PET imaging of oestrogen receptors in patients with breast cancer

Review

PET imaging of oestrogen receptors in patients with breast cancer Michel van Kruchten, Elisabeth G E de Vries, Myles Brown, Erik F J de Vries, Andor W J M Glaudemans, Rudi A J O Dierckx, Carolien P Schröder, Geke A P Hospers

Oestrogen receptors are overexpressed in around 70% of all breast cancers, and are a target for endocrine therapy. These receptors can be visualised on PET with use of 16α-[¹⁸F]-fluoro-17β-oestradiol (¹⁸F-FES) as a tracer. Compared with biopsy, which enables assessment of individual sites, whole-body ¹⁸F-FES-PET enables quantification of oestrogen-receptor expression in all metastases. In several studies, measurement of tumour protein expression in oestrogen receptors by ¹⁸F-FES-PET, concurrent with biopsy, detected oestrogen-receptor-positive tumour lesions with a sensitivity of 84% and specificity of 98%. Roughly 45% of patients with metastatic breast cancer have discordant oestrogen-receptor expression across lesions (ie, ¹⁸F-FES-positive and ¹⁸F-FES-negative metastases). Low tumour ¹⁸F-FES uptake in metastases can predict failure of hormonal therapy in patients with oestrogen-receptor-positive primary tumours. Finally, ¹⁸F-FES-PET has shown that oestrogen-receptor binding capacity changes after intervention with hormonal drugs, but findings need to be confirmed. Factors other than oestrogen-receptor expression, including menopausal status and concomitant therapies, that can affect tumour ¹⁸F-FES uptake must be taken into account.

Introduction Around 70% of all patients with breast cancer have oestrogen-receptor-positive tumours, which makes targeted endocrine therapy an attractive treatment option in adjuvant and metastatic settings. The treatment success rate in breast cancer relies heavily on the tumour oestrogen-receptor status, which is currently assessed by immunohistochemistry. Although immunohistochemistry is well suited to test primary breast tumours, accuracy is lower in metastases. Oestrogen-receptor expression can change over time, and discordant expression between primary tumours and metastases is seen in up to 40% of the patients.1 Biopsy might be useful to reassess a patient’s oestrogen-receptor status, but is not always feasible. Moreover, heterogeneous oestrogenreceptor expression can lead to sampling errors.2 PET with 16α-[¹⁸F]-fluoro-17β-oestradiol (¹⁸F-FES) enables non-invasive visualisation and quantification of oestrogen-receptor expression in all tumour lesions within a patient.3 In addition, this imaging technique can potentially provide in-vivo information about oestrogenreceptor binding of endocrine drugs. Insight into factors that might affect ¹⁸F-FES uptake, such as endogenous oestrogen concentrations and concurrent treatments, are relevant to the optimum use of this technique. In this Review, we summarise the role of oestrogen receptors in breast cancer, address the potential of molecular imaging of oestrogen-receptor expression, and discuss factors that potentially influence the uptake of ¹⁸F-FES.

Oestrogen receptors When natural (eg, oestradiol) or synthetic ligands (eg, ethynyloestradiol, tamoxifen) bind to oestrogen receptors, dimerisation and binding to specific DNA sequences occur and mediate the transcription of oestrogen-responsive genes. There are two oestrogenreceptor subtypes, α and β, which are encoded by different genes (ESR1 and ESR2) and are located, www.thelancet.com/oncology Vol 14 October 2013

respectively, on chromosomes 6q25.1 and 14q23.4 Being largely homologous in their DNA-binding domains (94%), both isoforms bind to similar DNA sequences.5 Differences in ligand-binding and activation-function domains mean that oestrogen-receptors α and β can bind various ligands with different affinity and exert opposite actions. The role of oestrogen-receptor β, however, is not fully understood, and drugs that selectively target the oestrogen-receptor β isoform have not yet been studied in clinical trials of breast-cancer treatment.6,7 Therefore, in this Review we focus on imaging of oestrogen receptor α (hereinafter referred to as oestrogen receptor). Three classes of endocrine treatments are available that prevent the activation of oestrogen receptors by oestrogens. Selective modulators competitively bind to oestrogen receptors and induce conformational changes that alter transcriptional activity. Aromatase inhibitors exert indirect effects on oestrogen receptors by inhibiting the conversion of androgens into oestrogens. Finally, fulvestrant, a selective oestrogen-receptor downregulator, competitively binds to and degrades receptors by increasing turnover rate. The sole predictive factor for response to endocrine therapy is the tumour oestrogen-receptor status.8 Patients with even marginally oestrogen-receptor-positive tumours have a survival advantage.9 Patients with oestrogenreceptor-negative tumours are unlikely to benefit from endocrine therapy.10 Absence of progesterone receptors in oestrogen-receptor-positive tumours is suggested to be a marker of poor response to endocrine therapy, but might in fact be related to poor prognosis in progesteronereceptor-negative disease. The benefit derived from endocrine therapy compared with placebo in patients with oestrogen-receptor-positive, progesterone-receptornegative disease is similar to that in oestrogen-receptorpositive, progesterone-receptor-positive disease.9 Thus, progesterone-receptor status has strong prognostic value but is of little predictive importance.

Lancet Oncol 2013; 14: e465–75 Department of Medical Oncology (M van Kruchten MD, Prof E G E de Vries MD, C P Schröder MD, Prof G A P Hospers MD) and Department of Nuclear Medicine and Molecular Imaging (E F J de Vries PhD, A W J M Glaudemans MD, Prof R A J O Dierckx MD), University of Groningen, University Medical Centre Groningen, Groningen, Netherlands; and Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA (Prof M Brown MD) Correspondence to: Prof Geke A P Hospers, Department of Medical Oncology, University Medical Centre Groningen, PO Box 30.001, 9700 RB Groningen, Netherlands [email protected]

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Discordant oestrogen-receptor expression In retrospective studies discordant oestrogen-receptor expression between the primary tumour and distant metastases was found in 14·5–40·0% of patients.11 Prospective data have shown loss of oestrogen-receptor expression in distant metastases in three (12%) of 25 patients with oestrogen-receptor-positive primary tumours12 and in 11 (16%) of 69 patients in another study.1 Gain of oestrogen-receptor expression was seen in the distant metastases of four (16%) of 25 patients with oestrogen-receptor-negative primary tumours.1 The clinical relevance of change in oestrogen-receptor status is supported by a retrospective study in 459 patients, in which oestrogen-receptor expression in metastases predicted overall survival independent of the status of the primary tumour.13 The researchers concluded that patients in whom a status switches from negative to positive might benefit from endocrine therapy.

