Human Cancer BiologySee related article by Segal and Dowsett, p. 1724
Emergence of Constitutively Active Estrogen Receptor-aMutations in Pretreated Advanced Estrogen Receptor–Positive Breast Cancer
Rinath Jeselsohn1,2,3, Roman Yelensky6, Gilles Buchwalter1,2,3, Garrett Frampton6, Funda Meric-Bernstam7,Ana Maria Gonzalez-Angulo8, Jaime Ferrer-Lozano9, Jose A. Perez-Fidalgo10, Massimo Cristofanilli11,HenryG�omez12, Carlos L. Arteaga13, JenniferGiltnane13, JustinM.Balko13,Maureen T.Cronin6,Mirna Jarosz6,James Sun6, Matthew Hawryluk6, Doron Lipson6, Geoff Otto6, Jeffrey S. Ross6, Addie Dvir14,Lior Soussan-Gutman14, Ido Wolf15, Tamar Rubinek15, Lauren Gilmore4, Stuart Schnitt4, Steven E. Come5,Lajos Pusztai16, Philip Stephens6, Myles Brown1,2, and Vincent A. Miller6
AbstractPurpose: We undertook this study to determine the prevalence of estrogen receptor (ER) a (ESR1)
mutations throughout the natural history of hormone-dependent breast cancer and to delineate the
functional roles of the most commonly detected alterations.
Experimental Design: We studied a total of 249 tumor specimens from 208 patients. The specimens
include 134 ER-positive (ERþ/HER2�) and, as controls, 115 ER-negative (ER�) tumors. The ERþ samples
consist of 58 primary breast cancers and 76 metastatic samples. All tumors were sequenced to high unique
coverage using next-generation sequencing targeting the coding sequence of the estrogen receptor and an
additional 182 cancer-related genes.
Results: Recurring somatic mutations in codons 537 and 538 within the ligand-binding domain of ER
were detected in ERþ metastatic disease. Overall, the frequency of these mutations was 12% [9/76; 95%
confidence interval (CI), 6%–21%] in metastatic tumors and in a subgroup of patients who received an
average of 7 lines of treatment the frequency was 20% (5/25; 95%CI, 7%–41%). These mutations were not
detected in primary or treatment-na€�ve ERþ cancer or in any stage of ER� disease. Functional studies in cell
line models demonstrate that these mutations render estrogen receptor constitutive activity and confer
partial resistance to currently available endocrine treatments.
Conclusions: In this study, we show evidence for the temporal selection of functional ESR1mutations as
potential drivers of endocrine resistance during the progression of ERþ breast cancer.Clin Cancer Res; 20(7);
1757–67. �2014 AACR.
IntroductionEstrogen receptora (ERa) is a nuclear transcription factor
that drives proliferation and growth of luminal-type breastcancers and is the target of endocrine therapies in thisdisease. Although such antiestrogen treatments are highlyeffective, a major clinical limitation is the development of
acquired resistance to these therapies. Despite the fact thatapproximately 50% of patients with ERþ breast cancerbenefit from adjuvant endocrine treatment, a significantfraction of them recur with metastatic disease (1–3). Fur-thermore, all patients with ERþ metastatic breast cancerwho respond to endocrine treatment will eventually
Authors' Affiliations: 1Center for Functional Cancer Epigenetics,2Department of Medical Oncology, Dana-Farber Cancer Institute;3Department of Medicine, Brigham and Women's Hospital; Depart-ments of 4Pathology and 5Breast Medical Oncology Program, BethIsrael Deaconess Medical Center, Harvard Medical School, Boston;6Foundation Medicine, Cambridge, Massachusetts; 7Departments ofInvestigational Cancer Therapeutics, Surgical Oncology, and 8SystemsBiology and Breast Medical Oncology, The University of MD AndersonCancer Center, Houston, Texas; 9Fundacion de Investigacion INCLIVA-Institute for Health Research; 10Departments of Hematology-Oncology,Hospital Clinico Universitario de Valencia, Valencia, Spain; 11JeffersonBreast Care Center, Kimmel Cancer Center, Thomas Jefferson Univer-sity, Philadelphia, Pennsylvania; 12Instituto Nacional de EnfermedadesNeopl�asicas (INEN), Lima, Per�u; 13Breast Cancer Program, Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville,Tennessee; 14Teva Pharmaceuticals, Petach Tikva; 15Oncology Divi-
sion, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel; and 16Section ofBreast Medical Oncology, Yale School of Medicine, New Haven, Con-necticut
Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).
R. Jeselsohn, R. Yelensky, G. Buchwalter, and G. Frampton contributedequally to this work.
Corresponding Author: Myles Brown, Dana-Farber Cancer Institute, 450Brookline Ave, D730, Boston, MA 02215. Phone: 611-631-3948; Fax: 611-581-8501; E-mail: [email protected]
progress with antiestrogen-resistant, hormone-indepen-dent disease.
In most cases of acquired endocrine resistance, ERcontinues to be expressed. Proposed mechanisms of resis-tance include activation of growth factor receptor, cellsurvival and cell-cycle signaling pathways, as well as stress-induced pathways (4, 5). In addition, aberrant expressionof ER coactivators and corepressors has been implicated inendocrine resistance (6, 7). ER mutations have also beenexplored as a potential mechanism of drug resistance andinitially it was postulated that loss-of-function mutationscould contribute to resistance. However, several preclin-ical studies analyzed the effects of point mutations in ERand found a number of mutations that can actuallyenhance ER function similar to androgen receptor muta-tions in castrate-resistant prostate cancer (1–10). Onesuch functional mutation in ESR1 leads to the substitutionof tyrosine by serine or alanine at position 537 in theligand-binding domain (LBD) and results in ligand-inde-pendent ER transcriptional activity that does not respondto endocrine manipulation (11–13). Despite these pre-clinical findings, the frequency of ERmutations in primarybreast cancers was found to be extremely rare. In one earlystudy of 118 ERþ and 70 ER� primary breast cancers, onlytwo ESR1 mutations of unknown significance were foundand both were in ER� breast cancers (14). More recently,several studies describing next-generation sequencing(NGS) in hundreds of primary breast cancer samplesdetected multiple significant somatic alterations, but nonewere detected in ESR1 (11–17). Two small studies con-ducted in the 1990s detected mutations in the ER LBDwith a frequency of 1% to 10% in metastatic breast cancersamples, suggesting that the frequency of ER mutations inmetastatic disease may be higher (18, 19). However, thesestudies were small and had not been validated with moresensitive sequencing technology and/or integrated withclinical correlations. Therefore, in this study, we sought tocomprehensively study the frequency and functional sig-
nificance of ER mutations in both primary and metastaticbreast cancer using targeted NGS.
