The Oncologist-2014-Adaniel-1019-27.pdf

Christina Adaniel, Komal Jhaveri, Adriana Heguy and Francisco J. EstevaGenome-Based Risk Prediction for Early Stage Breast Cancer

doi: 10.1634/theoncologist.2014-0124 originally published online September 3, 20142014, 19:1019-1027.The Oncologist

http://theoncologist.alphamedpress.org/content/19/10/1019located on the World Wide Web at:

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Breast Cancer

Genome-Based Risk Prediction for Early Stage Breast CancerCHRISTINA ADANIEL,a KOMAL JHAVERI,a ADRIANA HEGUY,b FRANCISCO J. ESTEVAaaDivision of Hematology/Oncology, Laura and Isaac Perlmutter Cancer Center, and bGenome Technology Center, New York UniversityLangone Medical Center, New York, New York, USADisclosures of potential conflicts of interest may be found at the end of this article.

Key Words. Breast cancer x Gene expression x Genomic profiling

ABSTRACT

Tests to better characterize tumor genomic architecture arequickly becoming a standard of care in oncology. For breastcancer,theuseofgeneexpressionassays forearlystagediseaseis already common practice. These tests have found a place inrisk stratifying the heterogeneous group of stage III breastcancers for recurrence, forpredicting chemotherapy response,and for predicting breast cancer-relatedmortality. In the last 5years, more assays have become available to the practicing

oncologist.Given the rapiditywithwhich this fieldhasevolved,it is prudent to review the tests, their indications, and thestudies from which they have been validated. We presenta comprehensive review of the available gene expressionassays for early stage breast cancer. We review data forseveral individual tests and comparative studies looking atrisk prediction and cost-effectiveness. The Oncologist 2014;19:10191027

Implications for Practice: Gene expression assays are an important component in the management of early stage breastcancer. Understanding their utility as well as their drawbacks is critical for the practicing oncologist to make informed decisionsregarding care.

INTRODUCTION

Breast cancer is one of the leading causes of cancer-relatedmorbidity worldwide [1]. In the U.S., 235,030 new cases areexpected to be diagnosed in 2014 [2]. Of these, approximately164,000 cases (70%) will be classified as early stage (stage III)breast cancer, for which the potential for cure is excellent.Although surgery is the major curative modality, adjuvantchemotherapy plays an important role in increasing cure ratesfor a selected group of patients. Approximately 15%30% ofpatients with stage I breast cancer and up to 40% of stage IInode-negative patients will have a recurrence with localtherapy alone. The disparity in outcomes within groups ofsimilar histopathologic characteristics speaks to the incredibleheterogeneity of this disease [3, 4]. How to better define andidentify these higher risk individuals has been an area ofintense investigation for more than a decade [5].

This clinical conundrum has resulted in a scientific race ofsorts to more precisely prognosticate risk of recurrence (ROR)in early stage breast cancer. The investigation has focusedmainly on patients with hormone receptor-positive (HR1)HER2-negative (HER22), and lymph node-negative (LN2)disease, encompassing the majority of patients with earlystage breast cancer. HER2-positive tumors are considereda separate group that benefit from adjuvant HER2-basedchemotherapy [68]. Axillary lymph node involvement isassociated with poor outcome, and those patients generally

receive adjuvant chemotherapy. However, efforts are ongoingto identify patients with one to three positive lymph nodes forwhom chemotherapy is not indicated. This is particularlyimportant because although the benefits of chemotherapymay be great for a subset of patients, chemotherapy may alsoconfer significant, potentially life-long toxicities for others.These include peripheral neuropathy; premature ovarianfailure; and, rarely, cardiac dysfunction [9, 10]. Short-termtoxicities are also substantial. Risk for infection, alopecia, andfatigue, in addition to the emotional toll that chemotherapycan often take on patients, are important side effects, and allmay substantially affect quality of life.These secondary effectsare common to most standard adjuvant chemotherapyregimens for breast cancer [11].

