BIOMARKERS (S DAWOOD, SECTION EDITOR)

Biomarkers in Breast Cancer and the Implicationsof Their Discordance

Ashish Singh & Bhawna Sirohi & Sudeep Gupta

Published online: 19 October 2013# Springer Science+Business Media New York 2013

Abstract The analysis of biomarkers in breast cancer hasmade a large impact in treatment decisions and determiningprognosis. Information gained from the phenotypic and mo-lecular profile helps both in prognostic stratification as well astreatment selection.With the advent of newermore efficaciouschemotherapeutic and biological agents in the management ofmetastatic breast cancer, most women with this disease areexposed to multiple lines of therapy. Discordance in the re-ceptor status between the primary and metastatic sites isfrequently encountered and may play an important role indeciding subsequent therapy. In this review, we look at therole of current biomarkers in the management of breast cancer,which are promising and the therapeutic and prognostic im-plications of their discordance during the natural course ofdisease progression.

Keywords Biomarkers . Breast cancer . HER2overexpression . Hormone receptors ER/PR . Urokinaseplasminogen activator system (uPA) . Basal markers .

Circulating tumor cells

Introduction

Breast cancer is a heterogeneous [1] and phenotypically di-verse disease with varied presentation, morphology, and be-havior. It is composed of several biologic subtypes that havedistinct characteristics and responses to therapy.

Information provided by a complete routine pathologicevaluation of the surgical specimen remains the most impor-tant element in determining the prognosis of patients withbreast cancer. The most potent prognostic factors available

are lymph node status, tumor size and histologic grade, histo-logic tumor type, and lymphatic vascular invasion [2].

Molecular biomarkers are required to improve predictionof response to specific agents, to aid tumor classification, andovercome the inherent subjectivity involved in histopatholo-gy. Recent microarray-based expression profiling studies haveattempted to classify breast cancer into biologically and mo-lecularly distinct groups based on their molecular profile [3].Other larger studies including immunohistochemical studyresults have demonstrated that the molecular biomarker ex-pression is best utilized in combination with other biomarkersrather than individually [4].

Evidence of discordance in receptor status between prima-ry and metastatic breast cancer lesions is a phenomenon that isbeing reported more frequently. Discordance between theresults of the tumor biomarkers between the primary andmetastatic site and 2 different metastatic sites is likely toinfluence treatment decisions. Certainly, a change from anegative to a positive receptor status will imply that a targetedtherapy may then be incorporated into the treatment plan.Conversely, a change from a positive to a negative statuswould not only avoid the use of these agents and their relatedside effects but also would reduce cost of therapy [5].However, evidence to suggest discordance leading to alter-ations in patient outcomes is limited, highlighting the need forfurther research in this area.

Her2 neu Overexpression

HER2 overexpression is regarded as a negative prognosticfactor based on the available data [6]. In most studies, HER2overexpression in primary tumor tissue is associated with aworse prognosis in untreated patients. HER2 overexpressionhas also been shown to correlate with other factors associatedwith a poor prognosis (such as tumor grade, size, and nodal

A. Singh :B. Sirohi (*) : S. GuptaTata Memorial Center, Parel, Mumbai 400012, Indiae-mail: bhawna.sirohi13@gmail.com

Curr Breast Cancer Rep (2013) 5:266–274DOI 10.1007/s12609-013-0126-8

status) [7]. HER2 overexpression in tumor tissue as a markerof poor prognosis in patients with node-positive disease iswell established. In women with node negative disease arecently reported series where almost 90% of the tumors inwere >1.0 cm in size and 70% did not receive adjuvantsystemic therapy, HER2 overexpression was independentlyassociated with significantly worse 10-year relapse free andbreast cancer-specific survival [6]. However, AmericanSociety of Clinical Oncology (ASCO) expert panel on tumormarkers in breast cancer does not recommend the use ofHER2 for determining prognosis, largely because in the eraof targeted agents outcomes are determined by subsequenttherapy [8]. There is insufficient evidence to support theclinical use of serum HER2 extracellular domain testing foreither prognostication or predictive value [9].

The level of discordance in HER2 status in paired primaryand metastatic breast cancers range between 2% and 25%, butaverages about 10% [10–13]. In a recent review on this issue,Turner et al. [14••] compiled data on the discordance foundacross studies in reported literature. Many studies report arelatively consistent pattern of 5%–10%HER2 discordance rate.Gain and loss of HER2 is relatively equally reported, in contrastwith ER/PgR, where loss is more common than gain (Table 1).