PET imaging PET can be used to acquire functional information, such as oestrogen-receptor expression, by use of ligands labelled with positron-emitting isotopes. ¹⁸F-FES is the preferred tracer for visualisation of oestrogen-receptor expression in clinical studies. This tracer is intravenously injected and allowed to accumulate in tissues expressing oestrogen receptors for around 60 min before PET is done. The accumulated ¹⁸F-FES decays by emitting a positron that annihilates after collision with an electron, which results in two γ-ray photons of 511 keV that travel in opposite directions. The PET camera detects these photons as they arrive simultaneously at opposite detectors.14 The events are converted into threedimensional images by use of mathematical algorithms (figure 1). PET images can be combined with CT or MRI scans to link PET findings to anatomical information. Additionally, multimodal scanners have become available, such as PET-CT and PET-MRI. These scanners acquire anatomical information about the location, density, and size of lesions simultaneously with the functional information obtained by PET. Current PET-CT cameras have

See Online for appendix

A

B

γ

e– β+

γ

β+emission [18F] v

Neutrino

Figure 1: Principles of PET (A) A positron collides with an electron nearby and annihilates, resulting in two antiparallel photons. (B) Accumulated ¹⁸F-fluoro-oestradiol is detected by the γ camera.

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integrated CT cameras of between 16 and 256 slices and a PET camera with resolution as high as 2 mm. For wholebody PET imaging, cameras scan for 1–3 min per bed position. Images from seven to eight bed positions (skull to mid-thigh) are obtained. In whole-body PET studies, tracer uptake is typically quantified as the standardised uptake value (SUV), which is calculated as radioactivity in the volume of interest (kBq/mL) divided by the injected dose per kg bodyweight (MBq/kg).15 SUV can be affected by the partial volume effect, which is especially prominent in lesions with sizes that approach the spatial resolution of the PET camera. MRI and CT can correct for this effect. Accuracy of oestrogen-receptor-density measurements can be improved with kinetic modelling studies. These studies, however, require arterial blood sampling, analysis of ¹⁸F-FES metabolism, and dynamic imaging during 60–90 min in one bed position, which precludes wholebody imaging. Thus, kinetic modelling studies are not feasible in routine clinical practice, but should be done to validate SUV for oestrogen-receptor expression.

PET tracers The most potent endogenous oestrogen-receptor agonist is oestradiol (figure 2). Only a small percentage (1–3%) of total oestradiol circulates in a biologically active form; most is bound to plasma carrier proteins, such as sex hormone binding globulin (SHBG) and albumin. The development of PET tracers that target oestrogen receptors has focused on radiolabelling of oestradiol and structural analogues. Around 20 ¹⁸F-labelled oestrogen analogues have been described. Of these, ¹⁸F-FES (figure 2) is the most extensively characterised and is most frequently used in clinical studies (appendix). ¹⁸F-FES has a 60–100% relative binding affinity for oestrogen receptor. Affinity for oestrogen-receptor α is 6·3 times higher than that for oestrogen-receptor β, and greater specificity has been shown in vivo in knockout mouse studies.16 Binding characteristics are affected by substituents and by the position of the ¹⁸F atom. For example, the addition of ethynyl at the 11β or 17α position of ¹⁸F-FES increases oestrogen-receptor binding affinity,17 but 11β-methoxy substitution decreases affinity.18,19 The increased affinity of ethynyl oestrogens for ER coincides with raised nonspecific binding owing to increased lipophilicity.20 In animal studies, various oestrogen-receptor tracers showed specific uptake in the uterus and ovaries that could be blocked by the addition of unlabelled oestradiol.21 In mice, oestrogen-receptor-positive murine mammary adenocarcinomas specifically take up ¹⁸F-FES, whereas uptake is absent in oestrogen-receptor knockdown tumours.22 In rats with carcinogen-induced mammary tumours, ¹⁸F-FES uptake is seen in tumour lesions that correlates with ex-vivo quantitative measurements of oestrogen-receptor concentrations.23,24 All radiolabelled oestrogens are rapidly metabolised in the liver and excreted via the gastrointestinal tract in bile and via the kidneys in urine. To decrease the rate of www.thelancet.com/oncology Vol 14 October 2013

Review

metabolism, substituents can be added at the A-ring, where most of the conjugation of ¹⁸F-FES occurs. In rats, an ¹⁸F atom in the A-ring of ¹⁸F-FES resulted in lower liver uptake and slightly increased uterus-to-blood ratios, which were ascribed to decelerated metabolism.25 Whether the tracer’s affinity for SHBG affects tumour uptake is not entirely clear. SHBG regulates the bioavailability of steroids and, therefore, tracers with low affinity for SHBG were suggested for oestrogenreceptor-targeted PET.26,27 In 239 patients with metastatic breast cancer, ¹⁸F-FES uptake in tumours decreased with increasing SHBG concentrations in serum and binding of ¹⁸F-FES to SHBG.28 Paradoxically, ¹⁸F-fluoromoxoestrol, an oestrogen-receptor tracer with 256 times lower binding affinity for SHBG than ¹⁸F-FES, did not improve results in a clinical study. Excellent oestrogenreceptor-mediated uptake was seen in rodents that lack SHBG, but in a later clinical study, none of three oestrogen-receptor-positive primary breast tumours could be visualised with ¹⁸F-fluoro-moxoestrol PET.29,30 These findings could not be explained by resolution limitations of the PET camera because the tumours were larger than 15 mm. To ensure delivery of the tracer to tumours in human beings, therefore, oestrogenreceptor tracers must have good binding affinity for SHBG. Development of other oestrogen-receptor tracers deserves further exploration. For PET imaging, those that are less extensively cleared by the liver might improve visualisation of liver metastases. Oestrogenreceptor tracers with increased binding affinity may well improve tumour-to-background ratios.31 Labelling of PET tracers specific for the oestrogen-receptor-β isoform might be of interest in other tumour types.32 Singlephoton emission CT, which is widely available, with ⁹⁹mT and iodine-labelled oestrogen-receptor ligands, have been assessed in small clinical studies.33–35 Finally, nonradioactive oestrogen-receptor tracers for use in MRI or near-infrared fluorescent optical imaging to guide surgery could be of clinical use.36 ¹⁸F-FES is deemed an investigational new drug and, therefore, relevant documents might be needed for its use in clinical trials. Quality-control tests and modifications might also be needed to match local requirements.

