exploring breast cancer estrogen disposition: the basis ...review exploring breast cancer estrogen...

Review

Exploring Breast Cancer Estrogen Disposition:The Basis for Endocrine Manipulation

Per E. Lønning1,2, Ben P. Haynes3, Anne H. Straume1,2, Anita Dunbier3, Hildegunn Helle1,2,Stian Knappskog1,2, and Mitch Dowsett3

AbstractAlthough normal breast tissue and breast cancer estrogens are known to be elevated compared with

plasma estrogen levels, the mechanism behind this phenomenon has been an issue of debate for

2 decades. If local estrogen aromatization were to be confirmed as the main estrogen source in breast

cancer tissue, tissue-specific inhibition of estrogen production, avoiding systemic side effects, would

become a potentially attractive option for breast cancer treatment and prevention. Based on recent

results from our groups exploring tissue estrogens, together with estrogen-synthesizing and estrogen-

regulated gene expression levels, we propose a new model to explain elevated breast tissue estrogen

levels. Although local estrogen production may be important, the local contribution is overruled

by rapid plasma-to-tissue equilibration, including active uptake of circulating estrogens or enhanced

tissue binding. As for breast cancer tissue levels, elevated levels of estradiol may be explained to a

large extent by estrogen receptor binding and local conversion of estrone into estradiol. This model

indicates that effective suppression of benign and malignant tissue estrogens as a treatment for ERþ

breast cancer requires systemic suppression and will not be markedly affected by local enzyme targeting.

Clin Cancer Res; 17(15); 4948–58. �2011 AACR.

Introduction

Estrogensplay a pivotal role in breast cancer developmentand sustained tumor growth. Observational studies con-ducted before the era of estrogen replacement therapyrevealed that early loss of ovarian function reduced breastcancer riskby60%to70%(1, 2).More recently, anumberofgroups reported that highpostmenopausal plasma estradiol(E2) levels are associated with a subsequent risk of breastcancer development (see ref. 3 for references to originalwork). Whether this relates only to growth stimulation ofalready transformed cells or includes promotion of carci-nogenesis (4, 5) is not fully understood. Of note, oophor-ectomy reduces subsequent breast cancer incidence by 50%(6) among BRCA1 carriers; however, about 80% of thebreast cancers that arise in this group are estrogen recep-tor–negative (ER�) basal-like (7, 8). This finding supports arole for estrogen during the early stages of cancer develop-

ment. The notion that estrogens sustain the growth of ER-positive (ERþ) cells is confirmed by the dramatic effectsachieved through antihormonal therapy for ERþ tumors inboth adjuvant (9) andmetastatic (10) settings. The stimula-tion of ERþ breast cancer cell proliferation in vitro byestrogens, particularly E2, strongly suggests that these clin-ical growth effects result from direct effects of estrogens onERþ breast cancer and not by intermediary pathways.

Although multiple estrogen metabolites expressing vary-ing estrogen-agonistic activity have been identified, ingeneral 4 estrogen hormones are considered of interest:E2, estrone (E1), estriol (E3), and the estrone conjugateestrone sulfate (E1S). Although E3 plays a major role inpregnancy, it is of little importance in nonpregnantwomen and will not be further considered here. Estronesulfate is derived by sulfate conjugation of E1 and may bedeconjugated by sulfatases, leaving conjugated and uncon-jugated estrogens at equilibrium (11, 12). Estradiol is theactive hormone that binds the ER (13) with a high affinity(kDa < 1 nmol/L). Although E1 and E1S are biologicallyinactive, many tissues express enzymes (sulfatase andreductases) that convert E1S and E1 into active E2 (14, 15).

In premenopausal women, the main estrogen source forbreast tissue is circulating E2, which is secreted by theovaries. At menopause, ovarian production of estrogenceases. In postmenopausal women, circulating E1, whichis produced in different tissues (e.g., skin, soft tissue,muscle, and liver) by aromatization of androstenedionetaken up from the circulation, is the main unconjugatedplasma estrogen. However, due to conflicting evidence, the

Authors' Affiliations: 1Section of Oncology, Institute of Medicine, Uni-versity of Bergen, and 2Department of Oncology, Haukeland UniversityHospital, Bergen, Norway; 3Department of Academic Biochemistry, RoyalMarsden Hospital, London, United Kingdom

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Per E. Lønning, Department of Oncology,Jonas Lies vei 26, Haukeland University Hospital, N-5021, Bergen,Norway. Phone: 47-5597-5000; Fax: 47-5597-2046; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-11-0043

�2011 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 17(15) August 1, 20114948

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relative contribution from circulating versus locally synthe-sized estrogens to intratumor E2 levels in postmenopausalwomen has been controversial for 2 decades. Althoughintratumor aromatase expression has been detected atmRNA (16) and protein (17) levels, and aromatase activityhas been detected in tumor specimens in vitro (18), thequantitative importance of local estrogen productionwithin tumors and adjacent benign tissues versus circula-tory uptake has not been defined.The contribution of locally synthesized versus systemi-

cally delivered E2 to intratumor hormone levels mayhave important therapeutic implications. First, if localaromatization is the main source of intratumor E2,intratumor aromatase expression should predict sensi-tivity to aromatase inhibitors (19). Second, althoughthe aromatase protein is similar in all body compart-ments, gene transcription is regulated by tissue-specificpromoters (20) that are sensitive to stimulation bydifferent ligands (see below). This raises the possibilityof using pharmacologic organ- or tissue-specific estrogendeprivation as an improved means of targeted therapy.For example, breast-specific estrogen deprivation couldavoid unwanted side effects in bone and genito-urethraltissue while suppressing breast cancer estrogen levels.Third, a strong quantitative contribution from alterna-tive pathways, such as E1S deconjugation, would incontrast indicate a role for other classes of drugs,such as sulfatase or dehydrogenase inhibitors, in breastcancer therapy.The aim of this review is to examine the evidence

evaluating the contributions from different sources tonormal breast tissue and intratumor E2. During this pro-cess, we focus on 4 topics. First, we discuss data from recent

sensitive and specific analyses of plasma, benign breast,and breast tumor estrogen levels. Second, we comparethese findings with previous data from in vivo tracer infu-sion studies. Third, we correlate the expression of hormonesynthesizing enzymes in normal breast and breast cancertissue with hormone levels, with the aim of identifying thekey synthetic pathways regulating tissue estrogen levels.Finally, we consider potential correlations between plasmaE2 values and intratumor expression of estrogen-regulatedgenes. An integration of these lines of evidence provides anew perspective on the importance of different compart-ments for the estrogenic stimulation of ERþ breast cancer.

Plasma Estrogen Levels, Production, andMetabolic Pathways

Although ovarian estrogen synthesis ceases at meno-pause, E2, E1, and E1S are all detected in the plasma ofpostmenopausal women.

Estrogens arise through aromatization (Fig. 1) of andro-gens only (21). The gene coding for the aromatase enzyme(CYP19) contains at least 10 promoters, each of which givesrise to mRNAs with unique untranslated first exons but toidentical proteins because the translational start site islocated in exon II (20). Of note, these promoters are differ-entially activated in different tissue compartments, andthere are differences between benign and malignant tissuesderived from the breast; thus, promoter 1.4 seems to act asthe major regulator of aromatase expression in benignbreast tissue, in contrast to breast cancers, in which promo-ters 1.3, 1.7, and PII seem to be the most active. Each tissuehas its own active promoters [e.g., 1.3, 1.4, andPI in adiposetissue; 1.1, 1.2, and 2a in the placenta; and 1.4 and 1.6 inbone (see ref. 20 fordetails and references tooriginalwork)].Although aromatase has been detected in most normaltissue compartments (see references in ref. 21), the quanti-tative contribution from individual organs and tissue typesto circulating estrogens has not been formally addressed.