Findings from neoadjuvant studies have suggestedtrastuzumab based therapy as 1 of the factors for loss of Her2 expression in tumor tissue [15]. Data supporting therapydriven Her2 loss is sparse. From 4 studies assessingtrastuzumab effect on HER2 loss at relapse, no correlationwas found [16–19].

Discordance could arise due to limited accuracy and repro-ducibility of receptor assays stemming from differences intissue fixation, antigen retrieval, and staining methods as wellas subjective scoring resulting in inter-observer variability.Intratumor heterogeneity that can subsequently result in sam-pling error is another possible explanation for the reporteddiscordance especially in the setting of preoperative chemo-therapy that incorporates targeted biological agents. A truegenetic switch in the biology of the disease, whereby themolecular profile and thus subtype of the disease changes, isalso possible since cancers are inherently genetically unstable.

Moreover, factors that could have led to a high false dis-cordance rate includes retrieval of HER2 status from medicalrecords, rather than retesting both primary and metastaticdisease, and analytical errors, including differing methods ofHER2 determination, and assays performed at different times,with different protocols, and/or by different pathologists, haslikely impacted on reported discordance rates [14••].

Perez et al. [20] have reported this analytical error whenHER2 expression was assessed by local and centralized labsin tumors from 2175 patients (86%) enrolled in the phase IIIN9831 trial. Despite analyses being performed on the sametumor, discordance rates of 12% for FISH and 18% for IHCwere found.

The effect of discordance on prognosis is subject to biasbecause of the confounding influence of change in treatmentfollowing the biopsy result. The majority of studies that in-cluded some assessment of discordance as a prognostic factorreported an association with worse outcome in patients withdiscordance vs concordance [14••]. In a study by Mittendorfet al. [21], 142 HER2-positive patients were treated withconcomitant trastuzumab and neoadjuvant systemic chemo-therapy—pCR was achieved in 72 (50.7%) patients. Eight(32.0%) post-treatment tumors were found to be HER2-negative by FISH. At a median follow-up of 37 months(range 8–56 months), the relapse free survival was signif-icantly better for patients with tumors that retained HER2amplification (87.5% vs 50%, P = 0.04).

Hormone Receptors ER/PR

While the predictive value of hormone receptor expression iswell accepted, its prognostic importance has been a matter ofdebate for many years.Womenwith stage I ER-positive breastcancer who receive no systemic therapy after surgery have a5%–10% lower likelihood of recurrence at 5 years than dothose who have ER-negative tumors [22]. However, as thelength of follow-up increases, the advantage of ER-positivityin terms of relapse and death grows smaller and ultimatelydisappears [23]. This might reflect sequential improvementsin adjuvant chemotherapy, with disproportionate benefits forwomen with hormone receptor-negative tumors. Expressionof hormone receptors is associated with a number of otherwell-established prognostic indicators, but not with nodalmetastases [24]. Thus, ER and PR status may be bettermarkers of growth rate than of metastatic potential.

ER-positive tumors are more likely to be histologicallywell differentiated, to have a lower fraction of dividing cells,less likely to be associated with mutations, loss or amplifica-tion of breast cancer-related genes such as p53, HER2, orHER1. Despite these associations, there is no consistent evi-dence that these factors influence response to endocrine ther-apy in patients with receptor-positive tumors [25]. ER-positive breast cancers are more likely metastasize in bone,soft tissue, or the reproductive tracts. ER-negative breastcancers commonly metastasize to brain and liver, sites thatare associated with shorter survival [26].