A

B

OH 11 C

D

H

H 16 H

2 A

OH

17

18

F

B HO

HO 4

Figure 2: Structures of 17β-oestradiol and 16α-[¹⁸F]-17β-oestradiol (A) 17β-oestradiol consists of four cycloalkane rings and two hydroxyl groups. The numbers indicate commonly used positions for substituents. (B) 16α-[¹⁸F]-17β-oestradiol.

¹⁸F-FES-PET in healthy volunteers Typically, 200 MBq ¹⁸F-FES is injected before PET, with a specific activity higher than 25 000 GBq/mmol, which comprises an injected mass of less than 8 nmol ¹⁸F-FES. ¹⁸F-FES is initially metabolised rapidly, and less than 20% of the injected dose remains circulating as unconjugated ¹⁸F-FES after 10 min. Glucoronide-conjugated and sulphatase-conjugated ¹⁸F-FES is excreted via the gut in bile. As a result of enterohepatic circulation, only a low amount of ¹⁸F-FES enters the colon. The reabsorbed conjugated ¹⁸F-FES is cleared mainly by the kidneys. After the initial rapid decline, the activity of unconjugated ¹⁸F-FES blood declines slowly, over around 50 min, and by 60 min reaches less than 5% of peak value. The presence of conjugated ¹⁸F-FES metabolites is unlikely to affect tumour ¹⁸F-FES uptake because they cannot bind oestrogen receptors and their polarity prevents penetration of cell membranes.23,59 Unconjugated ¹⁸F-FES is bound to carrier proteins SHBG (45%) and albumin (45%) in serum, and the remainder circulates in free form.56 The radiation burden for patients is 0·022 mSv/MBq, which results in 4·4 mSv per 200 MBq ¹⁸F-FES injection. The organs exposed to the highest radiation doses are the liver (0·13 mGy/MBq), gallbladder (0·10 mGy/MBq), and bladder (0·05 mGy/MBq).55 Oestrogen-receptorspecific uptake has been seen in the uteri of healthy premenopausal volunteers with SUVs of roughly 2·5 in the myometrium and 4·0–6·0 in the endometrium.50 Endometrial SUVs were higher in the proliferative phase of the menstrual cycle than in the secretory phase.

For the relevant documents see http://imaging.cancer.gov/ programsandresources/fesdocumentation

Sensitivity and specificity

Clinical studies Multiple studies of ¹⁸F-FES-PET have been done in several countries (Italy, Japan, the Netherlands, and the USA; table 1).3,37–62 As of May, 2013, 13 further continuing ¹⁸F-FES-PET studies in breast cancer plus one in ovarian cancer are registered with ClinicalTrials.gov—seven in the USA, three in the Netherlands, two in Canada, one in France, and one in South Korea. ¹⁸F-FES-PET studies have also been reported in endometrial cancer,43,44,51 uterine tumours,37,40,49 meningiomas,58 and ovarian cancer.45 www.thelancet.com/oncology Vol 14 October 2013

Four studies involving 114 patients have assessed sensitivity and specificity of ¹⁸F-FES-PET for oestrogenreceptor-positive breast-cancer lesions concurrently with in-vitro assay for oestrogen-receptor expression (table 2).3,47,60,61 Sensitivity was good overall and quantitative ¹⁸F-FES uptake correlated moderately to excellently with oestrogen-receptor expression (r=0·56–0·96).3,47 Metastases can be seen with ¹⁸F-FESPET, such as in the liver, bone, lung, and lymph nodes, and physiological uptake can be seen in organs such as the bile duct, intestinal tract, and bladder (figure 3). e467

Review

Disorder

Number of Study aim patients

Conclusions

Zhao et al, 201337

Uterine tumours

47

Assess relation between tumour ¹⁸F-FDG and ¹⁸F-FES uptake with ER, GLUT-1, and Ki-67

¹⁸F-FES uptake correlated with ERα and PR expression, and ¹⁸F-FDG-uptake with GLUT-1 and Ki-67

Van Kruchten et al, 201238

Breast cancer

33

Assess clinical value of ¹⁸F-FES-PET in patients with unresolved diagnosis after conventional workup

¹⁸F-FES-PET aided diagnosis and therapy decision making

Peterson et al, 201139

Breast cancer

239

Assess correlations between ¹⁸F-FES uptake and clinical and laboratory data, effects of previous treatments and ¹⁸F-FES metabolism

¹⁸F-FES uptake correlated positively with BMI and inversely with plasma SHBG levels and binding capacity

Yoshida et al, 201140

Suspected uterine sarcoma

24

Assess usefulness of ¹⁸F-FES-PET and ¹⁸FDG-PET to differentiate uterine sarcoma from leiomyoma

¹⁸F-FDG-to-¹⁸F-FES ratios >2·0 differentiated with 90·9% sensitivity and 92·3% specificity

Kurland et al, 201141

Metastatic breast cancer

91

Measure variability in ¹⁸F-FES uptake between and within patients

Substantial variation seen between patients (37% had low or absent uptake; roughly 40% had mixed uptake)

Linden et al, 201142

Metastatic breast cancer

30

Measure changes in ¹⁸F-FES uptake during treatment with aromatase inhibitors, tamoxifen, or fulvestrant

No effect with aromatase inhibitors, decreases of around 55% with tamoxifen and fulvestrant

Tsujikawa et al, 200943

Endometrial cancer

19

Assess correlation between ¹⁸F-FES-PET, ¹⁸F-FDG-PET, and ER-status on immunohistochemistry

