Molecular pathways and molecular imaging in breast cancer: An update

Nuclear Medicine and Biology xxx (2013) xxx–xxx

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Nuclear Medicine and Biology

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Molecular pathways and molecular imaging in breast cancer: An update☆

Anna Rita Cervino a, Marta Burei a, Luigi Mansi b, Laura Evangelista a,⁎a Radiotherapy and Nuclear Medicine Unit, Istituto Oncologico Veneto IOV-IRCCS, Via Gattamelata, 64 35128 Padova, Italyb Nuclear Medicine Department, Seconda Università di Napoli, Napoli, Italy

☆ Conflict of interest: none.⁎ Corresponding author. Tel.: +39 0498217997; fax:

E-mail address: [email protected] (L. Eva

0969-8051/$ – see front matter © 2013 Elsevier Inc. Alhttp://dx.doi.org/10.1016/j.nucmedbio.2013.03.002

Please cite this article as: Cervino AR, et al,http://dx.doi.org/10.1016/j.nucmedbio.2013

a b s t r a c t
a r t i c l e i n f o
Article history:Received 2 January 2013Received in revised form 13 March 2013Accepted 15 March 2013Available online xxxx

Keywords:Molecular imagingBiologyBreast cancerPETRadiotracers

Breast cancer is a heterogenic cancer being characterized by a variability of somatic mutations and inparticular by different receptor expressions, such as estrogen, progesterone and human epidermal receptor.These phenotype characteristics play a crucial role in determining tumour response to variouschemotherapies and other treatments and in the development of resistance to therapies. Positron emissiontomography (PET) as a nuclear medicine technique, has recently demonstrated the advantages indetermining the severity of disease and in evaluating the efficacy of treatments in a variety of neoplasm,including breast cancer. Because this procedure is able to pinpoint molecular activity within the body, it offersthe potential to identify disease in its earliest stages as well as a patient’s immediate response to therapeuticinterventions in a non-invasive way. In this paper we performed an extended view about the correlationbetween molecular factors of breast cancer and PET tracers; in particular, we focalized our attention on theirpossible advantages in terms of 1) early detection of primary or recurrent cancer; 2) as a guide for targettherapies and 3) for the evaluation of response to specific and now-available molecular treatments.

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Molecular pathways and molecular imaging.03.002

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1. Background

The hallmark of breast cancer (BC), likemost human neoplasm, is itsbiological heterogeneity that reflects the complexity and variability ofthe vast array of somatic mutations acquired during oncogenesis. Thisheterogeneity is concretely apparent in tumour with expression ofestrogen receptor (ER) or human epidermal receptor 2 (HER2) [1,2].

Nowadays, the manifestation of six essential alterations in cellphysiology that collectively dictate malignant growth of BC is known,in particular: 1) self-sufficiency in growth signals, 2) insensitivity togrowth-inhibitory (antigrowth) signals, 3) evasion of programmedcell death (apoptosis), 4) limitless replicative potential, 5) sustainedangiogenesis, and 6) tissue invasion and metastasis [3] (Fig. 1). It iswell established that these factors play a crucial role in determiningtumour response to various endocrine therapies and the developmentof resistance to these treatments in BC. Furthermore, distinctcharacteristics of BC can be exploited to help in determining theoverall prognosis and the likelihood of response to specific therapies[4]. Positron emission tomography (PET) is a nuclear medicineimaging that, such as with conventional nuclear medicine modalities,uses small amounts of radioactivematerial to diagnose and determinethe severity or the efficacy of treatments in a variety of diseases,including many types of cancers. Because this procedure is able to

pinpoint molecular activity within the body, it offers the potential toidentify disease in its earliest stages as well as a patient’s immediateresponse to therapeutic interventions in non-invasive way. In thispaper wewill make an overview of the correlation betweenmolecularfactors of the BC and PET tracers, already available for clinical practice.

2. Current clinical practice

In the last decades, the identification and development of newmarkers (such as HER2 receptor, ki67, estrogen and progesteronereceptors) have led to insight into BC biology aswell as that of existingtumor tags. The biological importance of these established markershas been reinforced over the last decade by the results from genomicclassification in which the presence or absence of these markersidentifies the three main groups [2]: luminal (estrogen receptorpositive), HER2-like (mainly estrogen receptor negative and HER2positive), and basal-like (mainly estrogen receptor negative, proges-terone receptor negative and HER2 negative) which approximate theso-called triple negative group of BC. The difference among threesubsets is related to the prognosis and the response to treatment,configuring different risk categories. Moreover, Cheang et al. [5]reported that ki67 and HER2 expression could be used to over-stratifythe risk for BC relapse and death among patients with luminal BC whowere treated with both tamoxifen and chemotherapy as theiradjuvant systemic therapy. The treatment scenario of BC is expanding,although it is variable according to risk categories. Triple negativecancers are sometimes referred as a refractory disease, but recently a

in breast cancer: An update, Nucl Med Biol (2013),

http://dx.doi.org/10.1016/j.nucmedbio.2013.03.002

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Fig. 1. The molecular pathway of breast cancer cells and targets for molecular imaging.

2 A.R. Cervino et al. / Nuclear Medicine and Biology xxx (2013) xxx–xxx

high efficacy to adjuvant chemotherapy has been reported. Unfortu-nately, patients with this type of tumor and who are refractory tochemotherapy will relapse early, with a peak of metastases occurringat 1 year [6]. Therefore the early identification of relapse can improvethe prognosis of this subset of patients.

HER2 expression was associated with a positive response to targettherapies (such as trastuzumab) and recently its amplification andoverexpression have been associated with benefit from standarddoses of doxorubicin-based adjuvant chemotherapy, also in luminalcategories [7,8]. The reliable advantages of performing chemotherapy(in adjuvant or neoadjuvant setting) in BC disease are the decrease incancer recurrence, cancer-related morbidity and disease-specificdeath. Most women with early-stage BC (stage I–II) receive adjuvantsystemic therapies with a positive effect on the overall survival. Thechoice of therapies depends from the biological characteristics of thetumors and by its extension, but their efficacy has been more deeplyanalyzed [9,10]. Locally advanced BC includes tumors with at least oneof the following features: size larger than 5 cm, extensive regionallymph node involvement, direct involvement of underlying chest wallor skin, tumors considered inoperable but without distant metastases,and inflammatory BC. Induction chemotherapy or endocrine or tissue-targeted therapies followed by local therapy (surgery, radiationtherapy, or both) are becoming the standard of care. Five-yearsurvival can be achieved in 55% of patients presenting with non-inflammatory locally advanced BC [11]. Moreover, patients whoachieve an excellent response to induction treatments have a similaroutcome than those patients with early stage BC. The identification ofthis subset of patients represents an important end-point in clinicalpractice that could be reached by the employment of molecularimaging modalities. In particular, the early detection of a distantinvolvement can deeply influence the choice of treatment.