Hormone receptor loss has been reported following endo-crine therapy [27–29]. In one of the largest studies to date,Lindstrom et al. [30] reported a retrospective analysis of 459patients, finding that ER loss was associated with poorer OScompared with concordant ER + disease (HR = 1.48, 95% CI1.08–2.05). In contrast, a prospective trial from Amir et al.[31], which included 121 patients with paired biopsies,looking at patients with any receptor discordance comparedwith patients with unaltered receptor status, post hoc analyses

Curr Breast Cancer Rep (2013) 5:266–274 267

Tab

le1

StudiesreportingHER2+/−

ER/PgR

discordanceassessed

ondistantm

etastasisonly

Author

Year

Totalp

ts(n)

Num

berof

paired

biopsies

assessingHER2

HER2

discordance*

(n)

HER2

discordance*

(%)

HER2gain

(n)

HER2loss

(n)

HRdiscordance

ER

(%)

ERgain

(n)

ERloss

(n)

PgR

(%)

PgRgain

(n)

PgRloss

(n)

Amir[31]

2012

121

838

106

216

411

404

34

Duchnow

ska[18]

2012

120

119

1714

107

2913

2229

1123

Botteri[64]

2012

100

605

82

211

48

--

-

Liu

[65]

2012

4646

511

14

307

754

1015

Curigliano

[19]

2011

255

172

2414

717

1515

2226

18106

Niik

ura[66]

2011

182

182

4324

-43

--

--

--

Sari[17]

2011

7561

915

63

3611

1654

1326

Chang

[67]

2011

5656

713

52

--

--

--

Gonzalez-Angulo

[68]

2011

5151

24

11

--

--

--

StR

omain[69]

2011

3434

13

01

201

5-

--

Hoefnagel[70]

2010

233

233

63

33

107

1730

1258

Koo

[71]

2010

1313

00

nana

8na

na8

nana

Simmons[32]

2009

4025

28

20

120

328

07

Low

er[72]

2009

382

382

128

3437

90-

--

--

-

Broom

[73]

2009

100

181

60

118

56

370

22

Tapia[74]

2007

105

105

66

24

--

--

--

Zidan

[12]

2005

5858

814

71

--

--

--

Lear-Kaul[75]

2003

1212

00

00

--

--

--

Vincent-Salom

on[76]

2002

4444

25

02

--

--

--

Gancberg[10]

2002

100

685

72

3-

--

--

-

Lacroix

[77]

1989

5353

713

nana

--

--

--

268 Curr Breast Cancer Rep (2013) 5:266–274

demonstrated no difference in either OS or time totreatment failure in patients with discordant, comparedwithconcordant, receptor expression (median OS 27.6 vs 30.2months for concordant vs discordant groups, respectively,HR = 0.94, P = 0.85).

In a prospective pilot study from Simmons et al. [32], thatenrolled 40 patients, 25 biopsies confirmed recurrent breastcancer. Receptor discordance was reported in a total of 10patients (40%), 2 patients presumably had change in morethan 1 receptor, although this is not specifically reported.Treatment was altered at the discretion of the treating oncol-ogist in 6 patients (17% of all patients with biopsy performed).

Thompson and colleagues [33] reported on the BRITS(Breast Recurrence in Tissue Study) study that enrolled 205patients for rebiopsy at time of relapse and found discordancerates in 137 cases as 10%, 25%, and 3% for ER, PgR, andHER2, respectively. Changes in HER2 or hormone receptorstatus were considered important for change in treatmentstrategy. As per the accruing clinicians, 24 out of 137 patients(17%) had a clinically significant change in receptors.

Importantly, in all studies, retrospective and prospectivealike, treatment decisions have been made at the discretionof the treating clinician, confounding the results. Nonetheless,from the available data, receptor discordance appears not toalways lead a change in management.

Markers of Proliferation

The proliferative rate of breast tumors may be assessed by avariety of methods, including mitotic counts, thymidine label-ing index, bromodeoxyuridine (BrdU) labeling, S-phase frac-tions as determined by flow cytometry, IHC using monoclonalantibodies (MoAbs) to antigens found in proliferating cells(eg, Ki-67 or proliferating cell nuclear antigen [PCNA/cy-clin]), and the assessment of argyrophilic nucleolar organizerregions (AgNOR).