Correlations for tumour ERα status good

Tsujikawa et al, 200944

Endometrial cancer

31

Assess correlation between ¹⁸F-FES-PET, ¹⁸F-FDG-PET, and clinicopathological features

¹⁸F-FES-to-¹⁸F-FDG ratios correlated with tumour aggressiveness

Yoshida et al, 200945

Ovarian cancer

Review role of PET in ovarian cancer (plus preliminary results of ¹⁸F-FES-PET in three patients)

¹⁸F-FES uptake was seen in ER-positive tumours

Dehdashti et al, 200946

Metastatic breast cancer

59

Investigate whether ¹⁸F-FES-PET and serial ¹⁸F-FDG-PET (plus Baseline tumour ¹⁸F-FES uptake and metabolic flare after oestradiol challenge) predicts response to endocrine therapy oestradiol challenge both predictive of treatment response

Peterson et al, 200847

Primary and metastatic breast cancer

17

Assess correlation between ¹⁸F-FES uptake and immunohistochemistry

Good correlation for ERα

Tsujikawa et al, 200848

Endometrial hyperplasia

Assess effect of tamoxifen on ¹⁸F-FES uptake

Take endocrine therapy into account when using ¹⁸F-FES-PET to assess ER status

Tsujikawa et al, 200849

Benign and malignant uterine tumours

38

Assess relation between ¹⁸F-FES and ¹⁸F-FDG uptake in benign and malignant uterine tumours

¹⁸F-FES-to-¹⁸F-FDG ratios aided differentiation of diagnosis of uterine tumours

Tsuchida et al, 200750

Healthy volunteers

16

Assess relation between ¹⁸F-FES uptake, menstrual cycle, and endogenous oestrogen concentrations

Changes in ¹⁸F-FES uptake were consistent with changes in ER concentrations on immunohistochemistry

Yoshida et al, 200751

Endometrial cancer

1

Use ¹⁸F-FES-PET to assess response to medoxyprogesterone in endometrial cancer

Focal uptake, confirmed by histology

Kanne et al, 200752

Metastatic breast cancer

1

Investigate use of ¹⁸F-FES-PET and ¹⁸F-FDG-PET in a patient with gastric linitis plastica from metastasis of ER-positive lobular breast cancer

¹⁸F-FES-PET confirmed regional ER binding in metastases

Linden et al, 200653

Metastatic breast cancer

47

Quantify tumour ¹⁸F-FES uptake as predictor of response to endocrine therapy

Absence of uptake predicts failure of endocrine therapy

Mortimer et al, 200154

Breast cancer

40

Assess serial ¹⁸F-FES-PET and ¹⁸F-FDG-PET to predict response to tamoxifen

Increase in ¹⁸F-FDG uptake and decrease in ¹⁸F-FES uptake after the start of tamoxifen predicted response

Mankoff et al, 200155

Breast cancer

49

Radiation dosimetry of ¹⁸F-FES-PET

Radiation dose is similar to commonly used nuclear medicine tests

Tewson et al, 199956

Breast cancer

18

Assess interaction between SHBG and ¹⁸F-FES

Around 45% of ¹⁸F-FES was bound to SHBG and could affect tracer uptake

Dehdashti et al, 199957

Metastatic breast cancer

11

Asses serial ¹⁸F-FES-PET and ¹⁸F-FDG-PET to predict response to tamoxifen

Increase in ¹⁸F-FDG uptake and decrease in ¹⁸F-FES uptake after the start of tamoxifen predicted response

Moresco et al, 199758

Meningioma

Assess ER-status of meningiomas by means of ¹⁸F-FES-PET

Four of six patients showed focal ¹⁸F-FES uptake, and uptake correlated with immunohistochemistry status in five of six patients

Mankoff et al, 199759

Primary or metastatic breast cancer

15

Assess clearance of labelled ¹⁸F-FES metabolites

Rapid clearance

Mortimer et al, 199660

Primary or metastatic breast cancer

43

Assess correlation between ¹⁸F-FES-PET and ¹⁸F-FDG and in-vitro assays for response to therapy

¹⁸F-FES-PET had a sensitivity of 76% and specificity of 100% compared with immunohistochemistry

Dehdashti et al, 199561

Primary or metastatic breast cancer

53

Compare ¹⁸F-FES-PET with ¹⁸F-FDG-PET and immunohistochemistry

¹⁸F-FES-PET 88% agreement with immunohistochemistry and provided information not obtained by ¹⁸F-FDG-PET

McGuire et al, 199162

Metastatic breast cancer

16

Assess use of ¹⁸F-FES-PET in ER-positive metastatic postmenopausal breast cancer

¹⁸F-FES-PET sensitivity 93%

Mintun et al, 19893

Primary breast cancer

13

Assess feasibility of ¹⁸F-FES-PET to detect primary ER-positive Focal uptake seen in all patients with ¹⁸F-FES and uptake breast-tumour lesions and correlation with in-vitro ER status correlated well with in-vitro assays (r=0·96)

3

2

6

¹⁸F-FES=16α-[¹⁸F]-fluoro-17β-oestradiol. ¹⁸F-FDG=2-deoxy-[¹⁸F]fluoro-D-deoxyglucose. ER=oestrogen receptor. PR=progesterone receptor. BMI=body-mass index. SHBG=sex-hormone-binding globulin.

Table 1: Overview of clinical ¹⁸F-FES-PET studies

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In one study, ¹⁸F-FES-PET visualised only six of ten oestrogen-receptor-positive primary breast tumours,61 although the four patients with false-negative findings were aged 34–45 years and were probably premenopausal; the ages of the six patients with true-positive ¹⁸F-FESPET scans ranged from 56 to 71 years. Premenopausal patients might have impaired uptake of ¹⁸F-FES because of competitive binding by endogenous oestrogens, but this hypothesis needs to be tested in a large sample. Other studies have included either only postmenopausal patients or have not calculated the correlation between menopausal status and ¹⁸F-FES-PET results. As well as variations in selection criteria for patients, the studies used different PET cameras, definitions of ¹⁸F-FES positivity, and methods of quantification analysis (tumour-to-background ratio vs SUV). High specificity of ¹⁸F-FES for oestrogen receptors is illustrated by the absence of ¹⁸F-FES uptake in benign lesions and oestrogen-receptor-negative breast-cancer lesions. Combined data from three studies showed that 51 of 52 histologically benign or oestrogen-receptornegative lesions were also ¹⁸F-FES negative (SUV lower than 1·0) and, therefore, overall specificity is 98% (table 2).47,60,61

Larger studies are needed to determine the predictive value of ¹⁸F-FES-PET. So far, differences between studies in predictive values for response to endocrine therapy Biopsy results

Sensitivity or specificity (95% CI) Number of lesions

Sensitivity Dehdashti et al61

ER-positive tumour

69% (44–86)

Mintun et al3

ER-positive tumour

100% (77–100)

16 13

Mortimer et al60

ER-positive tumour

76% (55–89)

21

Peterson et al47

ER-positive tumour

100% (76–100)

12

Overall

..