In metastatic patients, the five-year survival is attained in only23.3% [12]; therefore, it is important to understand the patient’s

Please cite this article as: Cervino AR, et al, Molecular pathways and mohttp://dx.doi.org/10.1016/j.nucmedbio.2013.03.002

treatment goals and targets. The variety of tracers available in nuclearmedicine represents an obvious advantage in advanced BC disease, fordetermining the therapeutic options. Nevertheless until now, onlyFDG PET, as recommended in European and American guidelines, isemployed for the evaluation of distant metastases in locally advancedBC and for monitoring the efficacy of treatment on the primaryinoperable tumor and in patients with metastases.

The NCCN guidelines address only approved radiopharmaceuticaland widely accepted imaging procedures supported by evidence formclinical trials. Therefore, the current NCCN guidelines (version 3.2012)reported the use of PET/CT for the initial staging of locally advanced BC(stage III; optional, category 2B) or in the evaluation of metastatic BC(stage IV, category 2B). Only 18 F-Fluorodeoxyglucose (FDG) and18 F-Fluoride as PET tracers are mentioned in the current guidelines,but as often reported, widely accepted standards of disease activityassessment are missed. No data about the utility of PET or PET/CTwithother tracers are reported, probably due to the absence of specifictrials. Moreover, although 99mTc-sestamibi for scintigraphic studiesis approved for breast imaging, it is not part of the guidelines due to alack of evidence of its efficacy and consensus for its use.

As known, the objective of guidelines development is to assistphysicians and patients in making optimal health care decisions,which in turn should improve the quality of clinical practice [13]. Theinclusion of new probes into clinical practice and therefore in theguidelines requires some steps that meet the Good Clinical Practicethus providing assurance of the safety and the efficacy of the newlydeveloped compounds. Several sophisticated molecular imagingagents and technologies are currently available for clinical use. Theirutility and promise are evident in the substantial number that hasbeen studied in the clinical trials. None of these agents, however, isofficially approved and reimbursed. In fact, only 2 new imaging agentshave been approved by Food and Drug Administration (FDA) in morethan 10 years. Three barriers should be overpassed: 1) clinical

lecular imaging in breast cancer: An update, Nucl Med Biol (2013),


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development; 2) regulatory approval and acceptance and finally 3)clinical acceptance. The first step requires research funding for thedevelopment of new imaging agents and the design of clinical trialsfor extended the process to the clinical field; the second one is time-consuming and needs of regulations that sometimes are unresolved,such as the definitions for qualifying or to validate an imagingbiomarkers, the efficacy and safety of imaging agents as therapeuticdrugs. The last process, issues related to funding, clinical acceptanceand expansion of indications requires of well-designed and poweredimaging studies, the standardization of the imaging protocols and thesupport of other physicians. As proposed by Barrio et al. [14], a newregulatory approach for PET molecular imaging probes including: 1)the positioning of PET molecular imaging probes in “no significantrisk” category, 2) the oversight of all diagnostic research with theseprobes and 3) a fast approval by competent organism (e.g. FDA),should be established.

Nowadays, contrast-enhancedMRI, MR spectroscopy, scintigraphywith 99mTc-sestamibi or PET techniques provide information beyondthat of structural imaging by displaying tumor neoangiogenesis,tumor metabolism and tumour cell mitochondrial activity. Muchneeds to be learned at themolecular level of normal cellular pathwayseither suppressed or enhanced by tumor-specific molecular changes.The available imaging devices can lead to the characterization ofbiological patterns of BC and an early assessment of treatmentresponse to target-therapies, but the clinical practice is distant fromthis realization.

3. Receptor tracers

Endocrine therapy targeting steroid receptors remains the mosteffective form of systemic therapy in BC. About 70%–80% of BCpatients have hormone receptor-positive tumours, and therefore arecandidates for endocrine therapy. ER expression is considered one ofthe most important biomarkers in BC, both providing prognosticinformation and predicting responsiveness to endocrine treatment.The expression of progesterone receptors (PR) is strongly dependenton the presence of functional ER. There is evidence that metastatic BCexpressing both ER and PR responds better to antiestrogen therapythan those that show ER positivity (ER+) but lack PR expression [15].A strong prognostic value for PR expression also has been reported inadjuvant trials comparing tamoxifen treatment [16]. Only a smallfraction of tumours are ER negative (ER−) and PR positive, and theydemonstrate an intermediate response to endocrine therapy [17].

The ER/PR concentration in tumour tissue is determined by in vitroassays. In recent years, immunohistochemical (IHC) receptor assayshave increasingly replaced quantitative radioligand binding assays[18,19]. Evaluation of ER/PR status is performed by means of IHCstaining of the primary tumour. This golden standard has somelimitations. Firstly, the technique is semiquantitative, which can resultin interobserver variation, and ER/PR scoring depends on the antibodyused and delay-to-fixation time [20,21], secondly as demonstrated bya recent systematic review of the American Society of ClinicalOncology (ASCO) and the College of American Pathologists (CAP),up to 20% of all IHC determinations worldwide are inaccurate [22] andtherefore altering the prediction of response to antihormonal therapyin 50%–60% of the patients [23,24]. The knowledge of the receptorstatus of recurrent or metastatic disease may be more predictive ofresponse to hormone therapy, but it can not be easily determinablebecause metastatic lesions often are not amenable to biopsy.Discordance between primary tumour and metastatic lesions occursin 18%–55% of the patients [25,26]. Although guidelines of theEuropean Society for Medical Oncology and National ComprehensiveCancer Network recommend repeated biopsies, they are oftenethically impractical and therefore therapeutic decisions for meta-static BC are most often based on the profile of the primary tumour.Even with metastatic disease, the median survival in patients with


ER+ tumours is 3 times longer than that in patients with ER− BC[17]. A method that can reliably determine both the quantity and thefunctional status of tumour ER and PR in individual lesions would beof critical importance in identifying patients likely to benefit fromhormonal therapy. Imaging with radiolabeled steroid can be used forcovering this latter request and, in particular, it can be helpful for thedetection of BC patients with resistance to anti-endocrine therapieswho are candidates to novel drugs, such as mTOR inhibitors (i.e.Everolimus or Tacrolimus) of Src-inhibitors (i.e. Dasatinib).