Once thought in error to be a major source of circulatingestrogens in postmenopausal women, the adrenal cortex isthe main source of circulating androgens, although con-flicting evidence suggests someminor ovarian contributionto circulating testosterone (22–24). Plasma androstene-dione (A) and testosterone (T) levels average 5 to 7nmol/L and 0.5 to 1 nmol/L, respectively, in postmeno-pausal women (25). Overall, about 1% to 4% of circulatingA (and <1% of circulating T) undergoes aromatization (seereferences to original works in refs. 21 and 26); thus, E1 andE2 synthesis represents the major and minor pathways,respectively, of estrogen production in postmenopausalwomen. Although to a substantial degree E2 may besynthesized by reduction of E1 (27), the relative contribu-tion from each of these 2 pathways (aromatization of T toE2 versus reduction of E1 to E2) to plasma and tissue E2 hasnot been formally assessed (Fig. 1).

Except during the first few days of the menstrual cycle, E2constitutes the major unconjugated plasma estrogen inpremenopausal women. In postmenopausal women,

Translational Relevance

The contribution of local estrogen production tonormal breast and breast cancer tissue estrogen levelsremains controversial. If local production is the mainfactor regulating normal breast and breast cancer tissueestrogen levels, tissue-specific inhibition of estrogenproduction may become an attractive option for breastcancer treatment as well as prevention. Tissue estrogenmeasurements obtained by highly sensitive assays,together with studies correlating estrogen-regulatingenzymes and estrogen-regulated gene expression pro-files with tissue estrogen levels, are providing newevidence on the subject. Although local estrogen pro-duction may be important, the data suggest a state ofrapid plasma-to-tissue equilibrium, including activeuptake of circulating estrogens or enhanced tissue bind-ing. As for breast cancer tissue levels, intratumor estra-diol correlates with the estrogen receptor level and localconversion of estrone into estradiol. This model indi-cates that effective suppression of benign andmalignanttissue estrogens as a treatment for ERþ breast cancerrequires systemic suppression.

Breast Cancer Estrogen Levels

www.aacrjournals.org Clin Cancer Res; 17(15) August 1, 2011 4949




plasma E1 levels average about 70 to 90 pmol/L, with E2levels of about 15 to 25 pmol/L, although there is substan-tial variation among individuals (25, 28). In contrast, aftermenopause, levels within individuals vary only modestlyover a number of years (29). In addition, circulating E1Sconcentrations may average from 5 to 600 pmol/L (30).Estrone sulfate is synthesized by sulfate conjugation of E1(31); nodirect secretionof estrone sulfate is known tooccur.

Estrogens are metabolized and eliminated by a 2-stepprocess (Fig. 2). The first part involves hydroxylationsexecuted by multiple cytochrome P450 enzymes (CYP)(32, 33). The estrogen steroid nucleus may be exposedto hydroxylations at multiple positions (32). The mainmetabolic pathways involve 2-hydroxylations and (to alesser degree) 4-hydroxylations, generating catechol estro-gens, and hydroxylations at the 16a position, generating16a-hydroxyestrone followed by reduction into E3 (34,35). Although these enzymes have both E1 and E2 assubstrates, some of them express a selective substrate pre-ference for either E1 or E2 (32, 36, 37). Of note, suchhydroxylations take place in different body compartments,including breast cancer and liver tissue, partly executed bydifferent members of the CYP family. Thus, whereas estro-gen-metabolizing CYPs such as CYP1A2 and CYP3A5 play amajor role in metabolizing estrogens in the liver (2- and16a-hydroxylation in particular), in the breast CYP1A1 and

CYP1B1 are the main executors of 2- and 4-hydroxylations,respectively (38, 39). In a second step, thesemetabolites aresubjected to conjugation (Fig. 2). Although a minoramount is sulfate-conjugated and excreted in the bile,followed by substantial intestinal deconjugation and recy-cling (40), the bulk is subsequently glucuronidated andexcreted through the urine (41). Similarly, circulating E1Smay be deconjugated by sulfatase and subsequently meta-bolized as unconjugated E1 (34).

So far, we have gained only a limited understanding ofwhich factors determine plasma and tissue estrogen con-centrations in postmenopausal women. Several growthfactors and cytokines have been shown to regulate aroma-tase expression in vitro (42), but little is known with respectto aromatase activity regulation in vivo. No feedbackmechanisms regulating plasma estrogen levels have beenidentified. Although in vivo aromatization of androstene-dione correlates with body weight (see references in ref.21), the correlation between plasma estrogen levels andin vivo aromatase activity is moderate (43), probably due tointerindividual variation in the estrogen elimination rate.Germline mutations in the aromatase gene are rare buthave severe consequences. Although a single germline gainof function aromatase mutation has been described (44),aromatase deficiency due to inactivemutations that reveal aclinical phenotype resembling inactivating mutations that

© 2011 American Association for Cancer Research

DHEA sulfate

17-OH-progesterone

ProgesteronePregnenolone

17-OH-pregnenolone Hydrocortisone

DHEA

Aldosterone

O

OAndrostenedione

OH

OTestosterone

9

4

10 10

3

Cholesterol

1

3

4

2

911

2

5 6

5 6

7 82

Estrone sulphate

Peripheral conversion

Adrenal cortex

Estrone

O

HOEstradiol

HO

OHO

O

S

Figure 1. Pathways of estrogensynthesis in postmenopausalwomen. The numbers in the figureindicate the following enzymes:1 ¼ desmolase, 2 ¼ 3b-ol-dehydrogenase-d-5 to 4isomerase, 3 ¼ 17a hydroxylase,4 ¼ C17,20-lyase, 5 ¼ 21-hydroxylase, 6 ¼ 11b-hydroxylase, 7 ¼ 18-hydroxylase,8 ¼ 18-hydroxysteroid oxidase,9 ¼ different 17b-hydroxysteroiddehydrogenases, 10 ¼aromatase, and 11 ¼ estronesulfatase and sulfotransferase.

Lønning et al.

Clin Cancer Res; 17(15) August 1, 2011 Clinical Cancer Research4950




affect the ER receptor gene (45) has been described in a fewfamilies. Multiple aromatase polymorphisms have beendetected, but few of these affect the coding region (46).Of interest, one of these intronic variants was reported tobe associated with improved prognosis in patients treatedwith letrozole (47), and recently 2 variants, locatedupstream of the promoter area, were found to be associatedwith enhanced aromatase activity (48).The different CYPs are subject to multiple polymorphic

variants (33). However, although plasma E1 and E2 clear-ance rates vary substantially (31, 49), so far no distinct CYPpolymorphism has been correlated with plasma estrogenlevels (33).

Analyzing Plasma and Tissue Estrogen Levels

Before discussing data on estrogen concentrations indetail, we must consider certain methodologic limitationsrelated to plasma and tissue estrogen measurements in

postmenopausal women. Measuring estrogen in plasmaand particularly in tissue is not trivial, and nonoptimizedmethodologies have contributed significantly to the vari-able data in the literature. Circulating estrogen levels inpostmenopausal women are among the lowest of all ster-oids assessed in clinical research or practice. Thus, plasmaestrogen assessment requires radioimmunoassays (RIA)specifically derived for that purpose.