Materials and MethodsBreast cancer human tissue specimens and clinical data
Paraffin-embedded blocks from formalin-fixed ERþ/HER2� primary and in-breast local recurrence/metastaticbreast cancer specimens were obtained from the PathologyDepartments of three institutions (MD Anderson CancerCenter, Houston, TX; Beth Israel Deaconess Medical Center,Boston,MA; andHospital ClUnicoUniversitario inValencia,Spain), including samples from the NCT00780676 trial (aphase II trial for patients with advanced metastatic breastcancer treated with dasatinib or selumetinib based on theexpression profile of their metastatic tumor). ERþ/HER2þ
tumors were excluded as they are known to be resistant toendocrine therapy.
All samples were stained for ER, progesterone receptor(PR), and HER2 and reviewed by a pathologist at eachinstitution but were not centrally reviewed. ER positivitywas defined as more than 1% of cells with strong staining.Anti-ERa antibodies were from Thermo Scientific and Ven-tana (clone SP1). HER2 was defined as positive either byimmunohistochemistry of 3þ or FISH HER2/CEP17 ratioof greater than 2.2. The anti-HER2 antibody was purchasedfromDakoor Ventana. Clinical datawere also collected. Forcomparison, additional ER� (either ER�/HER2� or ER�/HER2þ) primary breast cancer samples were obtained (MDAnderson Cancer Center and Instituto Nacional de Enfer-medades Neopl�asicas), as well as a small number of speci-mens from ER� metastatic disease (MD Anderson CancerCenter). Brief descriptions of all studied cohorts can befound in Table 1, and more detailed information about theERþ metastatic cohort is in Supplementary Table S1. Alltissue collections were done with the approval of the corre-sponding institutional review boards.
Sequencing and primary sequence data analysisTumor tissue sections of 40 mm were macrodissected
using hematoxylin and eosinophil staining to identify areasof at least 80% cellularity. Genomic DNA was extractedusing the Maxwell 16 FFPE Plus LEV DNA Purification Kit(Promega) and quantified using a PicoGreen fluorescenceassay (Invitrogen); �50 ng and up to 200 ng of extractedDNA was sheared to approximately 101–400 bp by soni-cation, followed by end repair, dA addition and ligation ofindexed, Illumina sequencing adaptors. Sequencing librar-ies were hybridization captured using RNA-based baits(Agilent), targeting a total of 3,320 exons of 182 cancer-related genes (most commonly altered in cancer, fromhttp://www.sanger.ac.uk/genetics/CGP/cosmic/) plus 37introns from 14 genes often rearranged in cancer (Supple-mentary Table S1). Paired-end sequencing (49� 49 cycles)was performed using the HiSeq2000 (Illumina). Sequencedata from gDNA were mapped to the reference humangenome (hg19) using the BWA aligner (20). PCR duplicateread removal and sequencemetric collectionwasperformedusing Picard (http://picard.sourceforge.net) and SAMtools
Translational RelevanceThis study demonstrates an overall 12% frequency of
somatic ESR1mutations in metastatic ERþ breast cancerand absence of detectable mutations in the primarytumors. In preclinical models, we show that these muta-tions render constitutive activity and relative resistanceto endocrine therapy. Taken together, these results sug-gest clonal evolution as the mechanism of resistance.Thus, we propose that these mutations are biomarkersand drivers of endocrine resistance in ERþ metastaticbreast cancer. As such, these mutations can assist inclinical decision making as disease progresses, under-scoring the value of serial biopsies and profiling ofmetastatic recurrences. In addition, further studies arewarranted to investigate therapeutic strategies to circum-vent the resistance conferred by these mutations.
Jeselsohn et al.
Clin Cancer Res; 20(7) April 1, 2014 Clinical Cancer Research1758
(21). Local alignment optimization was performed usingGATK (22).
Genomic alteration detectionBase substitution detection was performed using a Bayes-
ian methodology, which allows detection of novel somaticmutations at lowmutation annotation format and increasedsensitivity for mutations at hotspot sites (23) through theincorporation of tissue-specific prior expectation: P (Muta-tionpresentjReaddata "R")¼ P(Frequency ofmutation"F">0jR)/ 1 -P(RjF¼ 0)P (F¼ 0),whereP(RjF) is evaluatedwithamultinomialdistributionof theobservedallele countsusingempirically observed error rates and P(F ¼ 0) is the priorexpectation of mutation in the tumor type. To detect indels,de novo local assembly in each targeted exon was performedusing thede-Bruijn approach(24).Candidate calls arefilteredusing a series of quality metrics, including strand bias, readlocation bias, and a custom database of sequencing artifactsderived from normal controls. Germline alterations are iden-tified and filtered using dbSNP (version 135, www.ncbi.nlm.nih.gov/projects/SNP/) and subsequently annotated forknown and likely somatic mutations using the COSMICdatabase (version 62, http://cancer.sanger.ac.uk/cancergen-ome/projects/cosmic/). Detection of copy number altera-tions was performed by obtaining a log-ratio profile of thesample by normalizing the sequence coverage obtained at allexons against a process-matched normal control. The profileis segmented and interpreted using allele frequencies ofapproximately 1,800 additional genome-wide SNPs to esti-mate tumor purity and copy number based on establishedmethods (21–27) by fitting parameters of the equation
copy lrseg � N log2p�Csegþ 1�pð Þ�2
p�tumorploidyþ 1�pð Þ�2� �
, where lrseg andCseg
are numbers at each segment and sample purity, respectively.Focal amplifications are called at segments with �6 copies(1–8 in high aneuploidy samples) and homozygous dele-tions at 0 copies, in samples with purity >20%.