Monoclonal antibodies that are reactive with the Ki-67antigen in formalin-fixed, paraffin-embedded specimens (eg,MIB-1, the most frequently used) have been studied exten-sively and are most commonly used test today. In a meta-analysis of 4 Ki-67/MIB-1 positivity was associated with asignificantly higher risk of relapse and worse breast cancersurvival in both node-positive and node-negative disease [34].Comprehensive recommendations are available on assess-ment, interpretation, and scoring of Ki67 with a goal toharmonized methodology, create greater comparability be-tween the labs and subjects, and allow earlier valid applica-tions of this marker in clinical practice [35]. In a recent studyby Denkert et al. [36] the authors investigated the impact ofpretherapeutic Ki67 levels as a predictive marker for responseto neoadjuvant chemotherapy as well as a prognostic markerfor progression-free and overall survival. One thousand, one

hundred and sixty-six pretherapeutic core biopsies from theneoadjuvant Gepartrio trial were evaluated for Ki67 by im-munohistochemistry. Samples were divided into 3 subgroupsfor Ki67 (0%–15% vs 15.1–35% vs >35%) The pCR rates inthese 3 groups of Ki67 expression were 4.2%, 12.9%, and29.0%, respectively (P < 0.0005).

Due to difficulty with methodology and inconsistency ofthe data most of the methods have not gained wide spreadacceptance in clinical practice to assign patients to prognosticgroupings and is not considered a routine part of breast cancerevaluation.

Urokinase Plasminogen Activator System

Urokinase plasminogen activator (uPA) is a serine proteaseinvolved in the process of cancer invasion and metastases.Inhibitors of uPA (plasminogen activator inhibitors [PAI]types 1 and 2) have been identified. PAI-1 levels are high intumor tissue and plasma, and PAI-1 is inactivated when boundto uPA. PAI-2 is usually present in low levels except for someconditions such as pregnancy or myeloid leukemia. Highlevels of uPA, uPAR, and PAI-1 have been associated withshorter survival, while high levels of PAI-2 were associatedwith better outcomes in retrospective reports. uPA and PAI-1levels determined in primary tumor tissue extracts has shownto be the strongest predictors of disease-free and overallsurvival, after nodal status in a pooled analysis of individualpatient data from 8377 women treated in clinical trials spon-sored by the EORTC [37].

Levels of uPA and/or PAI-1 may allow patients to bestratified into prognostically relevant subgroups. Low riskwas defined in the N0 trial as levels of uPA (≤3 ng/mg ofprotein) and PAI-1 (≤14 ng/mg of protein). High risk patientshad significantly higher risk of recurrence and lower rates ofoverall survival compared with low-risk patients [38••].

Requirement of ELISA to be carried out on large on at least300 mg frozen tissue sections removed at the time of resectionis a major hindrance to the widespread acceptance of thisassay. Reports of immunohistochemical staining to determineuPA and PAI-1 status require further evaluation andvalidation.

Multigene Predictors of Clinical Outcome

Gene expression profiling has identified molecular signatures,such as the 21-gene recurrence score (RS, Oncotype Dx) [39],the Amsterdam 70-gene prognostic profile (Mammaprint)[40], and the Rotterdam/Veridex 76-gene signature [41].These tests help the clinician to predict breast cancer outcomeand response to treatment with information in addition to theconventional prognostic indicators.

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The 21-gene recurrence score (RS, Oncotype Dx) is an RT-PCR assay that measures the expression of 21 genes—16cancer-related genes and 5 reference genes—in RNA extract-ed from FFPE samples of tissue from primary breast cancer. Itis among the best-validated prognostic assays, and also ap-pears to predict response to systemic chemotherapy in womenwith node-negative, estrogen receptor positive breast cancer.For women treated with Tamoxifen, the recurrence scorepredicts locoregional and distant recurrence [42]. In theNSABP B-20 [43] trial the addition of CMF chemotherapyto tamoxifen resulted in a higher rate of distant disease freesurvival at 10 years among patients with a high RS comparedwith treatment with tamoxifen alone (88% vs 60%, respec-tively; relative risk [RR] 0.26, 95% CI 0.13–0.53) [9]. Thebenefit was not seen in low RS (96% vs 97%; RR 1.31, 95%CI 0.46–3.78) or an intermediate RS (90% vs 91%; RR 0.61,95% CI 0.24–1.59).

It is unclear at which RS cut-off chemotherapy should beoffered to women with early breast cancer. The recentlycompleted TAILORx trial [44] will hopefully provide betterdata to inform the role of the RS in identifying those node-negative patients who may and those who may not benefitfrom chemotherapy.