Over the years, several studies have evaluated plasma andtissue estrogen levels (see below). Due to the relatively lowspecific activity of 3H-labeled estrogens (50–150 mCi/mmol), assays based on 3H-isotopes in general do notprovide the sensitivity required for such purposes. Ourgroups have made considerable efforts to develop 125I-based RIAs (specific activity about 2,000 Ci/mmol) forestrogen measurement in the low postmenopausal con-centration range (28, 50–53). With such methods, we haveachieved sensitivity limits in the range of 0.67 pmol/L, 1.14pmol/L, and 0.55 pmol/L for plasma E2, E1, and E1S,

© 2011 American Association for Cancer Research

17β-HydroxysteroiddehydrogenasesSulphatase

Sulfotransferase

Estrone sulphate

2-Hydroxyestrone

Glucuronide

Glucuronide Glucuronide

Glucuronide

Glucuronide

16-Hydroxyestrone 2-HydroxyestradiolEstriol

Estrone

Minor metabolicpathways (including4-hydroxylation)

O

HOEstradiol

HO

OH

OHOH

OH

O

HO

OH

HO

OH

O

O

S

O

HO

HO

2-Methoxyestrone

O

HO

CH3O

2-MethoxyestradiolHO

CH3O

HO

HO

Sulphate

Sulphate Sulphate

Sulphate

Sulphate

COMTCOMT

CYPCYPCYPCYP

CYPCYP

Figure 2. The main pathways of estrogen metabolism. Although multiple CYPs and hydroxylations in different sites of the estrone and estradiolnucleus are involved, the key pathways involve 2-hydroxylations and, to a lesser degree 4-hydroxylations, generating catechol estrogens, and the16a-hydroxylation pathway, generating 16a-hydroxyestrone, and estriol. Following these hydroxylations (which occur mainly in the liver tissue), estrogenmetabolites undergo either glucuronidation followed by urinary excretion (the major pathway), or sulfoconjugation followed by biliary excretion, afterwhich they may be subject to enterohepatic circulation.






respectively (52). Although these methods for plasma (52)and tissue (51) estrogen measurement are highly sensitive,precise, and devoid of cross-reactivity, they are labor-inten-sive because they require multiple prepurification steps.The need for such sensitive assays, particularly for assessingestrogen levels in patients undergoing treatment with aro-matase inhibitors, is illustrated by recent findings (53)revealing that the majority of patients who receive treat-ment with the third-generation aromatase inhibitors ana-strozole or letrozole achieve plasma estrogen levels belowthese sensitivity limits. Recently, commercial assays such asthe DSL-8700 E1 RIA applying 125I-labeled E1, and theSpectria estradiol-sensitive RIA and DSL-4800 ultrasensi-tive RIA using 125I-labeled E2 have become available. Suchkits in general do not involve any chromatographicprepurification procedures, highlighting the need forhigh specificity of the antibody applied. The theoreticalsensitivity limit for DSL-8700 is 4.44 pmol/L, and theore-tical sensitivity limits for E2 kits are in the range of 5 to8.14 pmol/L. Although such assays may determine plasmaestrogen levels reliably in the majority of postmenopausalwomen, some individuals reveal plasma E2 concentrationseven below 5 pmol/L.

Many investigators consider methods such as gas chro-matography/tandem mass spectroscopy (GC/MS-MS) andliquid chromatography/tandem mass spectrometry (LC/MS-MS) as gold standards with respect to estrogen levelmeasurements. However, some MS methods lack sensitiv-ity for determining plasma E2 levels in many postmeno-pausal women (54), particularly during treatment withestrogen-suppressing compounds. Nonetheless, usingGC/MS-MS, Santen and colleagues (55) reported averageplasma levels of E2 in postmenopausal women similar tothose recorded by our RIA, and others have lower valuesthan most of the sensitive RIAs (56). Although such meth-ods, including assays for plasma E1S and tissue estrogenlevels, are in rapid development (57–59), it is clear that thechoice and validation of assays based on MS are as impor-tant as for those based on RIA.

Method sensitivity problems are even greater when mea-suring tissue estrogen concentrations. As mentioned above,breast tissue contains enzymes involved in estrogen meta-bolism. Although major catechol estrogens, such as 2-hydroxyestrone, are rapidly cleared from the circulation(60), the fact that these metabolites may account for about50% of total estrogen metabolites in the urine (61) indi-cates a high production rate. In general, we lack knowledgeregarding tissue concentrations of estrogen metabolites,but they are a potential source of interference in RIA thatmust be eliminated during sample preparation for assay.

Benign Breast Tissue Estrogen Concentrationsand Synthesis

Although our focus is on the source of estrogens forbreast cancers, a brief account of estrogen disposition inbenign breast tissue is merited because (i) this is potentiallya source of estrogens for the malignant tissue per se, (ii) it is

an important comparator for the arguments made withmalignant tissue, and (iii) it may be important in consider-ing prevention strategies for breast cancer.

It has been known for 3 decades that E2 levels in breastcancer tissue exceed those in plasma by a factor of about10; however, the mechanism behind this phenomenonremains obscure. In a previous study (30) we measuredtissue estrogen levels using a 125I-based RIA (51) afterprepurification through several steps, including high-performance liquid chromatography. Although wedetected lower tissue levels of the different estrogen frac-tions compared with previous studies that applied RIAswithout prepurification (62–64), we detected a benigntissue/plasma concentration ratio of E1, the main estro-gen product in postmenopausal women, of 6 to 7 (30).This is consistent with the benign tissue/plasma 14C E1ratio of 5 to 6 found by Larionov and colleagues (65)when they applied presurgical steady-state infusions of14C-labeled E1 to breast cancer patients.

The benign tissue/plasma gradient »1 (as will be seen fortumor tissue as well) has provoked much discussion aboutwhat the potential mechanism might be. Several explana-tions have been proposed, including active uptake from thecirculation, local estrogen synthesis, and enhanced tissuebinding. From their studies infusing 3H-androstenedioneand 14C E1 in concert, by determining the 3H/14C isotoperatio in the tissue compared with plasma E1 fractions,Larionov and colleagues (65) estimated that local tissueproduction (based on an elevated 3H/14C ratio) mayaccount for a median of 15% of local E1 concentrations.In light of the potential clinical implications of thesefindings, we have explored the robustness of a pharmaco-kinetic model (66, 67). Given the specialized nature of thecalculations, we provide a detailed analysis in the Supple-mentary Material and summarize the key findings here.

Overall, pharmacokinetic modeling is most consistentwith a model suggesting rapid plasma/benign tissueexchange of the estrogen compounds, in which case theactual local contribution may not be determined (66).Although an elevated tissue/plasma gradient may be dueeither to enhanced tissue binding or, alternatively, amechan-ism of active uptake of circulating estrogens in the tissue,regrettably there is no pharmacokinetic model by whichthese 2 processes may be evaluated separately in vivo (67).

