-free survival.

Tamoxifen + exemestane vs exemestane

FINHER trial,167 PIK3CA mutations were associated with small, ER-positive and grade 1 tumours, but they were not prognostic for clinical outcome in the entire p opulation analysed.168

It is possible that as the tumour evolves, an initial driver mutation might no longer drive tumour progression and viceversa. The PIK3CA mutation example shows that a single mutation in a cancer gene should not be considered on its own, but in the context of the dynamic evolution of the genomic landscape of a tumour. This complexity might have major implications for drug development. For example, the activity of an isoform-specific PI3K inhibitor in an ER-positive, PIK3CA-mutant breast cancer might depend on disease stage and ultimately on the genetic context in which the specific mutation is present.

Tumour heterogeneity

In addition to gene expression, the luminal tumours are also heterogeneous in terms of copy-number aberrations (CNAs) and several regions, such as the 8p1112, 11q13 and 20q13, are amplified in a subset of them.169171

In a recent study, genomic and transcriptomic data with long-term clinical follow-up from 2,000 patients were used to derive a new breast cancer classification with 10 breast cancer subtypes, called integrative clusters (IntClust 110).29 Of these 10 clusters, eight included luminal tumours. Among them, there was a high-risk, ER-positive 11q1314 cis-acting subgroup (IntClust 2) with particularly poor outcome exhibiting alterations in cell-cycle-related genes, and a subgroup devoid of CNAs (IntClust 4) that was characterized by increased l ymphocytic infiltration and good prognosis.

The advent of NGS has helped to better capture the intertumour and intratumour heterogeneity of these tumours. In a study that characterized 79 ER-positive and 21 ER-negative breast cancers using NGS, driver somatic CNAs or point mutations were identified in 40 cancer genes, but the number of mutated cancer genes in an individual cancer ranged from one to six, with 73 different combinations of mutated cancer genes.30 The majority of cancer genes (33 of 40) were mutated in <10% of the tumours. However, mutations in different cancer genes could be grouped into pathways and, for example, abrogation of the JUN kinase signalling pathway was suggested to occur in around half of patients with breast cancer.30 This study demonstrates the substantial genetic

diversity observed in breast cancer. The TCGA has recently provided compelling evidence of the extensive genetic and epigenetic heterogeneity within the major breast cancer molecular subtypes.33

Whole-genome sequencing of 21 breast tumours shed light onto the challenge of intratumour heterogeneity and mutational processes that operate during breast cancer development. The investigators of this study showed that every breast tumour had a dominant tumour subclone that represented more than 50% of the tumour cells, but also had other minor subclones with private mutations, suggesting considerable intratumour heterogeneity.32

Furthermore, the investigators identified five mutational signatures (patterns of mutations), with one of them being predominant in some ER-positive tumours. Interestingly, each tumour had more than one mutational signature, and different mutational signatures could be operative at different times during tumour development.31 Furthermore, single-cell sequencing revealed intratumour heterogeneity that was not appreciated by tumour mass sequencing.172 Further studies are needed to understand the processes that lead to these signatures. The association of intertumour, intratumour heterogeneity and treatment resistance is therefore an issue of ongoing and intense investigation.

A major challenge to resolve is how to move from making therapeutic decisions based on a primary tumour snapshot to capturing tumour heterogeneity both in space and time (Figure1). Several studies have shown discordance between primary and metastatic tumours with respect to commonly used biomarkers such as ER and HER2.173175 Currently, the biopsy of metastatic lesions is recommended when feasible to confirm the diagnosis of metastatic breast cancer and to re-evaluate these markers.176 NGS approaches, however, have suggested that a single tumour biopsy might significantly underestimate the tumour genomic landscape.32,177 Therefore, sequencing both the primary tumour and metastatic disease (metastatic biopsies, circulating tumour cells or circulating DNA),178181 together with functional imaging,182 is suggested for evaluation of tumour evolution.

Currently, two different approaches using NGS are used to evaluate tumour heterogeneity in the context of clinical research. The first is a targeted approach exemplified by Foundation Medicine in which formalin-fixed, paraffin-embedded (FFPE) tumours are screened using NGS for mutations in a panel of cancer genes. In this scenario, a

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patient report is provided with information about drugs and/or ongoing trials for actionable mutations.183 The

second is an unbiased approach that involves performing whole-genome sequencing, exome sequencing of the tumour and normal DNA and transcriptome sequencing (RNA-seq), with the available results being discussed by experts in an oncogenomic tumour board.184

In addition to the logistics associated with different prescreening strategies, not to mention financial and ethical issues,185 a major issue is the clinical interpretation of the available data. Indeed, in tumour boards, it will be challenging to assess whether identified m utationseven in known cancer genesare actionable, what the roles of actionable but subclonal mutations are, and, more importantly, whether the mutations identified in the particular genetic context can actually predict treatment response or resistance. Patient-derived xenograftsif availablemight complement the clinical approaches.186 Despite the challenges they pose, such initiatives are expected to alter the practice of oncology and to lead us ever closer to precision cancer medicine.

Conclusions

In luminal breast cancer, exciting clinical and translational research has recently been reported. Trials using commercially available gene signatures aim to better define which women can be safely spared adjuvant chemo therapy. Recent data suggest that extended treatment with adjuvant tamoxifen is improving clinical

outcome, but more work is needed to identify women who will really benefit from such hormone therapy. Subgroup analyses from several trials suggest that bisphosphonates reduce breast cancer recurrence in women with a low oestrogen environment. The role of detecting and targeting MRD as a way to improve clinical outcome for women with these tumours is currently being explored. A major challenge will be to address endocrine resistance, with the most exciting results coming from targeting the PI3K/AKT/mTOR axis. Finally, studies using NGS have revealed that luminal breast cancer is an extremely heterogeneous disease consisting of various combinations of driver mutations. The roles of some of these mutations (such as PIK3CA) might differ depending on the disease context. Todays major challenges include identifying and targeting epi-static gene interactions and tailoring therapy based on tumour evolution in space and time.

Review criteria

PubMed and MEDLINE were searched for articles in English published before May 2013 using the terms breast cancer, luminal, oestrogen receptor, endocrine resistance, genomics, next generation sequencing and liquid biopsy. Clinicaltrials.gov was searched for clinical trials in luminal breast cancer. Abstracts of the annual meetings of ASCO (20102012), AACR (20102013) and San Antonio Breast Cancer Symposium (20102012) were also considered.

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AcknowledgementsM. Ignatiadis and C. Sotiriou received grants from the Breast Cancer Research Foundation (BCRF), Fonds de la recherche scientifique (FNRS), Les Amis de Bordet, and the MEDIC foundation.

Author contributionsBoth authors researched data for the article and made a substantial contribution to discussions of the content. M. Ignatiadis wrote the article and both authors edited the manuscript before submission.

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