In the SWOG 8814 trial [45] which randomized 367 post-menopausal women with pathologically node-positive, hor-mone receptor-positive breast cancer to CAF chemotherapy vstamoxifen alone, the addition of CAF among women with ahigh RS resulted in improvements in 10-year disease-freesurvival (55% vs 43%, respectively; hazard rate [HR] 0.59,95% CI 0.35–1.01) and overall survival (73% vs 54%, respec-tively; HR 0.56, 95% CI 0.31–1.02). These results were notseen among women with a low or intermediate RS. Althoughthis data suggests a role for the RS in patients with patholog-ically involved lymph nodes, it is not yet approved for use inthis setting. Ongoing trials like RxPONDER [46] will try andmeasure just how much, if any, benefit these patients get fromchemotherapy. And it will try to determine where the cutoffscore is between patients who benefit from chemotherapy inaddition to hormonal therapy alone.

Similar findings have been reported with aromatase inhib-itors in postmenopausal women, suggesting that the RS iden-tifies relative endocrine insensitivity as a general phenome-non. In the TransATAC study 9-year disease recurrence ratesin low (RS < 18), intermediate (RS = 18 to 30), and high RS(RS > or = 31) groups were 4%, 12%, and 25%, respectively,in node negative patients and 17%, 28%, and 49%, respec-tively, in node positive patients [47].

EndoPredict assay is another tool to predict the risk ofrecurrence in women with estrogen receptor positive, HER2negative breast cancer treated with endocrine therapy alone[48]. This assay can be performed on formalin-fixed paraffin-embedded tissue. It provides additional prognostic informa-tion to standard clinic-pathological factors and improves risk

classification. It is designed to integrate clinical and genomicinformation and includes factors such as tumor size and nodalstatus [49].

The clinical relevance of molecular/clinical EndoPredicttest (EPclin) score was tested in 1702 (ABCSG-6: n = 378,ABCSG-8: n = 1324) ER-positive/HER2-negative postmen-opausal women from 2 large phase III trials treated only withendocrine therapy [50••]. The authors were able to dem-onstrate by combining EPclin one can separate a subsetof ER-positive, HER-2 negative, postmenopausal breastcancer patients with excellent prognosis, with distantrecurrence of ∼5% after 10 years of follow-up. Thestudy also showed that about 47%–57% of all womenassigned to intermediate/high risk by common clinicalguidelines could be spared chemotherapy.

Basal Markers

Expression of high molecular weight or ‘basal’ cytokeratin isreported to have a poorer prognosis independent of expressionof ER and Her2 [51]. The relationship between tumor size andproportion of lymph node positivity and outcome is also lessclear amongst tumors that express basal markers. The gener-ally accepted IHC markers used to define basal-like are Ck5/6and EGFR; in addition to these markers, Ck17 and Ck14 havealso been used in some studies. Using these markers in thetriple negative tumors identifies a biologically and clinicallydistinct subgroup (60%–90%) with poor response to treatmentand worse outcomes [52]. About 80% of breast cancers oc-curring in BRACA 1 mutation carriers are basal like howevermost of these cancers are sporadic. Basal-like subtype whichharbors some similarity in gene expression to that of the basalepithelial cells of normal breast tissue makes up about 15%–20% of breast cancers. It is characterized by low expression ofthe luminal and HER2 gene clusters. The discordance be-tween the classification methods of triple negative breastcancer and basal like is up to 30%.

Claudin low breast cancer comprises about 5%–10% ofbreast cancer and has a basal like or triole negative phenotype.It expresses epithelial-mesenchymal transition genes and char-acteristics reminiscent of stem cells. It is has low to absentexpression of epithelial cell-cell adhesion genes (claudin 3, 4,and 7, and E-cadherin), differentiated luminal cell surfacemarkers (EpCAM and MUC1), and enrichment forepithelial-to-mesenchymal transition markers, immune re-sponse genes, and cancer stem cell-like features (CD44+/CD24-and high ALDH1A1). In contrast to basal-like breastcancers, claudin-low tumors appear to be slower growing and,with features of mesenchymal and mammary stem cells,may be of different oncogenic origin. Metaplastic andmedullary breast cancers often overlap with the claudin-low subtype [53].

270 Curr Breast Cancer Rep (2013) 5:266–274

Disseminated Tumor Cells

In early stage breast cancer detection of metastatic cellsin bone marrow is reported to confer a higher risk forrelapse and worse survival [54, 55]. It is linked toother poor prognostic features such as tumor size,grade, and nodal status [56]. However, the associationbetween the presences of disseminated tumor cells withany other standard prognostic indicator was not con-firmed in other reports [57], Clinically apparent breastcancer metastases develops in approximately 30%–50%of patients who have disseminated tumor cells there-fore, not all detectable breast cancer cells in the bonemarrow are clinically relevant.