This model is supported by recent results from our ownstudies measuring plasma and benign breast tissue estro-gens in pre- and postmenopausal women (Tables 1 and 2).Average benign tissue levels of E1 and E2 in premenopausalwomen exceed those in postmenopausal women by a factorof about 3 and 15, respectively. On the other hand, thetissue/plasma concentration gradient is remarkably similarin both groups (E1 averaging 5–6, E2 averaging 1.5–2).These findings are consistent with both active uptake andenhanced tissue binding. In premenopausal women, thegreat majority of plasma E2 is ovarian in origin. Consider-ing the difference in tissue E2 concentrations between pre-and postmenopausal women and the similarity withrespect to the tissue/plasma concentration gradient, these

Lønning et al.





findings are consistent with a rapid equilibrium betweenplasma and benign tissue compartments.Another intriguing finding relates to the tissue/plasma

concentration ratios for the different estrogen fractions. Acomparison of E2, E1, and E1S reveals that E1 is the leastpolar (i.e., the most fat-soluble) compound, and E1S is themost polar (i.e., the most water-soluble). The tissue/plasma concentration ratios for E1, E2, and E1S are about5–6, 1.5–2, and 0.1, respectively. Lipophilicity and small-molecule size both predict for rapid membrane transfer(67). Because of its sulfate conjugation, E1S behaves as aweak acid, a group of compounds known in general toreveal a small volume of distribution as compared withbasic drugs (67). In contrast, E1 and E2 both behave asneutral compounds at physiologic pH. Taken together,these findings are consistent with (but do not definitelyprove) the notion that tissue/plasma estrogen concentra-tion ratios may be explained by the physiochemical prop-erties of the respective molecule.These results have potential implications for the future

clinical development of estrogen-modulating com-pounds. Because of its high plasma levels, the estrogenconjugate E1S has received much attention as a potentialsource of intratumoral estrogens (68). Thus, the findingsfrom previous studies of high levels of plasma (25) andtissue E1S (63) as compared with unconjugated E1 and E2suggest that circulating E1S, taken up by the tissue withsubsequent deconjugation and reduction, is an importantsource of intratumoral E2 (68). Although our own studies(69, 70) have confirmed high plasma levels of E1S, ourfindings of low tissue E1S and a low tissue/plasma con-centration ratio argue against the hypothesis that thisconjugate may be amajor contributor to tissue E2. Second,although we cannot directly assess benign breast tissueestrogen synthesis in postmenopausal women, rapid equi-libration between plasma and tissue compartments, whichis supported by the highly significant correlation betweenplasma and tissue estrogen concentrations for E1 and E2(Table 2), indicates that plasma estrogen levels predictbenign breast tissue levels well in both pre- and postme-nopausal women. Of even more importance, whatever themagnitude of local estrogen production within benignbreast tissue, rapid equilibration of breast tissue with theplasma compartment indicates the role of local synthesisto be of minor importance. Whether there is a substantiallocal synthesis (or not), this will have little impact on localestrogen levels in benign tissues as long as local concen-trations are at equilibrium with plasma estrogen levels.

Breast Cancer Estrogen Concentrations andSynthesis

In contrast to the finding that intratumoral concentra-tions of E2 in ER� tumors resembled concentrations inbenign tissue (30), we found substantially elevated levelsof E2 in ERþ tumors as compared with normal tissue fromthe same breast in postmenopausal women (Tables 1 and2; Fig. 3). Surprisingly, however, we detected tumor levels

Tab

le1.

Tissue

estrog

enleve

ls(fm

ol/g

wet

weigh

t)give

nas

geom

etric

mea

nleve

lswith

95%

confiden

ceintervalinben

ignan

dmaligna

ntbreas

tsa

mplesfrom

thesa

mebreas

tin

correlationwith

men

opau

sals

tatusan

dER

status

ER/P

GR

E2(ben

ign)

E2(tum

or)

E1(ben

ign)

E1(tum

or)

E1S(ben

ign)

E1S(tum

or)

Pos

tmen

opau

salw

omen

Totalg

roup

n¼

3029

.2(19.3–

44.1)

121.5e

(63.6–

232.0)

477.2(366

.7–62

0.9)

143.7e

(93.3–

221.3)

53.8

(32.0–

90.4)

99.5

e(53.8–

184.0)

ERþ

n¼

2131

.1(19.5–

49.7)

267.1b

,e(159

.9–44

6.2)

467.6(338

.2–64

6.4)

167.6e

(100

.1–28

0.5)

56.1

(29.3–

107.4)

98.6

c(44.8–

217.1)

ER�

n¼

925

.1(9.1–69

.6)

19.3

(6.1–61

.5)

500.3(285

.4–87

6.8)

100.3c

(39.9–

252.3)

48.8

(17.0–

140.5)

101.6c

(31.6–

327.2)

Premen

opau

salw

omen

Totalg

roup

n¼

1345

3.0(314

.3–65

2.8)

615.3(c)(230

.0–16

46.3)

1233

.1(817

.5–18

59.5)

194.6e

(109

.9–34

4.5)

394.4(213

.0–73

0.1)

612.6(c)(308

.6–12

16.2)

ERþ

n¼

739

8.7(243

.2–65

3.4)

1621

.8a,d(863

.2–30

47.9)

1144

.2(659

.0–19

86.6)

245.4c

(117

.0–51

4.5)

331.6(113

.7–96

7.4)

447.8(146

.2–13

71.8)

ER�

n¼

652

5.9(253

.2–10

92.4)

198.7(32.7–

1207

.5)

1345

.2(571

.5–31

67.4)

148.5c

(46.7–

471.6)

482.8(189

.5–12

30.3)

883.1(c)(303

.8–25

66.8)

Statis

tically

sign

ifica

ntdifferen

ceco

mpared

with

ER–su

bgrou

pof

patients:

aP<0.01

.bP

<0.00

1(M

ann–

Whitney

test).

Statis

tically

sign

ifica

ntdifferen

ceco

mpared

with

ben

igntis

sue:

(c)0.05

<P<0.10

,cP<0.05

.dP<0.01

.eP<0.00

1(W

ilcox

onsign

edrank

stest).

Datafrom

Lønn

inget

al.(30).






of E1 to be consistently lower as compared with tissueconcentrations of E1 in benign tissue from the same breastsindependently of tumor ER status. Consistent with thisobservation, in their tracer infusion studies, Larionov andcolleagues (65) found a lower concentration of 14C-E1 intumor than in benign tissue. Of interest, they recorded anelevated E1

3H/14C ratio in breast cancer compared withbenign breast tissue and plasma, indicating intratumoraromatization of 3H-labeled androstenedione. Althoughindividual variation was substantial, the results revealeda median contribution from local aromatization account-ing for about 30% of intratumor E1 levels, compared with amedian value of 15% in benign tissue. On the other hand,local synthesis was found to account for more than 50% ofintratumor E1 in only 3 of 24 tumors. The variationbetween tumors, however, was substantial. This may bedue not only to variable local synthesis but also to variationin diffusion rates between compartments (see Supplemen-tary Material). Similar results were obtained in a smallerstudy by Reed and colleagues (71). Although this differencebetween breast cancer and normal breast tissue couldindicate increased intratumor estrogen synthesis, alterna-tively it may be due to a slower uptake of E1 from thecirculation into the tumor compared with a normal breasttissue compartment (see details outlined in SupplementaryMaterial).

The potential impact of intratumor steroid synthesis ormodulation may have significant implications. Immuno-staining for aromatase protein expression remained a con-troversial subject for 2 decades. For several years,conflicting results were obtained with 2 antibodies: One(72) revealed major protein staining related to stromalcells, and the other antibody detected aromatase staining

in tumor cells (17). Based on these conflicting results, aninternational consortium was formed with the goal ofdeveloping and validating an antibody that could provideuniform results. This has now been achieved with theARO677 antibody, which has been validated by differentlaboratories (73).

No significant correlation of ARO677 immunostainingto either intratumoral E1 or E2 levels has been recorded.When ERþ tumors were analyzed separately, statisticallyhigher intratumoral E2 but not E1 levels were recordedin the group of ARO677þ tumors as compared withARO677� tumors (74). In the same material (75), wedetected low levels of aromatase mRNA in breast cancerand normal breast tissue. No correlations between benignand malignant tissue aromatase mRNA expression andeither E2 (Spearman rs ¼ �0.02 and 0.05, respectively) orE1 (Spearman rs ¼ �0.11 for both) levels were recorded.Although enzymatic aromatase activity may be detectedin most fresh breast cancer samples (19), these findings,in similarity to the data presented above, do not addressthe quantitative role of tumor aromatization in vivo.