84% (73–91)

62 10

Specificity Dehdashti et al61

Benign

100% (68–100)

Dehdashti et al61

ER-negative tumour

100% (82–100)

17

Mortimer et al60

ER-negative tumour

100% (84–100)

20

Peterson et al47

ER-negative tumour

80% (38–96)

5

Overall

..

98% (90–100)

52

ER=oestrogen receptor.

Table 2: Sensitivity and specificity of 16α[¹⁸F]-fluoro-17β-oestradiol PET

Prediction of treatment response Four studies reported the predictive value of ¹⁸F-FES uptake by tumours for response to endocrine therapy in 138 metastatic patients with breast cancer.46,53,54,57 All patients initially had oestrogen-receptor-positive primary tumours. ¹⁸F-FES-PET was done before a new line of endocrine therapy was started: 76 received aromatase inhibitors, 45 tamoxifen, and 17 fulvestrant. In one study, tamoxifen was discontinued at least 60 days before ¹⁸F-FES-PET to avoid competitive binding to the oestrogen receptor by tamoxifen and its metabolites. In the other studies, drug-free periods were not reported. Thresholds of 1·5 and 2·0 tumour ¹⁸F-FES SUV to select patients for endocrine therapy were assessed. 67 (49%) of 138 patients who received endocrine therapy showed clinical benefits according to response evaluation criteria in solid tumours (objective tumour response or stable disease for 6 months or longer) or clinical assessment. If the 1·5 threshold for tumour ¹⁸F-FES SUV had been used, 96 would have received treatment, of whom 62 would have experienced clinical benefits (positive predictive value 65%). In 42 patients with tumour ¹⁸F-FES SUV lower than 1·5, 37 showed no clinical benefit from endocrine therapy (negative predictive value 88%). Hence, in patients with a previously oestrogenreceptor-positive primary tumour, ¹⁸F-FES SUV lower than 1·5 predicts failure to respond to endocrine therapy. If denial of endocrine treatment had been based on a tumour ¹⁸F-FES SUV threshold of 2·0 instead of lower than 1·5, a notable proportion of patients who would have responded to treatment would not receive it (19 [31%] of 62, figure 4). www.thelancet.com/oncology Vol 14 October 2013

Physiological uptake

Bone metastasis

Liver metastasis

Nodal metastasis

Figure 3: Oestrogen-receptor-positive metastases on ¹⁸F-FES-PET Physiological ¹⁸F-FES uptake can be seen in the liver, bile duct, intestinal tract, and bladder. A limited number of oestrogen-receptor-positive bone, liver, and nodal metastases are also indicated. ¹⁸F-FES=16α-[¹⁸F]-fluoro-17β-oestradiol.

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F-FES >1·5 SUV 5 (4%)

F-FES >2·0 SUV

18

18

FES-positive responder FES- positive non-responder FES -negative responder FES -negative non-responder

19 (14%) 48 (35%)

37 (27%) 62 (45%) 43 (31%)

34 (25%)

28 (20%)

Figure 4: Predictive value of ¹⁸F-FES-PET (n=138 patients) In 138 patients, use of an SUV higher than 2·0 compared with a value of 1·5 did not increase the positive predictive value of ¹⁸F-FES-PET, 46,53,54,57 whereas the negative value dropped. ¹⁸F-FES=16α-[¹⁸F]-fluoro-17β-oestradiol. SUV=standardised uptake value.

and cutoff values, and the small number of patients studied have led to low-level evidence (level 3).63

Assessment of heterogeneous oestrogenreceptor expression One of the advantages of ¹⁸F-FES-PET is that it provides a whole-body indication of oestrogen-receptor expression across metastases. Most studies have so far used a cutoff SUV of 1·5 to classify ¹⁸F-FES-positive and ¹⁸F-FESnegative results. With use of this threshold, up to 37% of patients with a previously oestrogen-receptor-positive primary breast tumour develop ¹⁸F-FES-negative metastatic disease.41 Additionally, in three studies that compared ¹⁸F-FES-PET with 2-deoxy-¹⁸F-fluorodeoxyglucose PET in 107 patients, 15–47% of the patients had ¹⁸F-FES-positive and ¹⁸F-FES-negative tumour sites.60,61,64 Another study showed no ¹⁸F-FES uptake in one or more metastases in ten (45%) of the 22 patients with at least one ¹⁸F-FESpositive lesion.38 Furthermore, discordant oestrogenreceptor expression is supported by up to a six times difference in quantitative tumour ¹⁸F-FES uptake across metastases within an individual, and has a high coefficient of variance (30–68%).38,41,65 These data strongly suggest site-to-site variability in oestrogen-receptor expression across metastases within individual patients. Early results of a phase 2 study in 15 patients with newly diagnosed metastatic breast cancer in three (75%) of four patients with at least one metastasis and visually absent ¹⁸F-FES uptake showed disease progression. By contrast, eight (73%) of 11 patients without discordant sites showed clinical benefit with endocrine therapy.66 Final results of this and another prospective study are awaited (NCT00602043 and NCT00358098).