Cell and tissue culture293T andMCF7 cells (purchased from the American Type
CultureCollection and early passage used)weremaintained
in Dulbecco’s Modified Eagle Medium supplemented with10% FBS and penicillin/streptomycin. Hormone depletionwas carried out for 48 hours using phenol red–free DMEM(Cellgro) þ 10% charcoal dextran-treated FBS (OmegaScientific) and was followed by estradiol (E2, 10 nmol/L)stimulation. Transient transfections were accomplishedusing Lipofectamine 2000 (Life Technologies) as per themanufacturer’s protocol.
Luciferase assaysLuciferase reporter assay system (Promega) was used to
monitor luciferase activity in 293T cells as per the manu-facturer’s recommendations, using a single tube lumin-ometer (BD Monolight 2010). Briefly, 1 � 105 293T cellswere plated in 6-well plate in hormone-depleted medium.Transient transfections were accomplished using Lipofecta-mine 2000 (Life Technologies) with the following vectors:pcDNA-wt/mut ER (0.1 mg/well), ERE-TK-Luc (2 copies ofEstrogen Response Element upstream Luciferase reporter,minimal TK promoter, 1 mg/well), pCMV-bGal (internalcontrol normalization vector, 0.1 mg/well), pcDNA3.1(empty vector, 0.8 mg/well). Forty-eight hours after trans-fection, cells were treated with E2 or vehicle. For the doseresponse studies, doses ranged between 100 and 1,500nmol/L for the 4-hydroxytamoxifen (Sigma-Aldrich, cata-log # H7904) and between 50 and 2,500 nmol/L forfulvestrant (AstraZeneca). All transfection studies were per-formed in triplicates and luciferase results are reported asrelative light units (RLU) and normalized with b-galacto-sidase activity using theMammalianb-gal assay kit (ThermoScientific).
Site-directed mutagenesisThe GeneArt Site-Directed mutagenesis system (Life
Technologies) was used to generate Y537N, Y537C, andD538G mutations within ER LBD. Wild-type ER expres-sion vector (pcDNA-ER) was used as a template withthe following mutagenesis primers: Y537N: Forward, 50-AACGTGGTGCCCCTCAATGACCTGCTGCTGGAGA T-30
LMþ Metastases from patients with advanced ERþ disease that were heavily pretreatedbefore biopsy (participants in "Personalized Treatment Selection for MetastaticBreast Cancer" trial NCT00780676)
25 7
EMþ Metastases from patients with early metastatic ERþ disease 51 1–2Pþ Primary ERþ tumors 58 NAM� Metastases from patients with ER� disease 11 NAP� Primary ER� primary breast cancer disease 104 NA
Abbreviations: EMþ, early metastatic ERþ disease; LMþ, late metastatic ERþ breast cancer; M�, ER� metastatic disease; Pþ, ERþ
primary breast cancer; and P�, ER� primary breast cancer.aIncluding endocrine treatments and chemotherapy regimens.
ESR1-Activating Mutations in Advanced Breast Cancer
www.aacrjournals.org Clin Cancer Res; 20(7) April 1, 2014 1759
Western blot and real-time PCRThe following antibodies were used for ER (sc-543, Santa
Cruz Biotechnology) and b-Actin (A5441, Sigma) proteindetection. For mRNA level measurement, total RNA wasprepared using TRIzol reagent (Life Technologies), andcDNAwere synthesized using a high-capacity cDNA reversetranscription kit (Applied Biosystems). The following pri-mers were used for GREB1, pS2/TFF1, PR, CA12, b-ActinmRNAs detection, using ABI 7300 Real-time PCR system incombination with Power SYBR Green PCRMaster Mix (LifeTechnologies):
Forward: 50-GTG GTG TCC ATT TGG CTT TT-30, Reverse:50-GTG TCG CAA GTG TCC AGA GA-30.
ResultsSequencing studies
To examine the prevalence and clinical implications of ERmutations in human breast cancer, we assembled a datasetof 249 specimens, obtained from208 patients, representingboth ERþ/HER2� and ER� disease (Table 1). The cohortsselected enabled the analysis of the different stages of breastcancer, ranging from diagnostic biopsies of early breastcancers to samples from metastatic recurrences used forbiomarker assessment before enrollment in clinical trials.DNA sequencing was performed for 3,230 exons of 182cancer-related genes plus 37 introns of 14 genes commonlyfused on indexed hybridization-captured libraries to anaverage unique coverage of >500�.
Across the 249 specimens, a total of 16 ESR1 pointmutations (substitutions or indels) and 2 instances of locusfocal amplification were observed. Although matched nor-mal specimens were unavailable for definitive somaticstatus determination in our study, a computational assess-ment of variant allele frequencies supported the germlinehypothesis for 4 of 16 observed events (see methods,Supplementary Table S1). Of the remaining 12 somaticvariants (Table 2), 9 (75%) occurred at the known recurrentmutation sites in the ER LBD, codons 537/538, while therest were elsewhere in the LBD (Fig. 1A).
Focusing on the recurrent codon 537/538 LBD muta-tions, we observed a striking enrichment of these variants inERþ metastatic disease (Fig. 1B): Of the 76 ERþ metastaticspecimens profiled, the recurring ER LBD mutations werefound in 9 (12%) cases. The specific mutations include:Y537N (33%), D538G (33%), Y537S (22%), and Y537C(11%). The prevalence was even higher with a frequency of20% (5/25 patients) in heavily pretreated patients whoreceived an average of seven lines of treatment, includingat least two endocrine treatments formetastatic disease. Themutations were not found in any of the 58 ERþ primarytreatment-na€�ve tumors or the 115 ER� tumors.