A striking observation is the elevated tumor/benign-tissue E2 ratio in ERþ breast cancer tissue. In contrast,tumor tissue levels of E1 are reduced as compared withbenign tissue concentrations (Fig. 3). The balance betweenE2 and E1 is regulated through multiple dehydrogenaseisoforms. Some of these isoforms (e.g., HSD17B1, B5, B7,and B12) act reductively in catalyzing activation of E1 intoE2 (76–79), whereas others (e.g., HSD17B2, B10, and B14)favor oxidation, converting E2 into E1 in different tissues,including benign breast tissue (80–82). Of interest, both B2and B7 were found to be elevated in breast cancer ascompared with normal breast tissue (75), and intratumor

Table 2. Ratios of diverse plasma and tissue estrogen fractions in subgroups of patients (geometric meanwith 95% confidence interval)

Postmenopausal women Premenopausal women

ER status ERþ

n ¼ 21ER�

n ¼ 9ERþ

n ¼ 7ER�

n ¼ 6

NT-E2/PL-E2 2.0 (1.3–3.2) 1.3 (0.5–3.2) 1.6 (0.7–3.5) 2.2 (1.7–3.0)NTE1/PL-E1 5.9 (5.0–7.0) 6.3 (4.4–9.0) 4.4 (2.8–6.9) 7.4 (4.7–11.5)NTE1S/PL-E1S 0.1 (0.0–0.2) 0.1 (0.0–0.4) 0.1 (0.0–0.3) 0.1 (0.0–0.3)TU-E2/PL-E2 16.4c (10.8–24.9) 1.2 (0.3–5.3) 6.6a (2.2–20.5) 0.5 (0.0–9.7)TU-E1/PL-E1 2.1 (1.5–3.0) 1.4 (0.5–4.2) 1.0 (0.3–2.7) 0.7 (0.2–2.6)TU-E1S/PL-E1S 0.2 (0.1–0.4) 0.2 (0.0–0.8) 0.1 (0.0–0.3) 0.2 (0.1–0.6)TU-E2/TU-E1 1.5b (0.9–2.6) 0.2 (0.0–1.1) 7.1a (4.5–11.3) 0.8 (0.1–8.5)

Abbreviations: E1, estrone; E2, estradiol; E1S, estrone sulfate; ER, estrogen receptor; NT, normal (benign) tissue of the same breast;PL, plasma; TU, tumor tissue.Statistically significant difference compared with ER� subgroup of patients:

aP < 0.05.bP < 0.01.cP < 0.001 (Mann–Whitney test).

Data from Lønning et al. (30).

Lønning et al.





levels of E2 were found to be negatively correlated withHSD17B2 expression but positively correlated with B7expression. These findings support an additional mechan-ism of intratumoral E2 synthesis, i.e., reduction of E1 intoE2, which would explain elevated levels of tumor E2 ascompared with benign tissue E2 levels.Finally, we observed a strong correlation between

intratumoral E2 concentration and ER expression levels,suggesting an affinity effect of the ER (75). Before immu-nostaining became standard practice for ER assessment, thegeneral cut-off limit for ER positivity in ligand-bindingassays was 10 fmol/mg protein (83). Assuming that 1fmol/mg protein may correspond to about 100 fmol/g

wet weight of tissue or 100 pmol/L, the ER concentrationof tumors considered positive by this cutoff would farexceed the concentration of E2 in ERþ tumors. The estradiolbinding affinity for the ER is less than 1 nmol/L (13); thus,at the concentrations measured, on average we may envi-sion the bulk of intratumor E2 to be receptor bound.

Taken together, the findings with respect to intratumorE1 and E2 versus plasma levels differ significantly fromwhatwe observed with respect to benign tissue hormonal con-centrations. Although plasma E1 levels in postmenopausalwomen correlated with intratumor E1 and E2 levels, therewere no significant correlations in pre- or postmenopausalwomen for plasma E2 and intratumor estrogen levels, or forplasma E1 in premenopausal women. This suggests that theconcentration equilibrium seen for benign tissue may notapply to intratumor levels. Although we cannot estimatethe quantitative contribution from intratumoral aromati-zation, it is notable that the lack of correlation betweenaromatase expression at the mRNA and protein levels(immunostaining) contrasts with the correlations betweenintratumor E2 and dehydrogenase enzymes as well as ERlevel expression. On the contrary, the observation thattumor E2 concentration correlated positively to HSD17B7but negatively to HSD17B2 expression strongly supports arole for intratumor conversion of E1 into E2 as a source ofintratumor E2.

No correlations between intratumor levels of either E1,E2, or E1S and plasma E1S levels or intratumor estronesulfatase levels were observed (30, 75). Furthermore, theaverage expression level of estrone sulfatase in tumor tissuewas about one third the level recorded in benign breasttissue. Taken together, these findings do not suggest amajor contribution from circulating E1S to intratumor E2levels.

Plasma Estrogen Concentrations andIntratumor Expression of Estrogen-RelatedGenes

The development of multigene expression assays (84)has allowed investigators to study tumor gene expressionprofiling in relation to estrogen suppression with aroma-tase inhibitors (85), as well as to explore potential correla-tions between circulating estrogen levels and expression ofestrogen-regulated genes within tumor tissue. Althoughgermline polymorphisms in estrogen-synthesizing andmetabolizing genes (33) should affect all body compart-ments, as stated above, this may also have tissue- orplasma-specific effects due to organ-specific expression.Although the CYP19 SNPs rs10046 and TCT� were bothfound to be significantly associated with plasma E2 levels,they explained only a small percentage of interindividual E2variation (86).

In a recent study, some members of our group (87)assessed intratumor expression profiles of genes underestrogen regulation in postmenopausal women. Genesupregulated or downregulated by estrogen stimulation ofERþ breast cancer cells were previously indentified from

0

2

4

6

8

Ratio

malig

nant/

benig

ntissue c

oncentr

atio

n

10

12

14

**

****

***A

E2

ER + n = 21 ER − n = 9

E1

E1S

0

1

2

3

4

Ratio

malig

nant/

benig

ntissue c

oncentr

atio

n

5

6

7

**

** **

*B

E2

ER + n = 7 ER − n = 6

E1

E1S

Figure 3. Ratio between intratumor and benign breast tissueestrogen levels related to ER expression and menopausal status.A, postmenopausal women. B, premenopausal women. *, P < 0.05,**, P < 0.01, and ***, P < 0.001. Reproduced from Lønning et al. (30) withpermission.






in vitro experiments (88). When 104 breast cancers werestudied (87), a strong positive correlation between plasmaE2 levels and intratumor ER expression on the one handand intratumor expression levels of several classical E2-regulated genes on the other hand were found. When genessuch as trefoil factor 1 (TFF1), GREB1, PDZK1, and PgRwere combined into an index, a correlation coefficient of(rs) of 0.51 indicated that plasma E2 levels on averagepredicted intratumor E2 levels by 27%. In this study,neither breast cancer nor benign tissue estrogen concentra-tions were available. The data, however, provide strongsupporting evidence for the hypothesis that plasma/benign-tissue estrogens exert a strong biological influenceon breast cancer tissue.