3.1. ER-tracer PET/CT

PET using 18 F-β-estradiol (FES) is a method for imagingfunctional ER expression in vivo and may be used as a quantitativemeasure of ER expression in BC [26–28]. FES PET may offercomplementary advantages to in vitro assay of biopsy material,including the measurement of ER binding, identification of hetero-geneous expression over the entire burden of disease and measure-ment of the pharmacodynamic effect of ER-directed therapy [29].Several studies have shown that FES PET can reliably detect ER+tumour lesions and that FES uptake is well correlated with IHCscoring for ER [26,27,30]. A list of publication about FES PET isreported in Table 1. Low uptake of FES PET is a strong predictor forfailure of antihormonal therapy, as reported in literature [31–34] butthe factors influencing FES uptake, however, are incompletelyunderstood. The determination of these factors, other than thedesired dependency on ER expression levels, contributes to furtherunderstanding of this novel diagnostic tool and its use to measureregional ER expression. van Kruchten et al. [35], in a recent study,recruited 33 patients with an ER+ primary tumour revealing thatloss of ER expression in distant metastases represented a predictorof poor response to antihormonal therapy. Accordingly to this latterreport, FES PET may be used as a surrogate for tissue biopsy whenlesions are difficult to access or it could be used to prove thepresence of ER+ metastases in the case of an equivocal conventionalwork-up. Anyway, FES PET cannot be used to exclude metastases ingeneral because ER− metastases may be present but not visible. Inlight of a possible conversion in ER phenotype, knowledge of ERexpression can potentially facilitate the choice between chemother-apy and antihormonal therapy. The consequence of a heterogeneousER expression for therapy management has received strikingattention in the clinic and deserves further exploration.

Other than ER expression, therapeutic and clinical factors mightaffect FES uptake. The presence of estrogens analogues, such astamoxifen, can block tumour FES uptake [36]. For this reason, vanKruchten et al. [34] in their study arbitrarily chose drug withdrawalperiod of 5 weeks for ER ligands. In fact, patients who discontinuedfulvestrant 5 weeks before FES PET showed a high rate of FES-negative lesions (14/20 metastases) probably due to a long half-life(40 days) of the drug. Only a premenopausal patient in this study hadFES uptake values well below the 95% confidence interval ofpostmenopausal patients. In a previous study that evaluated 10subjects with primary ER+ tumour, only 6 patients showed focal FESuptake [37]. The authors found that 40% of patients with a false-negative FES PET finding were most likely premenopausal (agesranged: 34 to 45 years), whereas the age of the 60% of patients with atrue-positive FES PET finding ranged from 56 to 71 years. Together,these data underline the possibility that background estrogen levelsinfluence FES uptake, which warrants further exploration. Conversely,according to the experience of Peterson et al. [38], higher serumhormone binding globulin (SHBG) levels correlated with lower FESuptake and should therefore be measured for each patient. Pre-menopausal levels of estradiol do not appear to affect FES uptake, nordoes FES metabolism, suggesting that FES metabolism may not needto be measured for individual patients. These findings should allow



Table 1Characteristics of studies about 18 F-Fluoroestradiol.

n Author, (ref) Year ofpub.

Journal N ofpts

PETscanner

Main goal Conclusions

1 Mintun et al.,[18]

1988 Radiology 13 PET - FES uptake in primary breast tumours - High correlation between FES uptake andER concentration was demonstrated.

2 McGuireet al., [22]

1991 J Nucl Med 16 PET - To evaluate the uptake of FES in metastatic BC PET with FES has high sensitivity and specificityfor detecting metastatic BC and provide additionalconfirmatory evidence that the tumour uptakeof this ligand is a receptor-mediated process

3 Dehdashtiet al., [30]

1995 J Nucl Med 32 PET - To evaluate the concordance between ER statusand FES uptake

Only FES PET can provide information aboutER status while FDG PET does not.

- To assess the relationship between FDG uptake,ER status, tumour aggressiveness and prognosis

4 Mortimeret al., [20]

1996 Clin CancerRes

43 PET - FDG and FES in relation to response to treatment - FDG PET is sensitive in staging- Correlation between ER status and FES uptake - FES is more accurate in evaluation of ER status

(sensitivity: 76% and specificity: 100%)5 Mankoff

et al., [14]1997 J Nucl Med 15 PET - To optimize FES PET imaging studies Imaging starting after 20–30 min after injection

can provide good visualization of ER+ tissue- To provide an input function for quantitative analysis6 Tewson

et al., [16]1999 Nucl Med

and Biol18 PET The extension of tissue enriched in ER by FES uptake -FES binds SBP in the circulation

-This binding can alter the quantitative evaluationof FES PET studies


1999 Eur J NuclMed

11 PET - To evaluate the prediction of response to hormonetreatment by FDG and FES PET

A metabolic flare by FDG PET and the degree ofER blockade by FES PET appear to predictresponsiveness to anti-estrogen therapy inER+ BC patients

8 Mortimeret al., [28]

2001 J Clin Oncol 40 PET - To investigate whether PET FDG and FES performedbefore and after tamoxifen

- FES PET and FDG PET can predict the functionalstatus of ER

- To predict the response to treatment by FES PET - PET can predict the responsiveness to tamoxifentherapy in patients with advanced ER BC

9 Linden et al.,[23]

2006 J Clin Oncol 47 PET - To test the ability of FES PET in predicting responseto salvage hormonal treatment in heavily pretreatedmetastatic BC patients.