In-vivo oestrogen-receptor binding In two studies, a change in ¹⁸F-FES uptake during endocrine therapy was investigated as an early predictor of treatment response. In a prospective study in 40 postmenopausal patients with locally advanced and metastatic breast cancer, a decrease in ¹⁸F-FES uptake was e470

seen 7–10 days after tamoxifen was started. Patients who showed clinical benefits had greater decreases in ¹⁸F-FES uptake (55% [SD 14%]) than did non-responding patients (19% [17%]).54 Whether the 7–10 day period is the optimum time to assess the effects of tamoxifen on oestrogenreceptor binding is unclear, since concentrations of tamoxifen metabolites take 4–6 weeks to become stable.67,68 Nevertheless, the results are interesting because response to endocrine therapy can take several months to be shown accurately by anatomical imaging methods. A retrospective study of ¹⁸F-FES-PET in patients with metastatic breast cancer receiving fulvestrant (n=11) or tamoxifen (n=5) showed 49% and 55% decreases in ¹⁸F-FES uptake from baseline, respectively, during treatment.42 Tumour ¹⁸F-FES uptake was decreased more with tamoxifen than with fulvestrant on the basis of post-treatment SUV values lower than 1·5 (tamoxifen five of five patients vs fulvestrant four of 11 patients; p=0·019). Fulvestrant, however, was given as 250 mg intramuscularly (with or without a loading dose of 500 mg) rather than 500 mg every 4 weeks plus a 500 mg loading dose on day 14, which has since been approved. Nevertheless, the inadequacy of fulvestrant 250 mg to block tumour FES-uptake remains of interest in light of the improved efficacy of the 500 mg dose.69 A trial is underway in 15 patients to investigate residual binding capacity during treatment with the 500 mg fulvestrant regimen (NCT01377324). ¹⁸F-FES-PET could offer the opportunity to assess binding of novel oestrogenreceptor antagonists in early clinical trials and at various dose levels. Absolute and relative decreases in ¹⁸F-FES uptake by oestrogen-receptor antagonists varies widely, which could be related to variability in serum and tumour drug concentrations and conversion of the drugs into more potent metabolites. For instance, tumour concentrations of the tamoxifen metabolite hydroxytamoxifen ranged from 0·4 to 564·5 ng/g in patients with oestrogenreceptor-positive tumours treated with 20 mg taxmoxifen daily for 28 days.67,68 Monitoring of drug pharmacokinetics together with molecular imaging could provide insight into the relevance of drug concentrations in plasma to achieve sufficient oestrogen-receptor binding in the tumour. Adequately powered prospective studies are warranted to find out whether serial ¹⁸F-FES-PET has a role during drug development or in the clinic as an early predictor of treatment response.

Use of ¹⁸F-FES-PET during treatment with aromatase inhibitors Treatment with aromatase inhibitors would theoretically be expected to increase tumour ¹⁸F-FES uptake by reducing competitive binding, but only a slight decrease (13%) was detected soon after the start of treatment in a retrospective study of 14 patients.42 A potential explanation for this finding is that a plateau in ¹⁸F-FES binding had been reached by the time of assessment. Thus, an www.thelancet.com/oncology Vol 14 October 2013

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increase in available oestrogen-receptor binding sites might not result in an increased maximum SUV. Moreover, results might be different after long-term exposure to aromatase inhibitors. Indeed, in the same study, patients who started fulvestrant had higher baseline tumour ¹⁸F-FES uptake than other patients (tumour ¹⁸F-FES SUV 3·8 vs 2·3) and, of these 11 patients, ten were already taking chronic aromatase inhibitors treatment when baseline ¹⁸F-FES-PET was done. Theoretically, therefore, this increased ¹⁸F-FES uptake could be the consequence of an increase in the percentage of free oestrogen-receptor binding sites, or an absolute increase in oestrogen-receptor expression, for instance as a cellular response to long-term oestrogen-deprivation.

Timing of ¹⁸F-FES-PET in relation to concomitant therapies Since oestrogen-receptor antagonists clearly affect tracer uptake, most studies require a drug-free period of 6–8 weeks before baseline quantitative measurements are taken. Whether this time is sufficient to completely eliminate competitive binding is unknown, particularly for fulvestrant, which has a half-life of 40 days and both blocks and degrades oestrogen receptors. A longer drugfree period, therefore, might be needed. Moreover, tumour drug concentrations could remain high for a long period despite normal concentrations in plasma. Several drug metabolites should also be taken into account (especially for tamoxifen). More work is required to define the optimum times to do ¹⁸F-FES-PET.

Effects on tumour ¹⁸F-FES uptake Tumour-cell-specific mechanisms Endocrine resistance, which can be intrinsic or acquired, is a common complication. Resistance can occur with lost, decreased, or preserved oestrogen-receptor expression. Lost or decreased oestrogen-receptor expression might be caused by various mechanisms. Among them are oestrogen-receptor-negative cancer stem cells that drive metastasis formation,70 clonal selection of endocrine resistant cells,71,72 and epigenetic changes.73,74 The fact that low or absent ¹⁸F-FES uptake is a good predictor of endocrine resistance in clinical studies makes this method relevant to assessment of drug resistance. Endocrine resistance with preserved oestrogenreceptor expression can be the result of ligandindependent activation of the oestrogen receptor via crosstalk with other receptors, such as EGFR and the downstream mTOR.75 In case of a preserved ligandbinding domain, ¹⁸F-FES will still bind to tumour oestrogen receptors. In these instances, oestrogenreceptor degradation by fulvestrant might remain effective. Endocrine therapy combined with other targeted therapies might also bypass ligand-independent activation of oestrogen receptors: mTOR inhibition combined with aromatase inhibition has shown promising results in patients with endocrine-resistant www.thelancet.com/oncology Vol 14 October 2013

breast cancer.76 Finally, mutations in the gene ESR1 can lead to alterations in function and ligand binding affinity. When point mutations are artificially introduced in the ligand domain, such as Leu536Asn and Gly525Leu, ligand-binding affinity is strongly reduced in vitro77,78 and, therefore, ¹⁸F-FES-uptake would also be reduced. Other mutations, such as Lys303Arg, have been identified in breast cancers79 and have been associated with resistance to endocrine therapy in preclinical models.80 Further studies are needed to address the relevance and frequency of oestrogen-receptor mutations during disease progression. Several mechanisms of endocrine resistance are purported, with various hypothesised effects on tumour ¹⁸F-FES uptake and possible consequences for treatment (figure 5). Serial ¹⁸F-FES-PET might provide some insight into changes in oestrogen receptors during disease progression and how endocrine resistance develops and, in combination with tumour biopsy and detailed genotyping, improve the selection of patients for treatment.