Our study included 37 matched primary and metastaticpairs. In two of these pairs, EM8 and EM11 the pointmutations Y537C and D538G were detected in the meta-static samples but not in the matching untreated primarytumors. For onepatientwith amutation (sample EM43),wewere able to sequence tissue from an earlier metastaticspecimen. Both biopsies were from the samemetastatic sitebut were attained at two time points from a patient who didnot receive adjuvant hormonal treatment. The first meta-static lesion, without a detectable ESR1 mutation, wasobtained before the initiation of tamoxifen and the secondone, found to have a Y537C mutation, was obtained at thetime of disease progression after 8 years of treatment.Finally, in 3 specimens, codon 537/538 mutations exhib-ited allele frequencies consistent with subclonal cell popu-lations. Collectively, these findings suggest a clonal selec-tion of an endocrine-resistant phenotype.
We then broadened our analysis to the genomic contextof ER alterations by examining the full spectrumof genomicalterations in primary andmetastatic ERþdisease. The list ofthe 182 genes that were sequenced can be found in Sup-plementary Table S3. All 25 advanced metastatic, as well as46 earlymetastatic andprimary, specimenswere consideredin this analysis. A similar analysis of the other 63 ERþ
specimens has been presented previously (28). ER altera-tionswere notmutually exclusivewith anyother commonlyaltered gene in ERþ breast cancer (P53, PIK3CA, CCND1,MYC, FGFR1, MCL1), although HER2 copy gains could notbe assessed in our dataset as clinical ERþ/HER2þ cases wereexcluded. Nonetheless, we believe more samples will beneeded to verify this observation (Supplementary Fig. S1).Of the most frequently altered genes (altered in >5 sam-ples), all but ESR1 alterations displayed similar frequen-cies across primary and metastatic specimens, suggestinga role for aberrant ER in the development of recurrentdisease (Fig. 2).
Functional studiesTo delineate the functional roles of the mutations in
codons 537/538 found in metastatic breast cancer patientsamples, we performed site-directedmutagenesis to preparethree ER expression constructs containing 2 different aminoacid substitutions at position 537 (Y537C and Y537N) andone amino acid substitution at positionD538 (D538G). AllER mutations were confirmed by sequencing. The consti-tutive activity of the Y537N and Y537S mutations has been
Jeselsohn et al.
Clin Cancer Res; 20(7) April 1, 2014 Clinical Cancer Research1760
establishedpreviously (11, 19). To assess the transcriptionalactivity of the Y537C, Y537N, and D538G mutants wetransiently transfected ER� 293T cells with wild-type (WT)ER or the mutant ER constructs and an estrogen-responsivepromoter-reporter construct, ERE-TK-Luc (Fig. 3). Cellswere treated with either control vehicle or estradiol (E2)and luciferase activity was measured. As expected, the
WT-ER construct exhibited activity onlywithE2 stimulation.In contrast, all three ER mutants possessed constitutiveactivity in the absence of E2 stimulation, whereas the addi-tion of E2 did not increase the reporter activity significantly.In addition, both the ligand and nonligand–dependentactivity in the mutants was higher than the ligand-stimu-lated activity of the WT-ER. We next treated the cells withincreasing doses of 4-hydroxytamoxifen or fulvestrant withor without E2. Cells expressing the WT-ER responded to 4-hydroxytamoxifen and fulvestrant with a significant anddose-dependent reduction in luciferase activity. The ERmutants also responded to 4-hydroxytamoxifen and fulves-trant in a dose-dependent manner; however, themutant ERdisplayed a significantly reduced response to all doses ofantiestrogens except for the highest dose of 4-hydroxyta-moxifen. Mutant ER exhibited decreased response to 4-hydroxytamoxifen and fulvestrant andhigher doses of theseantiestrogens were required to achieve the level of inhibi-tion seen in the WT receptor. To confirm the decreasedresponse of the mutant ER to fulvestrant, we examined ERdegradation after 24 hours with increasing doses of fulves-trant. As we expected, mutant ER degradation comparedwith WT-ER was less pronounced at all doses of fulvestranttested (Fig. 3C). In addition, since it has been shown thatendocrine-resistant breast cancer can respond to estrogentreatment (29, 30), we tested the response of the Y537NandD538G mutants to a wide range of E2 levels but did notdetect a significant change in the luciferase activity (Sup-plementary Fig. S2). These results suggest that the ERmutants would be relatively resistant to established clinicaldoses of tamoxifen and fulvestrant as well as estrogentreatment and higher doses of SERMS or newer selectiveestrogen receptor degraders (SERDS) with improved phar-macokinetics may be required to inhibit mutant ER activity.
To study the transcriptional activity of the ER mutant inERþ breast cancer cells, we transfected a WT-ER constructand the Y537N construct in MCF7 cells. WT andmutant ERsignaling was measured by determining the expressionlevels of ER regulated transcripts with or without E2. Asshown in Fig. 4A, PR, GREB1, CA12, and PS2 transcriptlevels were comparable between the WT and ER-mutant infull media, whereas in E2-deprived conditions these ER-regulated genes are induced by the Y537N mutant but notthe WT-ER. In addition, when we examined cell prolifera-tion in estrogen-deprived conditions, which mimic aroma-tase inhibitor treatment, as well as treatment with 4-hydro-xytamoxifen or fulvestrant, we found a significant growthadvantage for the Y537N mutant (Fig. 4B). These resultsdemonstrate that cells expressing the Y537Nmutant exhibitconstitutive transcriptional activity of ER target genes andan abrogated growth inhibitory response to E2 deprivation,tamoxifen, or fulvestrant treatment.