Integrative Model

The topic of the contribution of intratumor synthesisversus circulating estrogens to intratumor E2 levels andER stimulation in breast cancer has been controversial fordecades. We believe that the recent evidence as reviewedhere adds a significant contribution to our understandingof the topic. Although we are not formally able to assess(and probably will not be able to in the near future) thequantitative contribution from each individual organ toplasma estrogen levels in postmenopausal women, webelieve that a relatively straightforward model canexplain our recent observations and clarify previous con-tradictory evidence with respect to plasma as well asbenign and malignant breast tissue estrogen concentra-tions. As for benign tissue estrogen concentrations, due torapid equilibration between the tissue and plasma com-partments, plasma estrogens become good surrogatemarkers for benign tissue levels. Although a tissue/plasma gradient for E2 and E1 exists, the fact that this

gradient is similar in pre- and postmenopausal womensupports the hypothesis that this gradient may be dueeither to active tissue uptake or simply to enhanced tissuesteroid binding or depot (fat) storage. High intratumor E2levels may be due mainly to a concerted mechanismrelated to uptake of circulatory (and potentially benigntissue) E1 with intratumor reduction of E1 into E2 and, toa greater degree, an affinity effect of the ER on E2. On thecontrary, indirect evidence suggests that intratumor aro-matization plays a minor role, and there is no evidenceadvocating a role of E1S as a potential source of intratu-moral E2.

Our data suggest that local enzyme inhibition ofthe aromatase enzyme or sulfatase is less important thaninhibition of total-body estrogen synthesis. Althoughinhibition of local tumor dehydrogenases could poten-tially have a role in breast cancer therapy, the evidenceargues against targeting local aromatization or sulfatedeconjugation as a therapeutic strategy in breast cancer.

Disclosure of Potential Conflicts of Interest

P. E. Lønning received honoraria from Novartis, AstraZeneca, and PfizerInc. for advice and lecture fees. M. Dowsett received honoraria fromAstraZeneca for advice and lecture fees. He also received grants fromAstraZeneca and Novartis.

Grant Support

Norwegian Cancer Society; Western Health Region of Norway; InnovestProgram of Excellence; Breast Cancer Research Foundation; Mary-JeanMitchell Green Foundation; Breakthrough Breast Cancer; National Institutefor Health Research (to the Royal Marsden National Institute for HealthResearch Biomedical Research).

Received January 6, 2011; revised April 5, 2011; accepted April 15, 2011;published OnlineFirst July 26, 2011.

References1. Trichopoulos D, MacMahon B, Cole P. Menopause and breast cancer

risk. J Natl Cancer Inst 1972;48:605–13.2. Feinleib M. Breast cancer and artificial menopause: a cohort study. J

Natl Cancer Inst 1968;41:315–29.3. Key T, Appleby P, Barnes I, Reeves G Endogenous Hormones and

Breast Cancer Collaborative Group. Endogenous sex hormones andbreast cancer in postmenopausal women: reanalysis of nine prospec-tive studies. J Natl Cancer Inst 2002;94:606–16.

4. Russo J, Russo IH. Genotoxicity of steroidal estrogens. TrendsEndocrinol Metab 2004;15:211–4.

5. Clemons M, Goss P. Estrogen and the risk of breast cancer. N Engl JMed 2001;344:276–85.

6. Rebbeck TR, Kauff ND, Domchek SM. Meta-analysis of risk reductionestimates associated with risk-reducing salpingo-oophorectomyin BRCA1 or BRCA2 mutation carriers. J Natl Cancer Inst 2009;101:80–7.

7. Carey LA. Targeted chemotherapy? Platinum in BRCA1-dysfunctionalbreast cancer. J Clin Oncol 2010;28:361–3.

8. Sørlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, et al.Gene expression patterns of breast carcinomas distinguish tumorsubclasses with clinical implications. Proc Natl Acad Sci U S A2001;98:10869–74.

9. Abe O, Abe R, Enomoto K, et al. Effects of chemotherapy andhormonal therapy for early breast cancer on recurrence and 15-year

survival: an overview of the randomised trials. Lancet 2005;365:1687–717.

10. Lønning PE. Aromatase inhibitors in breast cancer. Endocr RelatCancer 2004;11:179–89.

11. Ruder HJ, Loriaux L, Lipsett MB. Estrone sulfate: production rate andmetabolism in man. J Clin Invest 1972;51:1020–33.

12. Longcope C. The metabolism of estrone sulfate in normal males. JClin Endocrinol Metab 1972;34:113–22.

13. Edwards DP, McGuire WL. 17 alpha-Estradiol is a biologically activeestrogen in human breast cancer cells in tissue culture. Endocrinology1980;107:884–91.

14. Suzuki M, Ishida H, Shiotsu Y, Nakata T, Akinaga S, Takashima S,et al. Expression level of enzymes related to in situ estrogen synthesisand clinicopathological parameters in breast cancer patients. J Ster-oid Biochem Mol Biol 2009;113:195–201.

15. Santner SJ, Feil PD, Santen RJ. In situ estrogen production via theestrone sulfatase pathway in breast tumors: relative importance versusthe aromatase pathway. J Clin Endocrinol Metab 1984;59:29–33.

16. Price T, Aitken J, Head J, Mahendroo M, Means G, Simpson E.Determination of aromatase cytochrome P450 messenger ribonucleicacid in human breast tissue by competitive polymerase chain reactionamplification. J Clin Endocrinol Metab 1992;74:1247–52.

17. Esteban JM, Warsi Z, Haniu M, Hall P, Shively JE, Chen S. Detection ofintratumoral aromatase in breast carcinomas. An immunohistochemical

Lønning et al.





study with clinicopathologic correlation. Am J Pathol 1992; 140:337–43.

18. Miller WR. Aromatase activity in breast tissue. J Steroid Biochem MolBiol 1991;39[5B]:783–90.

19. Miller WR, Anderson TJ, Jack WJL. Relationship between tumouraromatase activity, tumour characteristics and response to therapy. JSteroid Biochem Mol Biol 1990;37:1055–9.

20. Bulun SE, Sebastian S, Takayama K, Suzuki T, Sasano H, Shozu M.The human CYP19 (aromatase P450) gene: update on physiologicroles and genomic organization of promoters. J Steroid Biochem MolBiol 2003;86:219–24.

21. Lønning PE, Dowsett M, Powles TJ. Postmenopausal estrogen synth-esis and metabolism: alterations caused by aromatase inhibitors usedfor the treatment of breast cancer. J Steroid Biochem 1990;35:355–66.

22. Dowsett M, Cantwell B, Lal A, Jeffcoate SL, Harris AL. Suppression ofpostmenopausal ovarian steroidogenesis with the luteinizing hor-mone-releasing hormone agonist goserelin. J Clin Endocrinol Metab1988;66:672–7.

23. Couzinet B, Meduri G, Lecce MG, Young J, Brailly S, Loosfelt H, et al.The postmenopausal ovary is not a major androgen-producing gland.J Clin Endocrinol Metab 2001;86:5060–6.

24. Sluijmer AV, Heineman MJ, De Jong FH, Evers JL. Endocrineactivity of the postmenopausal ovary: the effects of pituitarydown-regulation and oophorectomy. J Clin Endocrinol Metab1995;80:2163–7.

25. Lønning PE, Helle SI, Johannessen DC, Adlercreutz H, Lien EA, TallyM, et al. Relations between sex hormones, sex hormone bindingglobulin, insulin-like growth factor-I and insulin-like growth factorbinding protein-1 in post-menopausal breast cancer patients. ClinEndocrinol (Oxf) 1995;42:23–30.

26. Geisler J, Lønning PE. Aromatase inhibition: translation into a suc-cessful therapeutic approach. Clin Cancer Res 2005;11:2809–21.

27. Lønning PE, Geisler J, Johannessen DC, Ekse D. Plasma estrogensuppression with aromatase inhibitors evaluated by a novel, sensitiveassay for estrone sulphate. J Steroid Biochem Mol Biol 1997;61:255–60.

28. Dowsett M, Goss PE, Powles TJ, Hutchinson G, Brodie AM, JeffcoateSL, et al. Use of the aromatase inhibitor 4-hydroxyandrostenedione inpostmenopausal breast cancer: optimization of therapeutic dose androute. Cancer Res 1987;47:1957–61.