Focus has been on the larger group of stage III, node-negative,HR1, HER22breastcancer patients for two reasons:to better communicatewith patients about true individual riskof recurrence and to better identify high-risk patients forwhom adjuvant chemotherapy may be more recommendedand low-risk patients for whom chemotherapy may offer littlebenefit.

Inorder tohave amoreconcreteunderstandingof the risksand benefits of adjuvant chemotherapy, several gene expres-sionassayshavebeendeveloped tobetterstratify this groupofdiversepatients.Theassaysevaluatevaryingnumbersofgenes

Correspondence: Francisco J. Esteva,M.D., Ph.D., Laura and Isaac Perlmutter Cancer Center, NewYorkUniversity LangoneMedical Center, 160East 34th Street, New York, New York 10016, USA.Telephone: 212-731-5657; E-Mail: [email protected] ReceivedMarch 25, 2014;accepted for publication July 30, 2014; first published online in The Oncologist Express on September 3, 2014. AlphaMed Press 1083-7159/2014/$20.00/0 http://dx.doi.org/10.1634/theoncologist.2014-0124

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in the breast tumor, to quantify their expression levels, andoutput a score that correlates with risk of recurrence. Thesetests, which are commercially available and in some cases arecovered by insurance in the U.S., are being used in clinicalpractice toassistwithprognosticationandoften toaiddecisionmaking regarding adjuvant chemotherapy.

Since the last time The Oncologist reviewed gene expres-sion assays for breast cancer, in 2008, more tests have becomecommercially available, and multiple validation studies havebeenpublished [12]. Inaddition,onesuchtestwasclearedbytheU.S. Food and Drug Administration (FDA) in September 2013,and others are actively seeking approval by regulatory agencies.Given the large number of options now available to practicingoncologists, it is pertinent to update this review with acomprehensive analysis of the available breast cancer geneexpression tests.We aim to provide a clearer understanding ofthese assays so that providers may make more informeddecisions in the clinic, avoid unnecessary use of cytotoxicchemotherapy,andreduce theburdenonthehealthcaresystem.

MATERIALS AND METHODSA comprehensive literature search of all relevant clinical trials,original research articles, review articles, and editorials in thePubMed database concerning gene expression assays in breastcancerwas performed. Original research publications pertainingto discovery-phase and validation studies for each of thespecified gene expression assays were reviewed. The searchtermsgeneexpressionprofiling, geneexpressionassay, geneexpression, Oncotype DX, MammaPrint, PAM50, BreastCancer Index, and breast cancer were used. Individual bio-technology company websites were also reviewed for additionalinformation regarding tissue requirements, technologyused, andavailability of tests. Assays based on gene expression profilingwere included. Those based on immunohistochemistry (IHC)were excluded. Tests that are clinically available in the U.S. wereincluded (Table 1). Those available only in Europe, such asMapQuant Dx and EndoPredict, were excluded.

GENE EXPRESSION ASSAYS

Oncotype DXThis 21-gene assay developed by Genomic Health (RedwoodCity, CA, http://www.genomichealth.com) is the most fre-quently used test in clinical practice in the U.S. [13]. Based onquantitative reverse transcription polymerase chain reaction(PCR) expression levels of 5 reference genes and 16 selectedgenes related mostly to the estrogen receptor (ER), HER2,proliferation, and invasion, the assay determines a recurrencescore (RS) that assigns patients into a low-, intermediate-, orhigh-risk category.The original training setwithwhich the assaywas designed used 447 patient specimens from three breastcancer clinical trials (a variety of patients, some with ER2 andLN1 disease) but was most heavily weighted toward thetamoxifen-only arm of the National Surgical Breast and BowelProject-B20 (NSABP-B20) cohort [14]. The training set included250 candidate genes that were eventually paired down to 21based on a best-fit model. The assay was validated in a studypublished by Paik et al. in theNew England Journal of Medicinein 2004 [14]. The validation cohort included 668 ER1, LN2patients from the NSABP-B14 study who were all treated with

tamoxifen. The study showed a significant difference in distantrecurrence at 10 years among the three groups, as defined bytheRS, and thescoreprovidedpredictivepower independentoftumor size and age.The distant recurrence rate at 10 years was6.8% for the low-risk group, 14.3% for the intermediate-riskgroup, and30.5% for thehigh-risk group.TheRSwas also shownto correlate independently with overall survival (OS).