Menopausal status Endogenous oestrogens should theoretically lessen tracer uptake by competitive binding. Owing to oestrogen-receptor-driven uptake, oestradiol concentrations are up to ten times higher in oestrogenreceptive-positive tumours than in plasma.81 Furthermore, intratumour oestradiol concentrations correlate with those in plasma, which suggests that tumour oestradiol concentrations are higher in premenopausal than in postmenopausal patients. Indeed, concentrations of oestradiol within the mastectomy tissue of 47 patients with oestrogen-receptor-positive breast cancers were six times higher in premenopausal patients than in postmenopausal patients (1621·8 vs 267·1 fmol/g).82 ¹⁸F-FES-PET sensitivity seemed to be poor in a study that included premenopausal patients. Despite this observation, whether endogenous oestrogen concentrations are sufficiently high to have a notable effect on tumour ¹⁸F-FES uptake is unclear. A retrospective study found no significant association between serum oestradiol concentrations and tumour ¹⁸F-FES uptake.39 Furthermore, neither low specific activity of ¹⁸F-FES nor higher injected mass of unlabelled oestradiol were associated with decreased tumour ¹⁸F-FES uptake, which suggests that the exogenous dose of ¹⁸F-FES does not saturate the oestrogen receptors.39 Finally, in 14 patients treated with aromatase inhibitors, which was expected to increase ¹⁸F-FES uptake by reducing competitive binding, serial ¹⁸F-FES-PET imaging showed no effect. In the absence of concurrent biopsies, and the broad variability between patients in oestradiol concentrations, tumour oestrogen-receptor expression and injected mass of ¹⁸F-FES makes precise analysis of the effects of background oestradiol concentrations in individual patients difficult. e471

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Primary tumour Mixed expression

Cross-talk with other receptors E2

Metastases at disease progression Changed AF domain: altered response to ligand

Preserved ligand binding

Loss of ligand binding

Result 18 F-FES-PET

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Effectiveness of endocrine therapy

Aromatase inhibitors, tamoxifen, fulvestrant effective

Fulvestrant potentially effective

Oestrogen-receptor-targeted therapies not effective

Aromatase inhibitors, tamoxifen, fulvestrant partly effective

Figure 5: Potential mechanisms of resistance to oestrogen-receptor-targeted therapy, expected ¹⁸F-FES-PET results, and consequences for treatment Oestrogen-receptor-positive tumour cells are indicated in blue and oestrogen-receptor-negative cells in green. AF=activation function. ¹⁸F-FES=16α-[¹⁸F]-fluoro-17β-oestradiol.

Body composition ¹⁸F-FES is lipophilic and, therefore, patients with increased fat mass could be expected to have lower tumour ¹⁸F-FES uptake than leaner patients because of an increased pharmacological distribution volume, as suggested by animal studies.25 Additionally, raised concentrations of oestrogen in serum in obese patients might be expected to further decrease ¹⁸F-FES uptake because of competitive binding to oestrogen receptors. Paradoxically, however, high body-mass index correlated with increased tumour ¹⁸F-FES uptake in a study.39 Likewise, in human dosimetry studies no notable uptake of ¹⁸F-FES in fat tissue was observed.55 98% of the circulating ¹⁸F-FES is bound to plasma carrier proteins and, therefore, its distribution volume is probably not affected by body mass in human beings, or ¹⁸F-FES accumulation in fat mass is of minor importance. The increased tumour SUV seen in patients with high bodymass index is possibly a result of adjustments for patients’ weights rather than an absolute increase.

Liver function and metabolism Despite the rapid hepatic uptake and metabolism of ¹⁸F-FES, liver function is unlikely to affect quantitative measurements of oestrogen-receptor expression. Correction for individual differences in blood clearance and metabolites did not improve the correlation between e472

¹⁸F-FES uptake and semiquantitative in-vitro assays.47 In a large retrospective study involving 278 patients with oestrogen-receptor-positive metastatic breast cancer, no association was found between the rate of ¹⁸F-FES metabolism and tumour ¹⁸F-FES uptake.39

Concentrations of sex-hormone-binding globulin in serum High SHBG concentrations in serum decrease the bioavailability of free circulating ¹⁸F-FES. Concentrations of SHBG can vary greatly between patients, ranging from 2 nmol/L to 200 nmol/L.39 Moreover, treatments can lead to changes in SHBG concentrations in serum. SHBG production by the liver is increased by 40–50% during tamoxifen treatment because of the oestrogenic effects of this drug and decreased by 40–50% because of the actions of aromatase inhibitors, but is unaffected by fulvestrant.67,83–85 Additionally, SHBG concentrations in serum are higher in patients with oestrogen-receptorpositive breast cancer than in those with oestrogenreceptor-negative tumours, and in lean patients than in obese patients.86 In a retrospective study of 284 scans that assessed the association between tumour ¹⁸F-FES uptake and SHBG concentrations, 5–7% reductions in ¹⁸F-FES uptake were observed for every 1·5 nmol/L increase in the square root of SHBG in serum.39 No biopsy data were available, however, so whether www.thelancet.com/oncology Vol 14 October 2013

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Search strategy and selection criteria We searched PubMed and Scopus with one or more combinations of the following terms: “antihormonal”, “breast cancer”, “(o)estradiol”, “(o)estrogen receptor”, “(o)estrogen receptor alpha”, “(o)estrogen receptor beta”, “FES”, “fluoroestradiol”, “positron emission tomography”, “PET”, “pharmacokinetics”, “SHBG”, and “sex hormone binding globulin”. The search results were manually screened for relevance and the reference lists of selected articles were checked for additional literature. We selected papers written in English and published between January, 1970, and May, 2013. We searched ClinicalTrials.gov for continuing clinical trials, up to May, 2013, with the search terms “FES PET” and “fluoroestradiol”.

correction for SHBG levels would result in improved quantification of oestrogen-receptor density is unclear.