DiscussionThe advances in sequencing technologies over the past
few years have led to a new paradigm in the understandingof the mutational landscape of breast cancer and tumor
Figure 1. Location of the ER mutations and frequencies per cohort.A, diagram of ER with detected predicted somatic point mutationsdesignated with a circle at the representative protein position. �, amutation found in a primary ER� breast cancer. B, frequency of the 538/537 substitution mutations and ER amplifications per cohort in ERþ
tumors showing an increase in frequency with the progression andincrease in number of treatment lines for ERþ breast cancer. Each barrepresents the percentage of patients with a mutation and the differentcolors within the bars represent the frequency of the indicated alteration.In the EMþ cohort one patient had both an ER amplification and a Y537Smutation. AF1, activation function 1; AF2, activation function 2; EMþ,early metastatic ERþ disease; LMþ, late metastatic ERþ breast cancer.
Figure 2. Genetic alteration in primary versus metastatic breast cancer.The frequencies of the common genomic alterations found in our cohortscomparing primary tumors versus metastatic samples shows asignificant increase in theESR1alterations inmetastatic samples.P valuecalculated using the Fisher exact test.
Jeselsohn et al.
Clin Cancer Res; 20(7) April 1, 2014 Clinical Cancer Research1762
heterogeneity. In this study, we performed targeted NGS,which enables high levels of sequencing coverage to detectlow frequency mutations within limited cellular popula-tions, in order to study ERmutations in breast cancer. In thisstudy, which is an international effort, we assembled a largesample set of breast cancers comprised primarily of ERþ
/HER2� metastatic samples and associated treatment his-tories as well as ERþ primary tumors and control ER�
tumors. The majority of the ERþ metastatic samples were
obtained from patients who received at least one form ofendocrine treatment, to focus on endocrine-resistanttumors.
In this report, we detected in 11 of the 76 (14%) meta-static tumors ESR1 mutations in the LBD. Nine of thesemutations are substitution mutations affecting Y537 andD538. In addition, we identified two mutations, 344insCand E380Q, that were not described in the literature pre-viously and further studies are needed to test their
Figure 3. Analysis of the transcriptional activity of the recurring mutant ER and dose response to tamoxifen and fulvestrant. A, transient transfection of 293Tcells with WT or mutant ER expression vectors in addition to ERE-TK-Luc reporter vector. ER expression levels were determined to be equivalent byWestern blot analysis. b-Actin expression level is shown as an internal loading control (left). Luciferase activity comparison of transfected 293T cells thentreated for 24 hours with 10 nmol/L estrogen (E2) or vehicle (ethanol) for 24 hours. Experiments were done in triplicates and repeated three times. Datarepresentmean�SD and are normalized by b-galactosidase internal control activity (left). B, luciferase activity comparison of 293 T cells transfectedwithWTormutant ER expression vectors in addition to ERE-TK-Luc reporter vector, then treatedwith vehicle (ethanol) or indicated dose of E2 and increasing doses of4-hydroxytamoxifen (4-OHT) or fulvestrant (Fulv) for 24 hours. Response to 4-OHT and fulvestrant is normalized to the response to E2 stimulation. Datarepresent mean � SD and are normalized by b-galactosidase internal control activity. An unpaired two-tailed t test was used to examine the statisticaldifferences between the WT and Y537Nmutant, and P values are shown. C, R levels determined by immunoblotting 24 hours after treatment with increasingdoses of fulvestrant.
ESR1-Activating Mutations in Advanced Breast Cancer
www.aacrjournals.org Clin Cancer Res; 20(7) April 1, 2014 1763
functional significance. In line with our data, other groupshave recently identified the Y537 and D538 mutations andadditional other mutations (31–34). The mutationsdetected in these studies are clustered in the LBD in asubstantial number of metastatic ERþ cancers Among thesethree studies, the largest cohorts consisted of 36 and 44metastatic tumors with a mutation frequency of 25% and11%, respectively, supporting the significance of thesemutations in advanced disease. We did not detect LBDESR1mutations in 58 primary ERþ tumors and is consistentwith the findings of The Cancer Genome Atlas ofmore than450 cases (35). In contrast, LBDmutations were detected in3%of the primary tumors from the BOLERO2 study, where
patients with ERþ metastatic breast cancer that had pro-gressed in the metastatic setting were randomized to exe-mestane � the TORC1 inhibitor everolimus (32, 36). Thisdiscrepancy may be due to the fact that the primary tumorsin the BOLERO2 represent a selective group of patients whoall developed resistance to endocrine treatment, amongthem approximately 20% developed resistance early afteradjuvant hormonal adjuvant treatment. Thus, the frequen-cy of the recurrent ESR1 LBD mutations in primary diseaseremains anopen and important question, as the existence ofthesemutations in early disease has important implicationsfor the selection of adjuvant treatment for thousands ofpatients annually.
Figure 4. Transcriptional activity and growth of mutant ER in a breast cancer cell line. A, transient transfection of MCF7 cells with empty vector (EV), wt,or mutant Y537N ER expression vectors, then grown in complete medium (gray bars), hormone-depleted medium (white bars), or hormone-depletedmediumwith 10 nmol/L E2 (black bars). RelativemRNA levels of ER target genesGREB1 andPR (left) and pS2/TFF1 andCA12 (right) were determined by real-time PCR. Bars, fold change� SD of two independent experiments. B, MCF7 cells were transfected with WT or Y537N ER. Cells were grown in E2-deprivedconditions alone, with 4-OHT or fulvestrant and monitored for 5 days. Histograms depict cell count at day 5. Unpaired two-tailed t test was used toexamine the statistical difference andP value is shown. C,Western blot analysis of ER levels inMCF7 cells after transfection of eitherWT-ERor Y537NmutantER showing similar expression levels.
Jeselsohn et al.