Quantitative FES PET can predict response tohormonal therapy and may help guide treatmentselection

10 Petersonet al., [19]

2008 J Nucl Med 17 PET Comparison between FES uptake and expressionER by IHC

Good agreement between FES and ER status


2009 Breast CancerRes Treat

51 PET - To predict the response to endocrine therapy FES PET and metabolic flare can predict the responseto therapy- To determine the flare phenomen

12 Kurlandet al., [26]

2011 J Nucl Med 91 PET The heterogeneity of FES uptake by site to site inpatients receiving endocrine therapy

- FES uptake is vary between in patients- FES uptake can be vary according to the ERexpression by site

13 Petersonet al., [31]

2011 Nucl Medand Biol

239 PET Which factors could affect the quantitativelevel of FES uptake

- Pre-menopausal estradiol does not affect FES uptake- SHBG can influence FES uptake- Higher injected mass per kilogram can influence onFES uptake

14 van Kruchtenet al., [27]

2012 J Nucle Med 33 PET To evaluate the value of FES PET in BC FES PET can be a valuable additional diagnostic toolwhen standard workup is inconclusive, exceptfor liver disease


simpler, more targeted protocols for FES imaging in BC patients withER+ disease.

3.1.1. LimitationsCurrent FES PET studies do not describe the capacity of FES to

detect liver metastases. The physiologic uptake in the liver due tometabolization well exceeds the FES uptake that is seen in the uterus[39] or most ER+ metastases, therefore the detection of livermetastases by FES PET had poor results. Further, the resolutionlimitations of PET may not show FES uptake in small metastases. Thecorrelation between body mass index and FES standardized uptakevalue (SUV) suggests a need for lean body mass correction whencomparing SUV values between patients. A non-significant trend wasobserved suggesting a negative relationship between injected massper kilogram patient weight and FES uptake at low specific activity. Itmay therefore be prudent to limit injected dose at lower specificactivities for smaller patients. How can we overpass this latterlimitation? As reported by Peterson et al. [38] concurrent chemo-therapy, body mass index and SHBG had significant association withFES uptake. The evaluation of body weight, the menstrual cycle periodand the measurement of SHBG can be useful for evaluating the correctdose for FES. As example, in a patient with a low body weight (i.e.40 kg), we should prefer to perform PET FES during the luteal or


follicular phase of menstrual cycle period (low values of estradiol),test the SHBG for identifying the lowest value and avoid theadministration during the chemotherapeutic treatment.

3.2. PR-tracer PET/CT

Dehdashti et al. [40] have recently synthesized and characterizedthe radio-fluorinated steroid compound 21-18 F-fluoro-16a,17a-[(R)-(19-a-furylmethylidene)dioxy]-19-norpregn-4-ene-3,20-dione(FFNP), which has high affinity and selectivity for PR. FFNP wasidentified to be the most promising progestin derivative for PET inpreclinical studies [41–44]. It is designed to be stable againstdefluorination, with resultant low bone uptake [41,42]. Low levelsof activity accumulate in the liver and fat, as its relatively lowlipophilicity translates into low non-specific binding in vivo. On thebasis of the tracer biodistribution, Dehdashti et al. [40] chose FFNP forthe first-in-human study to document safety, to estimate humandosimetry, and to correlate primary tumour FFNP uptake with tissuePR assays. They studied 15 postmenopausal and 5 adult premeno-pausal women with newly diagnosed BC. All patients except onesubject had the ER and PR status of their tumours confirmed byqualitative IHC staining of the primary BC. Tumour FFNP uptakeassessed qualitatively was significantly different in patients with PR+




and PR− cancers [40]. Dynamic data indicated that peak tumouruptake was achieved early after FFNP injection and stayed unchangedthrough 60 min. The tumour/normal (T/N) ratio indicated rapiduptake and no significant washout. The biodistribution data haveshown the highest activity in the liver, gallbladder, and smallintestine. The dose to the liver is approximately 40% of that reportedfor FES, corresponding to a liver residence time 38% of that reportedfor FES. FFNP and FES show a similar route of elimination. Assessmentof uterine activity, in only 3 premenopausal women, showedsignificant uptake. The authors have demonstrated that FFNP is asafe imaging agent for PET, with organ and total-body radiation dosescomparable to those from FES and other commonly used clinicalradiopharmaceuticals. They have shown that it can be used to assessthe PR status of individual BC lesions. As previously mentioned, theexpression of ER is important both for prognostic and therapeuticevaluation. Nevertheless, although the presence of progesteronereceptor is linked with the responsiveness to endocrine therapy[17], it has not established any implication on prognosis in in vivostudies. This limitation could be at first associated with the presenceof few data in literature on progesterone imaging. Labeled progester-one analogues for PET imaging could be useful for indirectly detectingER activation that is a key transcription factor for the activation of PRand for evaluating the expression of PR in order to plan an endocrinetherapy, particularly as salvage treatment.

4. DNA synthesis's tracers

Cell proliferation imaging has long been a goal of nuclear medicineresearch, and most of the effort has focused on radiotracers of DNAsynthesis. Tumour cell proliferation is an important prognostic factorand is a key factor to monitoring efficacy of anticancer therapy.Several agents have been proposed for PET imaging of DNA synthesis,including 18 F-fluorothymidine (FLT), 123I/124Iiodo-2′-deoxyuridine(IdUrd,), 76Br-bromodeoyxuridine, 11C-thymidine, and derivatives ofdeoxy-18 F-fluoroarabinofuranosyl such as 18 F-FAU, 18 F-FMAU,18 F-FBAU, and 18 F-FIAU. Among all these radiotracers mainly testedin clinical studies, FLT has emerged as the most promising PET tracerin recent studies [45] due to its intrinsic advantages: 1) it isbiologically stable and crosses the cell membrane by specificnucleoside transporters than 11C-thymidine or radio-IdUrd deriva-tives that are rapidly degraded in vivo and 2) it is a pyrimidineanalogue and after its uptake into the cell, is phosphorylated bythymidine kinase 1 (TK1) into FLT monophosphate, causing intracel-lular entrapment of the radioactivity. TK1 is a principal enzyme in thesalvage pathway of DNA synthesis. Its activity is increased during theS phase of the cell cycle. Therefore, FLT uptake reflects theproliferation rate of malignant tissues [46]. Some studies haveestablished that FLT uptake correlates with Ki-67 immunostainingproliferation assay in many cancers including BC [46–50].