The test has since been validated inmultiple other studies,especially for patients treatedwith endocrine therapy. Amongthese is another study by Paiket al., from2006, demonstratingthe use of the Oncotype DX assay not only for prediction ofrecurrence but also for prediction of chemotherapy benefit[15]. Again, using the NSABP-B20 cohort, in this study Paiket al. evaluated 227 patients randomized to treatment withtamoxifen alone versus 424 patients treated with tamoxifenplus chemotherapy (cyclophosphamide, methotrexate, and5-fluorouracil [5-FU] or methotrexate and 5-FU). The RSsignificantly correlated with benefit from chemotherapy asdetermined by 10-year disease-free survival (DFS) comparedbetween each RS group in the tamoxifen-only and tamoxifen-plus-chemotherapy arms. It was estimated that freedom fromdistant recurrence was increased from 60% to 88% when che-motherapy was added to tamoxifen in the high-risk RS group.The benefit of chemotherapy in the intermediate-risk groupwas less clear. However, it is important to note that, similar toMammaPrint, this study was not an independent validationbecause therewas duplication of samples from the training set.Onlyoneof theoriginal validationstudies showednoprognosticvalueinpatientswithnode-negativebreastcancerwhoreceivednoadjuvant systemic therapy [16]. However, the study includedpatients with ER1 and ER2 breast cancer, and the test wasoriginally designed to assess ER1 tumors. Nevertheless, giventhe aforementioned studies and similar data from othervalidations, the American Society of Clinical Oncology (ASCO)and the National Comprehensive Cancer Network guidelineshave recommended the use of Oncotype DX to risk-stratifypatients with ER1, HER22, LN2/N1mi (axillary metastases,2 mm) early stage breast cancer [1720].

PAM50Prosigna (NanoString Technologies Inc., Seattle,WA, http://www.nanostring.com) is the gene expression profile assaymost recently cleared by the FDA, gaining approval inSeptember 2013. The technology, unlike other assays, can beperformed in any CLIA-certified laboratory with the use ofNanostrings NanoCounter Analysis System. Briefly, geneexpression is measured in RNA extracted from formalin-fixedparaffin-embedded tissue using a novel, digital, color-coded,bar code technology that allows measurement of multipletranscripts with high sensitivity (less than one copy per cell). Apanel of 50 classifier genes and 5normal geneswas selected toclassify breast cancers into an intrinsic subtype (luminal A,luminal B, HER2-enriched, and basal-like). Using the intrinsicsubtypedefinedby the50-gene signature, alongwith standardprognostic parameters, multiple studies have shown PAM50simproved ability to prognosticate risk of recurrence. Thetraining set for PAM50 included 29 normal breast samples and189 tumors with approximately 50% LN1 disease and mostlyhigh-grade tumors [21]. The training set evaluated 1,906genes, and this number was ultimately reduced to 50 genes