Clin Cancer Res; 20(7) April 1, 2014 Clinical Cancer Research1764
The recurring LBD mutations in positions Y537 andD538 are at the start of helix 12, a highly conserved a-helixthat undergoes conformational changes during ER activa-tion. Therefore, it is not surprising that mutations in thesepositions would have functional implications. Similar toprevious studies that focused on the Y537N, Y537S, andD538A mutations (11, 19, 37), our functional studiesindicate that the Y537N, Y537C, and D538G mutationslead to ligand-independent activity that is relatively resis-tant to tamoxifen, fulvestrant, and estrogen deprivation.Prior studies led by Katzenellenbogen and colleagues haveshed light on the mechanisms of these findings by demon-strating that the ERmutants that confer constitutive activityadopt a conformation that resembles the WT ligand-boundER and the Y537Smutant has a decreased affinity to E2 withincreased binding to coactivators (12, 38). The mutant ERaffinity to tamoxifen and fulvestrant has not been tested;however, we hypothesize that it is also reduced and togetherwith increased coactivator binding leads to the relativeresistance to these drugs. Our studies show that higherdoses of tamoxifen or fulvestrant are able to partiallydecrease the mutant transcriptional activity, suggesting thepossibility that higher doses of these agents could be astrategy to circumvent this relative resistance. This mightexplain in part the results of the CONFIRM trial in which ahigher dose of fulvestrant improved progression-free sur-vival (39). Further experiments to understand the detailedmechanism of the endocrine resistance of these mutantsand test this hypothesis are under way.Previous studies in xenograft models of MCF7 cells dem-
onstrated the emergence of a D351Y LBD mutation in atamoxifen-resistant and stimulated tumor (40, 41). In thisstudy, we analyzed tumor specimens obtained at differentstages of ERþ breast cancer, including primary treatment-na€�ve primary disease, local recurrent and/or metastaticdisease with an average of two lines of treatment for met-astatic disease, and metastatic disease biopsied after anaverageof seven treatments. The frequencies of the recurringER-LBDmutations in these three cohorts were 0%, 8%, and20%, respectively, demonstrating a correlation between theincrease in the frequency of these mutations and tumorprogression. In addition, in a case from which serial biop-sies were available, we were able to detect the emergence ofthe LBD mutation as the patient developed resistance totamoxifen treatment. Taken together, these findings suggesta temporal evolution of ERmutations with emergence of anendocrine-resistant phenotype.In our study, we detected ESR1 amplifications in a pri-
mary and metastatic tumor. Multiple studies have investi-gated the frequency of ESR1 amplifications in ERþ primarybreast cancer though the data have been controversial. Twolarge studies found ESR1 amplifications in approximately20% of cancers, which correlated with an increasedresponse to tamoxifen, whereas in other studies, amplifica-tionswere detected in just 1% to3%of tumors (41–45).Ourstudy is consistent with the latter studies as we observed anESR1 amplification rate of 1.7% (1/58) in primary ERþ
tumors. Similarly, the frequency of amplifications in met-
astatic samples was also very low (1.3%, 1/76). This is thefirst study to report the frequency of ESR1 amplifications inmetastatic breast cancer.
In summary, our study demonstrates an overall 14%frequency of somatic ESR1 mutations in a large cohort ofmetastatic ERþ breast cancers though the retrospectivenature of our study limits our ability to precisely determinethe frequency. The absence of detectable mutations in theprimary tumors suggests clonal evolution as themechanismof resistance. Thus, we propose that these mutations are agenomic mechanism of endocrine resistance. As such, thedetection of these mutations can assist in clinical decisionmaking as disease progresses, underscoring the value ofserial biopsies and profiling of metastatic recurrences. Inaddition, since the frequencies of these mutations aresubstantial when sensitive testing methods are used inpatients with ERþ breast cancer, studies to identify alter-native dosing schedules of currently approved antiestro-gens and novel therapeutics that can overcome this resis-tance are warranted.
Disclosure of Potential Conflicts of InterestR. Yelensky, M.T. Cronin, M. Jarosz, M. Hawryluk, and D. Lipson have
ownership interest (including patents) in Foundation Medicine. G. Framp-ton andP. Stephens are employees of andhave ownership interest (includingpatents) in Foundation Medicine. M. Cristofanilli is a consultant/advisoryboardmember for Cynvenio. J. Sun is an employee of FoundationMedicine.J.S. Ross is an employee of, has a commercial research grant from, and hasownership interest (including patents) in Foundation Medicine Inc. A. Dviris an employee of Teva pharmaceuticals. L. Pusztai has received a commercialresearch grant from Foundation Medicine. M. Brown has received a com-mercial research grant from and is a consultant/advisory board member forNovartis Pharmaceuticals.Nopotential conflicts of interest were disclosed bythe other authors.
Authors' ContributionsConception and design: R. Jeselsohn, R. Yelensky, G. Buchwalter, A.M.Gonzalez-Angulo, M.T. Cronin, J.S. Ross, L. Soussan-Gutman, P. Stephens,M. Brown, V.A. MillerDevelopment of methodology: R. Jeselsohn, R. Yelensky, G. Buchwalter,G. Frampton, J. Ferrer-Lozano, M.T. Cronin, M. Jarosz, G. Otto, J.S. Ross,V.A. MillerAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): R. Jeselsohn, G. Buchwalter, F. Meric-Bernstam,A.M. Gonzalez-Angulo, J. Ferrer-Lozano, A. Perez-Fidalgo, M. Cristofanilli,H. G�omez, C.L. Arteaga, J.M. Balko, M. Hawryluk, J.S. Ross, A. Dvir,L. Gilmore, S. Schnitt, L. Pusztai, V.A. MillerAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): R. Jeselsohn, R. Yelensky, G. Buchwalter,G.M. Frampton, F. Meric-Bernstam, A.M. Gonzalez-Angulo, M.T. Cronin,J. Sun, M. Hawryluk, D. Lipson, J.S. Ross, S.E. Come, L. Pusztai, P. Stephens,V.A. MillerWriting, review, and/or revision of the manuscript: R. Jeselsohn,R. Yelensky, G. Buchwalter, G. Frampton, F. Meric-Bernstam, A.M. Gonza-lez-Angulo, J. Ferrer-Lozano, M. Cristofanilli, H. G�omez, C.L. Arteaga,J. Giltnane, J.M. Balko, M. Hawryluk, J.S. Ross, L. Soussan-Gutman, I. Wolf,T. Rubinek, S.J. Schnitt, S.E. Come, L. Pusztai, P. Stephens, M. Brown, V.A.MillerAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): R. Jeselsohn, G. Buchwalter,J. Ferrer-Lozano, J.A. Perez-Fidalgo, H. G�omez, C.L. Arteaga, J.M. Balko,J.S. Ross, L. Gilmore, L. PusztaiStudy supervision: R. Jeselsohn, M. Brown, V.A. Miller
Grant SupportThis study was supported in part by grants from Susan G. Komen for the
Cure (to M. Brown), the NCI (P01 CA080111; to M. Brown), and NIDDK(R01 DK074967; to M. Brown).