Nevertheless, some data from literature indicate that FLT isprobably not an ideal tracer for detection and staging tumours, butthat it shows potential in measuring the response to therapy also at anearly stage of disease [47]. In fact, in BC early change in FLT uptake,immediately after initiation of chemotherapy predicts tumourresponse to treatment [51].

Clinical and therapeutic factors can alter the FLT uptake, such asthe competition from the endogenous thymidine pool for cellularuptake of exogenous FLT, the efficacy of thymidine synthesis that mayvary from patient to patient andmay likely fluctuate depending on thenutritional status. While serum glucose level is well monitored inpatients undergoing FDG PET and subject to hormonal control,intracellular thymidine concentration cannot be measured beforeFLT PET. Such variations could lead to variable FLT tumour uptakeeven in cancers of similar proliferation activity. Hence, 5-fluoro-2′-deoxyuridine (FdUrd) could improve the FLT tumour uptake byuncoupling it from the endogenous thymidine pool. FdUrd directly


blocks the endogenous thymidine synthesis without any majorimpact on other nucleotide pathways. Viertl et al. [52] demonstratedthat a single intravenous (i.v.) injection of a non-therapeutic dose ofFdUrd, aimed at improving tumour uptake of FLT in order to use thisprocedure in diagnostic PET. Moreover, different research groups haveobserved variable effects of FLT cellular uptake also after exposure totherapeutic doses of chemotherapies such as 5-fluorouracil (5-FU)and others in vitro and in patients [53–55]. Since 5-FU is partiallyconverted to FdUrd, this could explain the previously describedenhanced uptake of FLT, considered as a “flare” phenomenon. Thiscondition should be evaluated in clinical practice for avoiding falsepositive findings in therapeutic assessment.

Despite the urgent need for a proliferation imaging agent, FLT PETremains confined to clinical studies in particular situations or ascomplement to FDG PET. Smyczek-Gargya et al. [56] for the first timedemonstrated increased FLT uptake in primary BC and locoregionalmetastases. Twelve patients with 14 primary BC lesions were studied.For comparison, FDG PET scans were performed in six patients. Themajority of primary tumour and axillary lymph node metastasesshowed focally increased FLT. In direct comparison to FDG PET, theSUVs of primary tumours and axillary lymph node metastases werelower in FLT PET, since its uptake in surrounding breast tissue waslower than with FDG. Surprisingly, they did not find a correlationbetween FLT uptake and the Ki-67 labelling index. This findingparallels recent results in thoracic tumours [57], which have alsorevealed lack of a correlation between Ki-67 and SUV of FLT. The well-known biological heterogeneity of BC, which is reflected by differentprognostic and predictive parameters for subgroups [58,59], mightthus also contribute to the lack of correlation.

BC expressing high levels of ki67 is associated with worseoutcomes [60–62], nevertheless, it is not included in routine clinicaldecision-making because of lack of clarity regarding how Ki67measurements. Recent studies [63,64] indicate that changes in Ki67expression after neoadjuvant endocrine treatment may predict long-term outcome. Actually, Ki67 labeling index may serve as a clinicallyvaluable biomarker for the luminal B subtype, as reported by Cheanget al. [5]. From a recent meta-analysis by Chalkidou et al. [65], theutility of FLT PET in cancer detection and its correlation with Ki67 arenot really defined. In particular, from few published reports, differentconclusions were reached: 1) no correlation between SUVs and Ki67according to Smyczek-Gargya et al. [56] and 2) a good relationbetween kinetic parameters by FLT PET in accordance with Kenny etal. [66]. In fact, FLT PET could be useful in the evaluation of earlyresponse to treatment, for example in studying the response tochemotherapy [51] or taxanes regimens [67]. This latter propertycould be considered of relevance in a clinical setting. In particular, FLTPET can reduce the false positive effects by inflammatory changes,thus increasing the specificity for the detection of proliferationprocesses in comparison with FDG PET. This capability could allowin the future a key role in early diagnosis of recurrence. Nevertheless,FLT PET cannot be considered as an exclusive tool for the imaging ofcell growth being unable to detect the other factors involved in thisprocess. Conversely, it can be used for guiding biopsy in case of highlyheterogeneous tissue and for evaluating the “interim” response tochemotherapy. In the future a better pharmacokinetic analysis of FLTand/or the availability of an alternative radiotracers more strictlyconnected with DNA synthesis could further improve the clinicalimpact of “cell growth”. Moreover, future research will need to befocused on measuring the response to neoadjuvant chemotherapy byFLT PET in patients with locally advanced BC.

5. Epidermal growth factor receptor tracers

Twenty percent to 30% of BCs exhibit amplification or over-expression of the HER2/erbB2 oncogene, which is associated withmore aggressive disease and poor prognosis [68,69]. HER2 is a




member of the epidermal growth factor receptor (EGFR) family oftyrosine kinases, which includes HER1/erbB1, HER2/erbB2, HER3/erbB3, and HER4/erbB4. These receptors regulate a wide range ofcellular processes, including proliferation, differentiation, motility,survival, angiogenesis, invasion, and antiapoptotic functions [69]. Todate, an endogenous ligand for the HER2 receptor has not beenidentified, but its activation is thought to occur through heterodimer-ization with other ligand-bound HER family members or by homo-dimerization when highly expressed [69]. Trastuzumab (Herceptin) isa recombinant, humanized monoclonal antibody Food and DrugAdministration-approved, that selectively binds to the extra cellulardomain of HER2 [70]. As emerging from literature, overexpression andamplification of HER2 in 15% to 20% of BCs are strong predictors ofbenefit from treatment with trastuzumab [71]. To show that adjuvanttreatment of patients with HER2-positive primary BC improvesoverall survival [72,73] requires all BC patients to be tested for thismarker. The importance of accurate testing for this bio-label to ensureappropriate application of trastuzumab has to lead to the creation ofthe ASCO/CAP guidelines on methodology for immunohistochemistryand in situ hybridization techniques for establishing gene copynumber for HER-2 as well as on test interpretation [73]. HER2expression was associated with benefit from standard doses ofdoxorubicin-based adjuvant chemotherapy [7,8]. ASCO/CAP guide-lines recommend HER2 testing on the surgical specimen because ofpotential artifact in biopsy specimens, which may lead to inaccuratereadings [73]. This latter condition represents a limitation in theneoadjuvant setting.