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of interestwith8 controls.This 50-geneclassifierwas testedon761 samples from patients who had not received adjuvantsystemic therapy and 133 who had received neoadjuvantchemotherapy (paclitaxel followed by 5-FU, doxorubicin andcyclophosphamide). In the first group of no adjuvant therapy,the majority of the samples were node-negative (710 of 761).The 50-gene classifier showed a clear difference in recurrence-free survival (RFS) in the 4 identified intrinsic subtype groups.These intrinsic subtypesdonotcorrespondcompletelywithERandHER2 statusbasedon IHC [22], suggesting that the subtypeis not simply a recapitulation of findings based on routinehistologic examination. The statistically significant differencein RFS remained after groups were stratified by ER status.The identified subtypes, however, were less successful atpredicting outcomes for HER21 tumors. Prediction for riskof recurrence was significantly improved when the subtypeclassificationwasadded toknownprognostic clinical variables.The combination of these parameters was coined ROR-C, riskof recurrence plus clinical variables. In addition, in the group ofpatients who received neoadjuvant therapy, risk of recurrencebased on intrinsic subtypes was able to predict pathologiccomplete response to chemotherapy with 94% sensitivity and97%negativepredictivevalue [21].Nielsenetal.validated thesefindings in an independent cohort of 786 patients with bothLN1 and LN2 disease who were treated with tamoxifen only[23]. The majority of the tumors were ER1. The intrinsic sub-types and the ROR scores identified by PAM50 were stronglyprognostic for RFS and disease-specific survival. An even largervalidation study was published more recently, in January 2014,lookingat1,478patients fromtheAustrianBreastandColorectalStudy Group 8 (ABCSG-8) trial population [24]. This cohortincluded postmenopausal ER1 early stage breast cancerpatients who received adjuvant hormonal therapy (tamoxifenalone or tamoxifen followed by aromatase inhibitor) withoutadjuvant chemotherapy. Again, the PAM50 ROR significantlypredictedRFS. Inall subgroups,except for theHER21group,theROR score and ROR risk groups added prognostic informationto the routinely used clinicopathologic parameters. Currently,PAM50 is approved for use in postmenopausal women withHR1 tumors with or without lymph node involvement.

MammaPrintMammaPrint (Agendia, Amsterdam, The Netherlands, http://www.agendia.com) was the first gene expression array for

breast cancer prognosis to be cleared by the FDA, back inFebruary 2007. MammaPrint is composed of a 70-genemicroarray. The original training set evaluated 25,000 genesin 78 LN2 sporadic early breast cancer tumors that were,5 cm in diameter and present in patients,55 years old [25].The genes were eventually narrowed to a 70-gene classifierthat showed a difference in expression patterns betweengood-prognosis and poor-prognosis groups, as defined by theauthors. However, the groupswere preselected based on theirclinical outcomes (one group with distant metastases at 5years, the other disease free at 5 years). A subsequentvalidation study assessed 295 patients with pT1 or pT2 tumorswith pN0 or pN1, with a median follow-up of 7.8 years [26].Sixty-one of the 295 patients were part of the training set;therefore, this was not a completely independent validationset, although a separate analysis was also completed leavingout the repeated tumors. The analysis showed a significantdifference between OS and metastasis-free survival in thegood- and poor-prognosis groups. Overall survival across allpatients. comparing the good-prognosis and poor-prognosisgroups, was 94% versus 54%, respectively, at 10 years.Whenevaluating the LN1patients alone, theMammaPrint signaturecontinued to be a significant prognostic marker. This 70-genesignature has since been validated in other studies, includingpatientswithup49positive lymphnodes [2730]. In thepast,limitations of the assay included the requirement for freshfrozen tissue and high-quality cDNA; however, since 2012,Agendiahasmadethetestavailable for formalin-fixedparaffin-embedded tissue [31].

Breast Cancer IndexThe Breast Cancer Index (BCI) (bioTheranostics, San Diego, CA,http://www.biotheranostics.com) incorporates two previ-ously established gene expression assays into one test. Itevaluates theratioof theexpressionof twogenes,HOXB13andIL17BR, based on qualitative PCR in combination with theMolecular Grade Index. The Molecular Grade Index is a five-gene expression assay looking at genes related to histologicgrade and tumor progression. In an initial analysis, Ma et al.demonstrated that in 60 postmenopausal ER1 patientstreated with 5 years of tamoxifen, the ratio of HOXB13 toIL17BR was an independent prognostic factor, with higherratios correlating with poorer outcomes [32]. The study alsoshowed that the ratio was able to predict for response to

Table 1. Breast cancer gene expression tests clinically available in the U.S.