ESR1-Activating Mutations in Advanced Breast Cancer
www.aacrjournals.org Clin Cancer Res; 20(7) April 1, 2014 1765
The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received August 25, 2013; revised December 9, 2013; accepted December13, 2013; published OnlineFirst January 7, 2014.
Koralewski P, et al. Anastrozole versus tamoxifen as first-line therapyfor advanced breast cancer in 668 postmenopausal women: results ofthe Tamoxifen or Arimidex Randomized Group Efficacy and Tolera-bility study. J Clin Oncol 2000;18:3741–57.
2. Nabholtz JM, Buzdar A, Pollak M, Harwin W, Burton G, Mangalik A,et al. Anastrozole is superior to tamoxifen as first-line therapy foradvanced breast cancer in postmenopausal women: results of a NorthAmericanmulticenter randomized trial. J Clin Oncol 2000;18:3751–67.
3. BaumM, Budzar AU, Cuzick J, Forbes J, Houghton JH, Klijn JG, et al.Anastrozole alone or in combination with tamoxifen versus tamoxifenalone for adjuvant treatment of postmenopausal women with earlybreast cancer: first results of the ATAC randomised trial. Lancet2002;359:2131–9.
4. Osborne CK, Schiff R. Mechanisms of endocrine resistance in breastcancer. Annu Rev Med 2011;62:231–47.
5. Garcia-Becerra R, Santos N, Diaz L, Camacho J. Mechanisms ofresistance to endocrine therapy in breast cancer: focus on signalingpathways, miRNAs and genetically based resistance. Int J Mol Sci2012;14:101–45.
6. Osborne CK, Bardou V, Hopp TA, Chamness GC, Hilsenbeck SG,FuquaSA, et al. Role of theestrogen receptor coactivator AIB1 (SRC-3)andHER-2/neu in tamoxifen resistance in breast cancer. J Natl CancerInst 2003;95:351–61.
7. LavinskyRM, Jepsen K, Heinzel T, Torchia J,Mullen TM, Schiff R, et al.Diverse signaling pathways modulate nuclear receptor recruitment ofN-CoR and SMRT complexes. Proc Natl Acad Sci U S A 1998;95:2921–5.
8. Veldscholte J, Ris-Stalpers C, Kuiper GG, Jenster G, Berrevoets C,Claassen E, et al. A mutation in the ligand binding domain of theandrogen receptor of human LNCaP cells affects steroid bindingcharacteristics and response to anti-androgens. Biochem BiophysRes Commun 1990;173:531–40.
9. Zhao XY, Malloy PJ, Krishnan AV, Swami S, Navone NM, Peehl DM,et al. Glucocorticoids can promote androgen-independent growth ofprostate cancer cells through a mutated androgen receptor. Nat Med2000;6:701–6.
10. HerynkMH, FuquaSA. Estrogen receptormutations in humandisease.Endoc Rev 2004;25:861–98.
11. Weis KE, Ekena K, Thomas JA, Lazennec G, Katzenellenbogen BS.Constitutively active human estrogen receptors containing amino acidsubstitutions for tyrosine 537 in the receptor protein. Mol Endocrinol1996;10:1381–98.
12. Carlson KE, Choi I, Gee A, Katzenellenbogen BS, KatzenellenbogenJA. Altered ligand binding properties and enhanced stability of aconstitutively active estrogen receptor: evidence that an open pocketconformation is required for ligand interaction. Biochemistry 1997;36:14891–905.
13. White R, Sjoberg M, Kalkhoven E, Parker MG. Ligand-independentactivation of the oestrogen receptor by mutation of a conservedtyrosine. EMBO J 1997;16:1421–35.
14. Roodi N, Bailey LR, Kao WY, Verrier CS, Yee CJ, Dupont WD, et al.Estrogen receptor gene analysis in estrogen receptor-positive andreceptor-negative primary breast cancer. J Natl Cancer Inst 1995;87:441–51.
15. Ellis MJ, Ding L, Shen D, Luo J, Suman VJ, Wallis JW, et al. Whole-genome analysis informs breast cancer response to aromatase inhi-bition. Nature 2012;486:351–60.
16. Stephens PJ, Tarpey PS, Davies H, Van Loo P, Greenman C, WedgeDC, et al. The landscape of cancer genes and mutational processes inbreast cancer. Nature 2012;486:401–4.
17. Banerji S, Cibulskis K, Rangel-Escareno C, Brown KK, Carter SL,FrederickAM, et al. Sequence analysis ofmutations and translocationsacross breast cancer subtypes. Nature 2012;486:401–9.
18. Karnik PS, Kulkarni S, Liu XP, Budd GT, Bukowski RM. Estrogenreceptor mutations in tamoxifen-resistant breast cancer. Cancer Res1994;54:341–53.
19. Zhang QX, Borg A, Wolf DM, Oesterreich S, Fuqua SA. An estrogenreceptor mutant with strong hormone-independent activity from ametastatic breast cancer. Cancer Res 1997;57:1241–9.
20. Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 2009;25:1751–60.
21. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. Thesequence alignment/map format and SAMtools. Bioinformatics2009;25:2071–9.