Circulating Tumor Cells

Circulating tumor cells (CTCs) in the peripheral blood ofwomen with both early and advanced breast cancer has beenassociated with a poor prognosis. Christofanilli et al. [58]reported in 177 patients with metastatic breast cancer,levels of circulating tumor cells both before the patientswere to start a new line of treatment, and at the firstfollow-up visit. Patients with levels of circulating tumorcells equal to or higher than 5 per 7.5 ml of wholeblood, had a shorter median progression-free survival (2.7months vs 7.0 months, P < 0.001) and shorter overall survival(10.1 months vs >18 months, P < 0.001) compared withwomen with higher levels. Meta-analysis by Zhang [59•]showed the presence of CTCs was associated with an increasedrisk of disease progression and mortality. Change in treatmentdecisions based on the CTC levels are not made currently andresults of ongoing prospective trials to inform the impact ofCTCs on treatment and survival is awaited [60].

Limitations in Outcome Prediction Using Biomarkers

There can be some pitfalls in outcome prediction of breastcancer. Due to inter-tumor and intra-tumor heterogeneity forvarious traits related to carcinogenesis such as invasion,growth, and metastatic potential the reported literature maynot represent the clear picture. This may have contributed tothe discrepancies in the literature and to the inconsistentconclusions seen in published studies assessing the samemarkers. Multifocality , multicentricity, mixed histologies,discrepancies between clinical and pathological tumor size ,bilateral presentation of tumors, and combining dataset fromdifferent point times are various primary tumor factors,which are not standard across studies. Prognosticationstudies using clinical trial materials should identify selec-tion and randomization criteria and ensure their relevance to

the study hypothesis under investigation and use suitableoutcome endpoints [61].

Newer techniques, such as receptor expression analysis ofcirculating tumor cells [62, 63] are being developed to deter-mine tumor receptor status based on a ‘liquid biopsy’. Suchtechnology if validated in future will improve feasibility,safety, and rapidity of tumor receptor evaluation and reducethe needs to rebiopsy.

Conclusions

TNM stage, axillary lymph node status, tumor size, grade, andhormone receptor status are the uniformly accepted prognosticmarkers. As per the guideline recommendation from ASCO[8] ER, PR, and HER2 overexpression should be evaluated onevery primary breast cancer. HER2 expression should directHER2-directed therapy use in the metastatic and adjuvantsetting. Oncotype DX assay is an important tool, which canhelp predict the risk of recurrence in women with newlydiagnosed, node-negative, ER+ breast cancer who will bereceiving endocrine therapy. Urokinase plasminogen activator(uPA) and plasminogen activator inhibitor-1 (PAI-1) byELISA on fresh or frozen tissue may be used for determina-tion of prognosis in patients with newly diagnosed, node-negative breast cancer. Low levels of both markers are asso-ciated with a sufficiently low risk of recurrence in womenwithhormone receptor positive disease who will receive hormonaltherapy.

The available evidence suggests that loss of HER2 and/orER may result in worse outcomes, although data were notconsistent across trials and were often confounded by lack oftreatment in the setting of receptor loss. Therefore theinformation available on the clinical impact of discor-dance is limited, inconclusive, and conflicting. It islikely that currently clinicians would continue to usetargeted therapy in the treatment of the patient at sometime point regardless of the rebiopsy result, and there-fore receptor positive primary breast cancer patientswould benefit less from rebiopsy. However, patientswith receptor gain would appear to benefit from additionof targeted treatment. Further prospective trials regarding theimpact of HER2 discordance and the impact of newer bio-markers in treatment outcomes are needed.

Compliance with Ethics Guidelines

Conflict of Interest Ashish Singh declares that he has no conflict ofinterest. Bhawna Sirohi declares that he has no conflict of interest. SudeepGupta declares that he has no conflict of interest.

Human and Animal Rights and Informed Consent This article doesnot contain any studies with human or animal subjects performed by anyof the authors.

Curr Breast Cancer Rep (2013) 5:266–274 271

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