29. Hankinson SE, Manson JE, Spiegelman D, Willett WC, Longcope C,Speizer FE. Reproducibility of plasma hormone levels in postmeno-pausal women over a 2-3-year period. Cancer Epidemiol BiomarkersPrev 1995;4:649–54.

30. Lønning PE, Helle H, Duong NK, Ekse D, Aas T, Geisler J. Tissueestradiol is selectively elevated in receptor positive breast cancerswhile tumour estrone is reduced independent of receptor status. JSteroid Biochem Mol Biol 2009;117:31–41.

31. Lønning PE, Kvinnsland S, Thorsen T, Ueland PM. Alterations in themetabolism of oestrogens during treatment with aminoglutethimide inbreast cancer patients. Preliminary findings. Clin Pharmacokinet1987;13:393–406.

32. Lee AJ, Cai MXX, Thomas PE, Conney AH, Zhu BT. Characterizationof the oxidative metabolites of 17beta-estradiol and estrone formedby 15 selectively expressed human cytochrome p450 isoforms.Endocrinology 2003;144:3382–98.

33. Zhou SF, Liu JP, Chowbay B. Polymorphism of human cytochromeP450 enzymes and its clinical impact. Drug Metab Rev 2009;41:89–295.

34. Bolt HM. Metabolism of estrogens—natural and synthetic. PharmacolTher 1979;4:155–81.

35. Martucci CP, Fishman J. P450 enzymes of estrogen metabolism.Pharmacol Ther 1993;57:237–57.

36. Lee AJ, Conney AH, Zhu BT. Human cytochrome P450 3A7 has adistinct high catalytic activity for the 16alpha-hydroxylation of estronebut not 17beta-estradiol. Cancer Res 2003;63:6532–6.

37. Hershcopf RJ, Bradlow HL, Fishman J. Differential hydroxylations ofestrone and estradiol in man. J Clin Endocrinol Metab 1986;62:170–3.

38. Huang ZQ, Fasco MJ, Figge HL, Keyomarsi K, Kaminsky LS. Expres-sion of cytochromes P450 in human breast tissue and tumors. DrugMetab Dispos 1996;24:899–905.

39. Cribb AE, Knight MJ, Dryer D, Guernsey J, Hender K, Tesch M, et al.Role of polymorphic human cytochrome P450 enzymes in estroneoxidation. Cancer Epidemiol Biomarkers Prev 2006;15:551–8.

40. Adlercreutz H, H€ockerstedt K, Bannwart C, Bloigu S, H€am€al€ainen E,Fotsis T, et al. Effect of dietary components, including lignans andphytoestrogens, on enterohepatic circulation and liver metabolism ofestrogens and on sex hormone binding globulin (SHBG). J SteroidBiochem 1987;27:1135–44.

41. Gurpide E. Metabolic influences on the action of estrogens: thera-peutic implications. Pediatrics 1978;62:1114–20.

42. Bulun SE, Simpson ER. Regulation of aromatase expression in humantissues. Breast Cancer Res Treat 1994;30:19–29.

43. Jacobs S, MacNeill F, Lønning P, Dowsett M, Jones A, Powles T.Aromatase activity, serum oestradiol and their correlation with demo-graphic indices. J Steroid Biochem Mol Biol 1992;41:769–71.

44. Shozu M, Sebastian S, Takayama K, Hsu WT, Schultz RA, Neely K,et al. Estrogen excess associated with novel gain-of-function muta-tions affecting the aromatase gene. N Engl J Med 2003;348:1855–65.

45. Smith EP, Boyd J, Frank GR, Takahashi H, Cohen RM, Specker B,et al. Estrogen resistance caused by a mutation in the estrogen-receptor gene in a man. N Engl J Med 1994;331:1056–61.

46. MaCX, Adjei AA, Salavaggione OE, Coronel J, Pelleymounter L, WangL, et al. Human aromatase: gene resequencing and functional geno-mics. Cancer Res 2005;65:11071–82.

47. Colomer R, Monzo M, Tusquets I, Rifa J, Baena JM, Barnadas A, et al.A single-nucleotide polymorphism in the aromatase gene is asso-ciated with the efficacy of the aromatase inhibitor letrozole inadvanced breast carcinoma. Clin Cancer Res 2008;14:811–6.

48. Wang LW, Ellsworth KA, Moon I, Pelleymounter LL, Eckloff BW,MartinYN, et al. Functional genetic polymorphisms in the aromatase geneCYP19 vary the response of breast cancer patients to neoadjuvanttherapy with aromatase inhibitors. Cancer Res 2010;70:319–28.

49. Lønning PE, Johannessen DC, Thorsen T. Alterations in the produc-tion rate and the metabolism of oestrone and oestrone sulphate inbreast cancer patients treated with aminoglutethimide. Br J Cancer1989;60:107–11.

50. Lønning PE, Ekse D. A sensitive assay for measurement of plasmaestrone sulphate in patients on treatment with aromatase inhibitors. JSteroid Biochem Mol Biol 1995;55:409–12.

51. Geisler J, Berntsen H, Lønning PE. A novel HPLC-RIA method for thesimultaneous detection of estrone, estradiol and estrone sulphatelevels in breast cancer tissue. J Steroid Biochem Mol Biol 2000;72:259–64.

52. Geisler J, Ekse D, Helle H, Duong NK, Lønning PE. An optimised,highly sensitive radioimmunoassay for the simultaneous measure-ment of estrone, estradiol and estrone sulfate in the ultra-low rangein human plasma samples. J Steroid Biochem Mol Biol 2008;109:90–5.

53. Geisler J, Helle H, Ekse D, Duong NK, Evans DB, Nordbø Y, et al.Letrozole is superior to anastrozole in suppressing breast cancertissue and plasma estrogen levels. Clin Cancer Res 2008;14:6330–5.

54. Xu X, Roman JM, Issaq HJ, Keefer LK, Veenstra TD, Ziegler RG.Quantitative measurement of endogenous estrogens and estrogenmetabolites in human serum by liquid chromatography-tandem massspectrometry. Anal Chem 2007;79:7813–21.

55. Santen RJ, Demers L, Ohorodnik S, Settlage J, Langecker P, Blan-chett D, et al. Superiority of gas chromatography/tandem massspectrometry assay (GC/MS/MS) for estradiol for monitoring of aro-matase inhibitor therapy. Steroids 2007;72:666–71.

56. Lee JS, Ettinger B, Stanczyk FZ, Vittinghoff E, Hanes V, Cauley JA,et al. Comparison of methods to measure low serum estradiollevels in postmenopausal women. J Clin Endocrinol Metab 2006;91:3791–7.

57. Corona G, Elia C, Casetta B, Da Ponte A, Del Pup L, Ottavian E, et al.Liquid chromatography tandemmass spectrometry assay for fast andsensitive quantification of estrone-sulfate. Clin Chim Acta 2010;411:574–80.






58. Blonder J, Johann DJ, Veenstra TD, Xiao Z, Emmert-Buck MR, ZieglerRG, et al. Quantitation of steroid hormones in thin fresh frozen tissuesections. Anal Chem 2008;80:8845–52.

59. Arai S, Miyashiro Y, Shibata Y, Kashiwagi B, Tomaru Y, Kobayashi M,et al. New quantificationmethod for estradiol in the prostatic tissues ofbenign prostatic hyperplasia using liquid chromatography-tandemmass spectrometry. Steroids 2010;75:13–9.

60. Longcope C, Femino A, Flood C, Williams KIH. Metabolic clearancerate and conversion ratios of [3H]2-hydroxyestrone in normal men. JClin Endocrinol Metab 1982;54:374–80.