Test Company Number of genes Tissue Technology Measure FDA-cleared

Prosigna Nanostring 501 22 ctla FFPE Digital bar-codedmRNA analysis

ROR: Low (,10), intermediate(1020), high (.20%) risk

Yes

Oncotype DX Genomic Health 161 5 ctl FFPE qRT-PCR RS: low (,18), intermediate(18-31), high (.31) risk

No

MammaPrint Agendia 70 Fresh frozen,FFPE

Microarray Good risk and poor risk Yes

Breast CancerIndex

bioTheranostics 51 2 gene ratio FFPE qRT-PCR Low, intermediate, and high risk No

aProsignameasures the expression levels of 50 genes used in the PAM50 classification algorithm, 8 housekeeping genes used for signal normalization, 6positive controls, and 8 negative controls.Abbreviations: ctl, control/housekeepinggenes;FDA,U.S. FoodandDrugAdministration; FFPE, formalin-fixedparaffin-embedded;qRT-PCR,quantitativereverse transcription-polymerase chain reaction; ROR, risk of recurrence; RS, recurrence score.

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tamoxifen therapy. Later, itwas shown that the combinationofthis two-gene ratio with the Molecular Grade Index providedbetter predictive value than either alone. This finding wasvalidated in a cohort of 588 postmenopausal early stage ER1breast cancer patients from the Stockholm trial of adjuvanttamoxifen versus placebo [33].This studywas used to developa continuous risk predictor, the BCI, using a scale of 0 to 10.TheBCI was prognostic in an independent population of node-negative patients who did not receive adjuvant therapy, withthe aim of risk stratifying for breast cancer death [34]. Thepredictive role of the BCI in patients receiving extended ad-juvant endocrine therapy was tested on tissue from patientswho participated in theMA.17 trial. In this study, high BCI wasassociated with a decrease in late recurrence in patientsreceiving extended letrozole therapy [35].The BCI test may beconsidered to assess the probability of distant recurrence inpatients diagnosedwithER1node-negativebreast cancer andto predict the likelihood of benefit from extended (.5-year)endocrine therapy in patientswho are recurrence free after aninitial 5 years of adjuvant endocrine therapy.

COMPARATIVE STUDIESGiven the wealth of data supporting each individual test, howdoes one choose which to use in clinical practice? Only ahandful of studies have compared the assays to one another.One such study evaluated 1,017 ER1 early stage breast cancerpatients from the Arimidex, Tamoxifen Alone or in Combina-tion (ATAC) trial [36]. In this study,OncotypeDXwas comparedwith PAM50 and IHC4 (an integrated score of the immunohis-tochemical markers Ki-67, ER, PR, and HER2). The primaryendpoint was distant recurrence after endocrine therapy.The authors concluded that the PAM50 ROR, compared withthe Oncotype DX RS, provided more prognostic informationregarding risk of recurrence and better differentiated theintermediate- and high-risk groups.

In the sameyear, the Lancetpublishedanarticle comparingthe BCI with Oncotype DX and IHC4, using the translationalarm of the ATAC (TransATAC) population. The study looked atboth early (within the first 5 years) and late (after 510 years)distant recurrence. The analysis included 665 patients. Theauthors suggestedthat theBCI,whenusedasa linearmodel,wasmore predictive than Oncotype DX (hazard ratio: 1.69) and thatonly theBCIwas able topredict for latedistant recurrence [37].