The use of antibodies alone against EGFR (e.g., Erbitux; cetuximab)and HER2 for the treatment of cancer gave only moderate results butwhen trastuzumab is used in combination with chemotherapy, itextends overall survival and slows disease progression in HER2+ BCpatients [73]. Approximately 50% of HER2+metastatic BCs exhibit denovo or acquired resistance to trastuzumab [74–77], therefore theassessment of early response to trastuzumab therapy remains anundeveloped objective. Although the primary mechanism(s) of actionof trastuzumab remains unclear, the importance of phosphatidylino-sitol 3-kinase (PI3K) signalling in HER2+ BC [74] implies thatperturbation of this pathway is important in order for HER2-directedtherapies to exert an anti-tumour effect. This also implies that PI3K-regulated processes such as tumour cell apoptosis, proliferation, andglucose metabolism may be useful biomarkers of response totrastuzumab therapy. Because trastuzumab inhibits PI3K activationin HER2+ BC cells, glucose metabolism may be altered by trastuzu-mab therapy. Similarly, one might also predict that tumour cellproliferation would be inhibited by trastuzumab therapy, but a recentstudy have demonstrated that the proliferation marker Ki67 did notchange in HER2+ tumours from patients treated with trastuzumab[78]. Although these and other ex vivo assays are potentially valuable,tumour tissues biopsies for the assessment of drug action are invasiveand their reliability is limited by sample bias stemming from tumourheterogeneity and other confounding factors such as inflammation.Serial biopsy required to assess the effects of therapy are also clinicallyimpractical in many cases. Therefore, non-invasive molecular imagingbiomarkers, able to assess numerous relevant biological processes,could be particularly indicated to support clinical evaluation andprediction of response to trastuzumab in patients with HER2+ BC.Geldanamycin, an ansamycin antibiotic, has a unique mechanism forinhibition of HER2 activity by inducing proteasomal degradation ofthe receptor. The antibiotic binds to heat shock protein 90 (Hsp90), achaperone protein that is responsible for the maturation and stabilityof a variety of proteins including HER2 [79]. The binding ofgeldanamycin to Hsp90 inhibits the maturation and stability ofHER2 and leads to degradation of the receptor. The geldanamycinderivative 17-N-allylamino-17-demethoxy geldanamycin (17-AAG)was shown 1) to exhibit anti-tumour activity, 2) sensitize tumours totaxanes (drugs that inhibit cell proliferation by binding to microtu-


bules), 3) induce degradation of HER2 and 4) inhibit expression of thereceptor [80]. This drug is being studied in several clinical trials forthe treatment of various cancers (www.clinicaltrials.gov). Thesecharacteristic activities of 17-AAG have been used to image thepharmacodynamic effects of the drug with the F(ab´) fragment of anantibody against EGFR. 68Ga-F(ab´) fragment of trastuzumab wasused to determine the alterations of HER2 expression in response to17-AAG [81]. This tracer had a 3 h half-life in serum and showed nodecrease in signal even 24 h after administration. 68Ga-trastuzumabF(ab´)2 fragments showed a relatively good tumour uptake. In afollow-up study, a reduction in 68Ga-trastuzumab tumour uptakepredicted 17-AAG-induced tumour growth inhibition earlier than areduction in FDG tumour uptake [82]. Dijkers et al. [82] have chosento use the antibody trastuzumab instead of smaller proteins todetermine HER2/neu expression. The choice of a radiopharmaceu-tical for HER2/neu imaging should depend on the question to beanswered. If only receptor expression is relevant for diagnosticpurposes, then imaging can be performed with small proteins,allowing the patients to be diagnosed in a single day. If evaluation(and response prediction) of trastuzumab therapy is desired, thenradiolabeled intact antibodies, which most likely mimic drugbehaviour more accurately, are preferable.

Another trastuzumab tracer is 64Cu-Herceptin that has a half-lifeof 12.7 h and effectively visualizes presumably HER2+ metastaticlesions in bone, liver and, to a lesser degree, lymph nodes.Administration of 50 mg Herceptin reduces liver uptake of 64Cu,permitting positive visualization of intrahepatic HER2+ lesions [83].

Chan et al. [84] have compared the accuracy of microSPECT/CT andmicroPET/CT with 111In- or 64Cu-DOTA-trastuzumab Fab fragments,tracers able to image human tumour xenografts in mice with low,intermediate, or high HER2expression. The intensity of the tumoursignal was dependent on HER2 expression of tumours with interme-diate or high HER2 density, most readily imaged by microSPECT/CT ormicroPET/CT. The results have demonstrated specific uptake of 64Cu-DOTA-trastuzumab Fab in tumours with intermediate HER2 density,but not for tumours with lower HER2 expression. Tumour uptake wasnot significantly different for 64Cu- and 111In-DOTA-trastuzumabFab, but blood radioactivity was threefold lower for 111In-DOTA-trastuzumab Fab. Thus, T/B ratios were threefold lower for 64Cu- than111In-DOTA-trastuzumab.

In an effort to develop a HER2 imaging agent with superiorspatial resolution and signal/noise ratio compared to 111In-trastu-zumab, Dijkers et al. [82] developed a new PET-tracer, 89Zr-trastuzumab that has a half-life of 78.41 h. The investigatorsobserved a good correlation in the tumour uptake of both radio-and bio-pharmaceuticals and concluded that, because of its highspatial resolution, 89Zr-trastuzumab could be used in the clinicalsetting after further evaluation [82]. Imaging with radiolabelledtrastuzumab could be useful for the initial evaluation, particularly inwomen with large cancer masses and therefore with a possibleheterogeneity in HER2 expression.