61. Lønning PE, Skulstad P. Alterations in the urine excretion of estrogenmetabolites in breast cancer women treated with aminoglutethimide.J Steroid Biochem 1989;33[4A]:565–71.

62. Vermeulen A, Deslypere JP, Paridaens R, Leclercq G, Roy F, HeusonJC. Aromatase, 17 b-hydroxysteroid dehydrogenase and intratissularsex hormone concentrations in cancerous and normal glandularbreast tissue in postmenopausal women. Eur J Cancer Clin Oncol1986;22:515–25.

63. Chetrite GS, Cortes-Prieto J, Philippe JC, Wright F, Pasqualini JR.Comparison of estrogen concentrations, estrone sulfatase and aro-matase activities in normal, and in cancerous, human breast tissues. JSteroid Biochem Mol Biol 2000;72:23–7.

64. van Landeghem AA, Poortman J, Nabuurs M, Thijssen JHH. Endo-genous concentration and subcellular distribution of estrogens innormal and malignant human breast tissue. Cancer Res 1985;45:2900–6.

65. Larionov AA, Berstein LM, Miller WR. Local uptake and synthesis ofoestrone in normal and malignant postmenopausal breast tissues. JSteroid Biochem Mol Biol 2002;81:57–64.

66. Gurpide E, MacDonald PC, Wiele RLV, Lieberman S. Measurement ofthe rates of secretion and of peripheral metabolism of two intercon-vertible compounds: dehydroisoandrosterone-dehydroisoandroster-one sulfate. J Clin Endocrinol Metab 1963;23:346–54.

67. Rowland M, Tozer TN. Clinical pharmacokinetics and pharmacody-namics. 4th ed. Baltimore: Lippincott Williams & Wilkins; 2011.

68. Santner SJ, Leszczynski D, Wright C, Manni A, Feil PD, Santen RJ.Estrone sulfate: a potential source of estradiol in human breast cancertissues. Breast Cancer Res Treat 1986;7:35–44.

69. Geisler J, King N, Dowsett M, Ottestad L, Lundgren S, Walton P, et al.Influence of anastrozole (Arimidex), a selective, non-steroidal aroma-tase inhibitor, on in vivo aromatisation and plasma oestrogen levels inpostmenopausal women with breast cancer. Br J Cancer 1996;74:1286–91.

70. Geisler J, Haynes B, Anker G, Dowsett M, Lønning PE. Influence ofletrozole (Femara) and anastrozole on total body aromatization andplasma estrogen levels in postmenopausal breast cancer patientsevaluated in a randomized, cross-over-designed study. J Clin Oncol2002;20:751–7.

71. Reed MJ, Owen AM, Lai LC, Coldham NG, Ghilchik MW, Shaikh NA,et al. In situ oestrone synthesis in normal breast and breast tumourtissues: effect of treatment with 4-hydroxyandrostenedione. Int JCancer 1989;44:233–7.

72. Sasano H, Harada N. Intratumoral aromatase in human breast,endometrial, and ovarian malignancies. Endocr Rev 1998;19:593–607.

73. Sasano H, Anderson TJ, Silverberg SG, Santen RJ, Conway M,Edwards DP, et al. The validation of new aromatase monoclonalantibodies for immunohistochemistry—a correlation with biochemicalactivities in 46 cases of breast cancer. J Steroid Biochem Mol Biol2005;95:35–9.

74. Geisler J, Suzuki T, Helle H, Miki Y, Nagasaki S, Duong NK, et al.Breast cancer aromatase expression evaluated by the novel antibody677: correlations to intra-tumor estrogen levels and hormone receptorstatus. J Steroid Biochem Mol Biol 2010;118:237–41.

75. Haynes BP, Straume AH, Geisler J, A’Hern R, Helle H, Smith IE, et al.Intratumoral estrogen disposition in breast cancer. Clin Cancer Res2010;16:1790–801.

76. Poutanen M, Miettinen M, Vihko R. Differential estrogen substratespecificities for transiently expressed human placental 17 beta-hydro-xysteroid dehydrogenase and an endogenous enzyme expressed incultured COS-m6 cells. Endocrinology 1993;133:2639–44.

77. Penning TM, Burczynski ME, Jez JM, Hung CF, Lin HK, Ma H, et al.Human 3alpha-hydroxysteroid dehydrogenase isoforms (AKR1C1-AKR1C4) of the aldo-keto reductase superfamily: functional plasticityand tissue distribution reveals roles in the inactivation and formation ofmale and female sex hormones. Biochem J 2000;351:67–77.

78. Krazeisen A, Breitling R, Imai K, Fritz S, M€oller G, Adamski J. Deter-mination of cDNA, gene structure and chromosomal localization of thenovel human 17beta-hydroxysteroid dehydrogenase type 7(1). FEBSLett 1999;460:373–9.

79. Luu-The V, Tremblay P, Labrie F. Characterization of type 12 17beta-hydroxysteroid dehydrogenase, an isoform of type 3 17beta-hydro-xysteroid dehydrogenase responsible for estradiol formation inwomen. Mol Endocrinol 2006;20:437–43.

80. Wu L, Einstein M, Geissler WM, Chan HK, Elliston KO, Andersson S.Expression cloning and characterization of human 17 beta-hydro-xysteroid dehydrogenase type 2, a microsomal enzyme possessing20 alpha-hydroxysteroid dehydrogenase activity. J Biol Chem1993;268:12964–9.

81. He XY, Wegiel J, Yang YZ, Pullarkat R, Schulz H, Yang SY. Type 1017beta-hydroxysteroid dehydrogenase catalyzing the oxidation ofsteroid modulators of gamma-aminobutyric acid type A receptors.Mol Cell Endocrinol 2005;229:111–7.

82. Lukacik P, Keller B, Bunkoczi G, Kavanagh KL, Lee WH, Adamski J,et al. Structural and biochemical characterization of human orphanDHRS10 reveals a novel cytosolic enzyme with steroid dehydrogen-ase activity. Biochem J 2007;402:419–27. Erratum in Biochem J2007;403:615.

83. Everson RB, Lippman ME, Thompson EB, McGuire WL, Wittliff JL, DeSombre ER, et al. Clinical correlations of steroid receptors and malebreast cancer. Cancer Res 1980;40:991–7.

84. Perou CM, Sørlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA,et al. Molecular portraits of human breast tumours. Nature 2000;406:747–52.

85. Miller WR, Larionov A, Renshaw L, Anderson TJ, Walker JR, Krause A,et al. Gene expression profiles differentiating between breast cancersclinically responsive or resistant to letrozole. J Clin Oncol 2009;27:1382–7.

86. Dunning AM, Dowsett M, Healey CS, Tee L, Luben RN, Folkerd E, et al.Polymorphisms associated with circulating sex hormone levels inpostmenopausal women. J Natl Cancer Inst 2004;96:936–45.

87. Dunbier AK, Anderson H, Ghazoui Z, Folkerd EJ, A'hern R, CrowderRJ, et al. Relationship between plasma estradiol levels and estro-gen-responsive gene expression in estrogen receptor-positivebreast cancer in postmenopausal women. J Clin Oncol 2010;28:1161–7.

88. Frasor J, Stossi F, Danes JM, Komm B, Lyttle CR, KatzenellenbogenBS. Selective estrogen receptor modulators: discrimination of ago-nistic versus antagonistic activities by gene expression profiling inbreast cancer cells. Cancer Res 2004;64:1522–33.

Lønning et al.





2011;17:4948-4958. Published OnlineFirst July 26, 2011.Clin Cancer Res Per E. Lønning, Ben P. Haynes, Anne H. Straume, et al. Endocrine ManipulationExploring Breast Cancer Estrogen Disposition: The Basis for

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