22. DePristo MA, Banks E, Poplin R, Garimella KV, Maguire JR, Hartl C,et al. A framework for variation discovery and genotyping usingnext-generation DNA sequencing data. Nat Genet 2011;43:491–8.
23. Forbes SA, Bindal N, Bamford S, Cole C, Kok CY, Beare D, et al.COSMIC: mining complete cancer genomes in the catalogueof somatic mutations in cancer. Nucleic Acids Res 2011;39:D941–50.
24. Compeau PE, Pevzner PA, Tesler G. How to apply de Bruijn graphs togenome assembly. Nat Biotechnol 2011;29:981–91.
25. Carter SL, Cibulskis K, Helman E, McKenna A, Shen H, Zack T, et al.Absolute quantification of somatic DNA alterations in human cancer.Nat Biotechnol 2012;30:411–21.
26. VanLoo P, Nordgard SH, Lingjaerde OC, Russnes HG, Rye IH, Sun W,et al. Allele-specific copy number analysis of tumors. Proc Natl AcadSci U S A 2010;107:16911–5.
27. Yau C, Mouradov D, Jorissen RN, Colella S, Mirza G, Steers G, et al. Astatistical approach for detecting genomic aberrations in heteroge-neous tumor samples from single nucleotide polymorphism genotyp-ing data. Genome Biol 2010;11:R92.
28. Meric-Bernstam F, Frampton G, Ferrer-Lozano J, Yelensky R, PalmerGA, Cronin MT, et al. Cancer gene profile of metastatic breast cancer.J Clin Oncology 2012;30 (suppl; abstract 1015).
29. SwabyRF, JordanVC. Low-dose estrogen therapy to reverse acquiredantihormonal resistance in the treatment of breast cancer. Clin BreastCancer 2008;8:121–33.
30. Ellis MJ, Gao F, Dehdashti F, Jeffe DB, Marcom PK, Carey LA, et al.Lower-dose vs high-dose oral estradiol therapy of hormone receptor-positive, aromatase inhibitor-resistant advanced breast cancer: aphase 2 randomized study. JAMA 2009;302:771–80.
31. Li S, Shen D, Shao J, Crowder R, Liu W, Prat A, et al. Endocrine-therapy-resistant ESR1 variants revealed by genomic character-ization of breast-cancer-derived xenografts. Cell Rep 2013;4:1111–30.
32. Toy W, Shen Y, Won H, Green B, Sakr RA, Will M, et al. ESR1 ligand-binding domain mutations in hormone-resistant breast cancer. NatGenet 2013;45:1439–45.
33. Robinson DR,WuYM, Vats P, Su F, Lonigro RJ, Cao X, et al. ActivatingESR1 mutations in hormone-resistant metastatic breast cancer. NatGenet 2013;45:1446–51.
34. Merenbakh-Lamin K, Ben-Baruch N, Yeheskel A, Dvir A, Soussan-Gutman L, Jeselsohn R, et al. D538G mutation in estrogen receptor-alpha: a novel mechanism for acquired endocrine resistance in breastcancer. Cancer Res 2013;73:6851–64.
35. The Cancer Genome Atlas Network. Comprehensive molecular por-traits of human breast tumours. Nature 2012;490:61–70.
36. Baselga J, CamponeM, Piccart M, Burris HAIII, Rugo HS, Sahmoud T,et al. Everolimus in postmenopausal hormone-receptor-positiveadvanced breast cancer. N Engl J Med 2012;366:521–9.
37. Pearce ST, Liu H, Jordan VC. Modulation of estrogen receptor alphafunction and stability by tamoxifen and a critical amino acid (Asp-538)in helix 12. J Biol Chem 2003;278:7631–8.
38. Lazennec G, Ediger TR, Petz LN, Nardulli AM, Katzenellenbogen BS.Mechanistic aspects of estrogen receptor activation probed withconstitutively active estrogen receptors: correlations with DNA and
Jeselsohn et al.
Clin Cancer Res; 20(7) April 1, 2014 Clinical Cancer Research1766
coregulator interactions and receptor conformational changes. MolEndoc 1997;11:1371–86.
39. Di Leo A, Jerusalem G, Petruzelka L, Torres R, Bondarenko IN,Khasanov R, et al. Results of the CONFIRM phase III trial comparingfulvestrant 250mgwith fulvestrant 500mg in postmenopausal womenwith estrogen receptor-positive advanced breast cancer. J Clin Oncol2010;28:4591–600.
40. Wolf DM, Jordan VC. The estrogen receptor from a tamoxifen stim-ulated MCF-7 tumor variant contains a point mutation in the ligandbinding domain. Breast Cancer Res Treat 1994;31:121–38.
41. Wolf DM, Jordan VC. Characterization of tamoxifen stimulated MCF-7tumor variants grown in athymic mice. Breast Cancer Res Treat1994;31:111–27.
42. Holst F, Stahl PR, Ruiz C, Hellwinkel O, Jehan Z, Wendland M, et al.Estrogen receptor alpha (ESR1) gene amplification is frequent in breastcancer. Nat Genet 2007;39:651–60.
43. Tomita S, Zhang Z, Nakano M, Ibusuki M, Kawazoe T, Yamamoto Y,et al. Estrogen receptor alpha gene ESR1 amplification may predictendocrine therapy responsiveness in breast cancer patients. CancerSci 2009;100:1011–7.
44. Adelaide J, Finetti P, Charafe-Jauffret E, Wicinski J, Jacquemier J,Sotiriou C, et al. Absence of ESR1 amplification in a series of breastcancers. Int J Cancer 2008;123:2971–2.
45. Brown LA, Hoog J, Chin SF, Tao Y, Zayed AA, Chin K, et al. ESR1 geneamplification in breast cancer: a common phenomenon? Nat Genet2008;40:801–7.
ESR1-Activating Mutations in Advanced Breast Cancer
www.aacrjournals.org Clin Cancer Res; 20(7) April 1, 2014 1767