Non-invasive molecular imaging of EGFR would provide valuableinformation for selection and management of patients for EGFR-targeted therapy [85]. Current preclinical studies have showed thatEGF has been labelled with various radioisotopes to image EGFR+tumours with nuclear imaging modalities such as SPECT and PET [86–88]. EGF presents several advantages over EGFR-specific antibodiesthat could lead to the design of a better imaging tracer. First, the muchsmaller size (about 6.4 kDa) facilitates more rapid kinetics of traceruptake and may allow repetitive imaging studies using radioisotopeswith short half-lives. Second, as a natural ligand of EGFR, EGF showshigh binding affinity and specificity to the receptor [89]. In addition,imaging with EGF may help to discriminate between active andinactive forms of EGFR, which may be a more relevant parameter forpredicting efficacy of anti-EGFR agents [90]. To develop EGF-basedtracers, a Cys tag with N-[2-(4- [18 F]fluorobenzamido)ethyl]




maleimide (FBEM) fused human EGF (cEGF) has been prepared tofacilitate site-specific labelling of 99mTc or 64Cu [91]. Imaging with99mTc and 64Cu-labeled cEGF showed improved image quality andlower kidney and liver uptake. Li et al. [92] had achieved site-specificlabelling of EGF with 18 F using a thiolspecific labelling agent withhigh efficiency, and demonstrated bymicroPET imaging that the 18 F-labeled tracer was able to accumulate in EGFR+ squamous cellularhead and neck carcinoma. They also confirmed that adding the properamount of nonradioactive EGF effectively increased the tumouruptake and decreased liver accumulation of FBEM-cEGF. Velikyanet al. [87] have used the positron emitter 68Ga to label EGF for PETimaging of EGFR in a preclinical model. Lapatinib is a dual tyrosinekinase inhibitor of EGFR and HER-2 receptors and is clinically effectiveagainst HER-2-overexpressing metastatic BC. As reported by Diaz etal., Lapatinib is able to induce a dramatic reduction in angiogenesis inlung cancer [93]. This latter effect could be assessed also in BC, alsowith the use of labeled EGFR PET.

6. Angiogenesis tracers

As recently stated by a review ofWicki and Rochlitz [94], one of themain obstacles for anti-angiogenetic therapy remains the lack ofreliable biomarkers. Despite significant research efforts to identify cellpopulations or serum proteins predictive of response to anti-angiogenetic treatment, suchmarkers have yet to be found. Molecularimaging (e.g. PET with labeled dimeric RGD peptide or analogue ofintegrines) could be useful in this case, guiding for target therapieswith anti-angiogenetic drug, such as Bevacizumab that is a monoclo-nal antibody that binds the vascular endothelial growth factor A(VEGF-A) or VEGRF kinase inhibitors. Integrins are a family of celladhesion receptors that play an important role in tumour angiogen-esis and metastases development, by facilitating the interaction oftumour endothelial cells with the extracellular matrix (ECM) bybinding to ligands with an exposed arginine-glycine aspartate (RGD)sequence [95]. The integrins consist of a and b subunits that formnoncovalent ab heterodimers [96] and are the major receptors bywhich cells attach to the ECM. The αvb3 integrin, which ispreferentially expressed on proliferating endothelial cells associatedwith neo-vascularization in both malignant tumours and normaltissue but not in quiescent blood vessels [97,98], has been identified asa target for imaging neovasculature. The tripeptide RGD-amino acidsequence is particularly interesting in this regard, as ligands contain-ing RGD have high affinity for αvβ3/5 integrins [96]. After beinglabelled with positron-emitting or gamma-emitting radioisotopes,RGD peptide ligands have been used for imaging integrin receptorexpression with PET and SPECT [97,98]. In the first clinical PET studiesof the RGD-based radioligand [18 F]fluciclatide (formerly known as[18 F]AH111585) were showed the safety, the biodistribution and thepreliminary diagnostic value of this tracer. The αvβ3/5 subclasses ofthe integrin family are of particular interest as they are upregulated intumour neo-vasculature and in several types of tumour cells such asBC, therefore making them a valuable diagnostic tool [98]. Further-more, an association between expression of αvβ3/5 and relapse-freesurvival in BC has been reported [99], suggesting a prognostic value ofimaging for such receptors. Targeting the αvβ3 integrin receptorcould provide a tool to visualize and to quantify integrin expressionlevels, early cellular and molecular effects on therapies, changes inblood perfusion and permeability and terminal morphologic changes.As demonstrated by Sun et al. [100], the response to Abraxane, ananti-angiogenetic drug can be effectively predicted by labeledanalogue of integrines PET, reducing the presence of infiltratingmacrophages in regressing tumors, as reported by FDG PET. In ouropinion, a further contribution in the field could arrive in the nearfuture by a wider diffusion of PET/MRI, allowing the acquirement ofinformation on neo-angiogenesis not only by PET, but also from


functional MRI [101] with a hypothetic contribution achievable alsoby MRS.

7. Apoptosis imaging

In patients with the mutation of BRCA genes or in metastatic triplenegative cancer, an inhibitor of poly-ADP-ribose polymerase (PARP)has been employed, reporting in a phase II trial, in combination withother drugs, an impressive increase of overall survival (from 7.7 to12.3 months; [102]. Unfortunately, the results of the following phaseIII study were sobering, and no benefit on overall survival was found[103]. Although the development of PARP inhibitors was slowed downby the negative phase III trial described above, the chances are thatthey will ultimately find their way into the clinic. In this situation,efforts to identify predictive biomarkers for PARP inhibition therapyare mandatory. Molecular imaging can be employed also in thissetting, for example using specific tracers that are able to determineapoptotic processes (such as 18 F-fuorobenzyl triphenyl phosphoni-um-FBnTP; 105). In the report by Madar et al. [104], the authorsdemonstrated a linear relationship between 18 F-FBnTP accumulationin viable cells of BC and paclitaxel treatment duration. Targetingprobes of apoptosis using 18 F-FBnTP or similar agents can be a viablestrategy for the early detection of apoptosis and for quantifying theevolution dynamics of apoptosis. A broad range of clinical apoptosis-targeting probes are currently available for non-invasive in vivoimaging by PET of SPECT: radiolabeled caspases, annexin A5,glucarate, sestamibi and, more recently 18 F-ML-10 and duramycin[105]. As example, Del Vecchio et al. [106] obtained a consistentevidence that BCs which fail to accumulate 99mTc-MIBI, have alteredapoptotic program due to the overexpression of the anti-apoptoticprotein Bcl-2. Anyway, a detailed description of other apoptosistracers is beyond the scope of this review.