Conclusions Targeted therapies and personalised management of patients are rapidly emerging elements of breast-cancer treatment. The classic point of view is to assume the characteristics of metastases are similar to those of the primary tumour, but evidence is increasingly pointing to changes in tumour phenotype and behaviour during disease progression. Molecular imaging of characteristics that might be suitable treatment targets, such as oestrogen receptors, have the advantages of being non-invasive, providing quantitative information on all tumour lesions, and assessing changes before, during, and after treatment. ¹⁸F-FES-PET has clear potential to improve therapeutic decision making. Quantitative measurements of ¹⁸F-FES uptake reflect the availability of oestrogen-receptor binding sites and, therefore, might improve prediction of response to endocrine therapy compared with standard immunohistochemistry. Validation of quantitative measurements by modelling studies with arterial blood sampling for bound and free ¹⁸F-FES and metabolites will be crucial to ensure refinement and reliability of SUVs. Factors to take into account are body composition, binding of ¹⁸F-FES to carrier proteins, hepatic ¹⁸F-FES clearance, and competition between ¹⁸F-FES and endogenous ligands. ¹⁸F-FES-PET also deserves further exploration as a method for monitoring oestrogen-receptor binding, which could improve dosing regimens, in-vivo assessment of binding characteristics in new targeted drugs, and prediction of treatment response. Whether the use of ¹⁸F-FES-PET lowers the costs related to invasive biopsies and avoids multiple imaging examinations should also be explored. Contributors GAPH and EGEdeV contributed to the conception and design of this Review, and supervised all activities. All authors contributed to the literature search and drafting and review of the paper, and approved the final version.

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Conflicts of interest GAPH has received a research grant from AstraZeneca. The other authors declare that they have no conflicts of interest. Acknowledgments This research was supported by the Dutch Cancer Society (grant RUG 2009-4529) and the Centre for Translational Molecular Medicine— Mammary Carcinoma Molecular Imaging for Diagnosis and Therapeutics (CTMM – MAMMOTH) Project (grant 03O-201). We thank Esther van Straten for her assistance with designing figure 5. References 1 Amir E, Miller N, Geddie W, et al. Prospective study evaluating the impact of tissue confirmation of metastatic disease in patients with breast cancer. J Clin Oncol 2012; 30: 587–92. 2 Chung GG, Zerkowski MP, Ghosh S, Camp RL, Rimm DL. Quantitative analysis of estrogen receptor heterogeneity in breast cancer. Lab Invest 2007; 87: 662–69. 3 Mintun MA, Welch MJ, Siegel BA, et al. Breast cancer: PET imaging of estrogen receptors. Radiology 1988; 169: 45–48. 4 Mosselman S, Polman J, Dijkema R. ERβ: identification and characterization of a novel human estrogen receptor. FEBS Lett 1996; 392: 49–53. 5 Ahmad N, Kumar R. Steroid hormone receptors in cancer development: a target for cancer therapeutics. Cancer Lett 2011; 300: 1–9. 6 Honma N, Horii R, Iwase T, et al. Clinical importance of estrogen receptor-β evaluation in breast cancer patients treated with adjuvant tamoxifen therapy. J Clin Oncol 2008; 26: 3727–34. 7 Younes M, Honma N. Estrogen receptor β. Arch Pathol Lab Med 2011; 135: 63–66. 8 Allred DC, Harvey JM, Berardo M, Clark GM. Prognostic and predictive factors in breast cancer by immunohistochemical analysis. Mod Pathol 1998; 11: 155–68. 9 Early Breast Cancer Trialists’ Collaborative Group (EBCTCG). Relevance of breast cancer hormone receptors and other factors to the efficacy of adjuvant tamoxifen: patient-level meta-analysis of randomised trials. Lancet 2011; 378: 771–84. 10 Samaan NA, Buzdar AU, Aldinger KA, et al. Estrogen receptor: a prognostic factor in breast cancer. Cancer 1981; 47: 554–60. 11 Foukakis T, Astrom G, Lindstrom L, Hatschek T, Bergh J. When to order a biopsy to characterise a metastatic relapse in breast cancer. Ann Oncol 2012; 23 (suppl 10): x349–53. 12 Simmons C, Miller N, Geddie W, et al. Does confirmatory tumor biopsy alter the management of breast cancer patients with distant metastases? Ann Oncol 2009; 20: 1499–504. 13 Lindstrom LS, Karlsson E, Wilking UM, et al. Clinically used breast cancer markers such as estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 are unstable throughout tumor progression. J Clin Oncol 2012; 30: 2601–08. 14 Jones T, Price P. Development and experimental medicine applications of PET in oncology: a historical perspective. Lancet Oncol 2012; 13: e116–25. 15 Boellaard R, O’Doherty MJ, Weber WA, et al. FDG PET and PET/ CT: EANM procedure guidelines for tumour PET imaging: version 1.0. Eur J Nucl Med Mol Imaging 2010; 37: 181–200. 16 Yoo J, Dence CS, Sharp TL, Katzenellenbogen JA, Welch MJ. Synthesis of an estrogen receptor β-selective radioligand: 5-[¹⁸F] fluoro-(2R,3S)-2,3-bis(4-hydroxyphenyl)pentanenitrile and comparison of in vivo distribution with 16α-[¹⁸F]fluoro-17β-estradiol. J Med Chem 2005; 48: 6366–78. 17 VanBrocklin HF, Liu A, Welch MJ, O’Neil JP, Katzenellenbogen JA. The synthesis of 7α-methyl-substituted estrogens labeled with fluorine-18: potential breast tumor imaging agents. Steroids 1994; 59: 34–45. 18 Pomper MG, VanBrocklin H, Thieme AM, et al. 11β-methoxy-, 11β-ethyl- and 17α-ethynyl-substituted 16α-fluoroestradiols: receptorbased imaging agents with enhanced uptake efficiency and selectivity. J Med Chem 1990; 33: 3143–55. 19 Benard F, Ahmed N, Beauregard JM, et al. [¹⁸F]Fluorinated estradiol derivatives for oestrogen receptor imaging: impact of substituents, formulation and specific activity on the biodistribution in breast tumour-bearing mice. Eur J Nucl Med Mol Imaging 2008; 35: 1473–79.

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