8. Metabolic tracers

8.1. 11C/18F-choline

Choline is an essential component of cell membranes and is thusnecessary for cell division. The initial step results in the formation ofphosphocholine from the phosphorylation of choline by the enzymecholine kinase (CK; in a reaction dependent on ATP and Mg2+),which is regulated by the mitogen-activated protein kinase (MAPK)pathway; phosphocholine is then effectively trapped intracellularly[107]. Humanmammary epithelial cells oncogenically transformed byHer2 [108], Ras [109], Src [110], and Mos [111] exhibit increasedlevels of phosphocholine compared with untransformed cells due toincreased constitutive activity of CKα. Furthermore, choline has beenproposed as a pathway marker of the extracellular receptor kinase/MAPK [106] that is also implied in the phosphorylation of ERαtogether with PIK3 pathways, causing resistance to antiendocrineagents. ERα is the dominant mediator of mammary development, isexpressed in 70% of all human BCs and is able to promote theproliferation and differentiation of BC cells. Tamoxifen is currentlyused to inhibit ERα activity, but it also impairs ERβ mediated genethat is implied in an anti-proliferative process and inhibits ERαmediated proliferations.

At first, Contractor et al. [111], employed choline labeled by11Carbonium in patients with ER + BC, correlating its uptake withclinically aggressive phenotype. The authors reported that theelevated levels of phosphatylcholine in BC cells were due to elevatedcholine kinase-α expression and therefore suggested that employingcholine PET in clinical practice could be useful for the evaluation ofcholine kinase-α inhibitors and thus tested potentially drugs that acton the MAPK pathway. Furthermore, the same group [112] tested theutility of choline PET in evaluating the effect of trastuzumab inpatients with a positive HER2 BC. They reported that the retention of




11C-choline by PET was attenuated through inhibition of Her2 and/orMAPK pathway by trastuzumab, being a decline in choline uptake inresponse to treatment. The advantage of Choline PET/CT in clinicalpractice is not already defined by the previous mentioned studies. Themain question is if choline image with MRI or PET can be interpretedas an assay for choline kinase-α enzyme activity or if it can be used forthe identification of the expression of ERα. The question remainsopen, but from the current literature, it emerged that both of theconditions could be satisfied. Firstly, choline kinase-α is sufficientlyevaluated by choline PET as also reported by other cancer studies[113,114] and secondly it is able to evaluate the MAPK-pathway asextensively demonstrated by Kenny et al. [112].

Based on the comparison between FDG and Choline PET in primaryBC, Tateishi et al. [115] showed that 11C-choline is more specific thanFDG, but both had a similar sensitivity. Therefore, in consideration ofthe wide availability of FDG in clinical practice, it should be preferredthan 11C/18 F-choline. A wider diffusion of 18 F-choline, showingsignificant practical and technical advantages with respect to 11C-choline, could stimulate in the future a deeper analysis justifying andfavouring a possible clinical role.

8.2. 18F-Fluorodeoxyglucose

FDG as a glucose analogue is internalized in cells and as tracer itallows the identification of cells with increased glucose metabolism.FDG incorporation has been found to be correlated with cellularproliferation [116] and, hence, has been considered an indicator oftumour aggressiveness [117]. It was later found that many, but not all,tumour cells and proliferating normal cells exhibited high rates ofaerobic glycolysis and that increased glycolysis is neither an essentialproperty of proliferating cells nor a distinct borderline of malignancyfrom benignancy. FDG uptake is not specific for tumour cells and canbe found in non-neoplastic lesions, notably in inflammation andrepair. Furthermore, high physiological cerebral glucose uptake limitsits usefulness in brain evaluation [118]. FDG PET has neither anestablished value nor a clear role in BC. In patients with locallyadvanced BC, FDG PET adds considerable information to conventionalimaging (such as CT and bone scan) in detecting sites of metastaticspread [119–124], and subsequent studies have shown that thecombination of PET and CT in PET/CT instruments further improvesperformance [121]. Studies showed that FDG PET has an impact ontherapeutic approach in many cases [125,126] and may reduce thenumber of different imaging studies needed, thereby simplifying theapproach to staging [127,128]. In particular, FDG PET/CT may beespecially helpful in inflammatory BC, where studies suggest that itmay detect extra-axillary nodal disease and/or distant metastases notfound by CT and bone scan in 25%–50% of patients [129]. FDG PET/CT istherefore recommended in the NCCN guidelines as an optional stagingstudy for patients with locally advanced, inflammatory, and recur-rent/metastatic BC – clinical scenarios in which systemic staging isindicated – especially when there are questions arising from standardstaging studies [130]. FDG PET has been employed in evaluation ofresponse to chemotherapy in patients with locally advanced BC [131],showing promising results. However, FDG showing uptake ininflammatory cells sometimes hampered the interpretation duringor shortly after therapy. Finally, FDG PET can change the therapeuticmanagement of BC patients in a range between 30% and 60% [132].

9. Conclusions

The overview about different PET and SPECT tracers shows thecomplexity and heterogeneity of molecular pathways in BC. As clearlydescribed, heterogeneous cancer population needs have tailoredtreatments, and for this purpose molecular imaging is useful. Thelimitation of sampling by biopsy and the selection of stronger clonesrepresent the basis for imagingmodalities that are able to characterize


each lesion. In fact, considering that the cancer genome is instable, itmeans that some characteristics may be lost/gained due to Darwinselection of the cellular clones with the best survival abilities, themain advantage obtained by nuclear tools is the detection of cellsresistant to conventional treatments. Therefore, the knowledge of BCbiology is important both for diagnostic end-point and for under-standing the best strategies of therapy with a final impact onprognosis. We can conclude that:

1- 18 F-FDG PET and, if preliminary data will be confirmed, 11C/18 F-Choline PET can be proposed for the risk stratification ofpatients due to the ability of detecting loco-regional or distantinvolvement;

2- 18 F-FLT or analogous radiotracers can play an important rolefor the evaluation of “interim” treatment response

3- 18 F-FES, 18 F-FNNPandother target imaging tracers canbeusedfor addressing specific and target therapies (as neoadjuvant,adjuvant or salvage therapy) and for testing the resistance tonovel treatments (both radiation therapy and chemotherapy).

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Molecular pathways and molecular imaging in breast cancer: An update

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