An interesting study performed years earlier evaluatedthe concordance between various gene expression arrays. In2006, Fan et al. compared 5 gene sets, among which were theMammaPrint70-geneset, theOncotypeDXRS21-geneset, theintrinsic subtypes as defined by Perou et al., and the ratio ofHOXB13 to IL17BR, which makes up part of the BCI [38]. Thestudy was based on an independent set of 295 breast cancercases for which gene expressionmicroarrays were performed.The investigators performed multiple microarrays on eachsample using the various gene sets mentioned above.Although the population used was a heterogeneous one,including ER2 cancers and a variety of adjuvant treatments,Fan and collaborators showed that for both the compositegroup and theER1patients alone, theRFS andOSaspredictedby the tests were similar in four of five of the gene expressionarrays. The two-gene ratio failed to distinguish outcomesbetween the two high-risk and low-risk groups it had

identified.The studyalso revealed that the70-geneprofile andthe 21-gene RS classify similar groups of patients into riskcategories (77% concordance for ER1 patients and 81% con-cordance overall).When looking at the intrinsic subtypes, the21-gene RS and 70-gene profile classify all of the basal-likesubtypes into thehigh-riskcategoryandhave similar trends forthe other subtypes (Fig. 1). This study suggests that althoughthe actual genes tested are not the same, similar biologicalcharacteristics are being tested in the gene expression assaysand generate similar outcomes in prognostication.

COST-EFFECTIVENESS ANALYSESWith recent cutbacks in health care expenditure in the U.S.,practitioners are increasingly called on to make cost-effectivedecisions. This can often be difficult, given the hefty price tagof genomic testing and insurance restrictions. Fortunately,several cost-effectiveness analyses have been performed toassess the available gene expression assays. Adjuvant! Online,a publicly available online risk predictor, is often used asa standard with which to compare the cost-effectiveness ofgene expression assays because it incorporates patient andtumor characteristics (e.g., age, LN status, tumor size) into itsprognostication model and is free.

In theU.S., OncotypeDX andMammaPrint have both beenshown independently to be cost-effective tests in guiding de-cisions regarding adjuvant therapy [39, 40]. More important,a cost-effectiveness analysis directly comparing OncotypeDXwithMammaPrintwas performed in 2012.The analysis com-pared the cost and quality-adjusted life years (QALYs) of eachtest. Based on a hypothetical cohort of ER1, LN2 early stagebreast cancer patients, the study concluded that MammaPrintis the most cost-effective test based on a willingness-to-paythreshold of $50,000 per QALY [13].

In the U.K., another cost-effectiveness analysis looked atnine different gene expression profiles and IHC-based testsincluding Oncotype DX, MammaPrint, the BCI, and PAM50[41].The study reviewedprior analyses anddevelopedamodelto evaluate the cost-effectiveness of adjuvant therapy guidedby the tests as comparedwith routine care guided by standardclinicopathologic criteria. Unfortunately, the authors foundthat data were insufficient to fully compare each of the tests.They suggested that IHC4 had the most potential to be cost-effective; however, evidence for its clinical utility equal to orbeyond the genomic-based tests was lacking.

UNRESOLVED QUESTIONS AND ANTICIPATED STUDIES USINGGENE EXPRESSION ASSAYSMultigene assays are currently being used in large part toestimate risk of recurrence in patients with ER1/HER22tumors; however, the definition of ER positivity is evolving.Traditionally, tumors were considered ER1 and/or PR1 if atleast 10%of the cells exhibitedprotein expressionusing IHC. In2010, a joint committee from ASCO and the American CollegeofPathology recommendeda1%cutoff for ERandPRpositivity[42]. This recommendation was based on data from a SWOGstudy conducted decades ago [43], and, consequently, theprognostic and predictive value of ER and PR between 1%9%remains poorly defined [44].

Given that these assays are already being used in clinicalpractice, several clinical trials have incorporated them into



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their designs to answer further questions about their utility(Fig.2).TheTAILORxtrial,whichhascompletedaccrual and is inthe analysis phase, will look at the utility of chemotherapyspecifically in patients with an intermediate RS (Oncotype DX)[45]. The results of this trial are highly anticipated becausethere is no standard of care for these intermediate-riskpatients, and decisions are largely dependent on shareddecision making between the provider and the patient.

A similar SWOG trial is ongoing for patients with ER1,HER22 breast cancer and one to three positive nodes. TheRxPONDER trial randomly assigns patients with an RS#25 toendocrine therapy alone or to chemotherapy plus endocrinetherapy [46]. This trial is currently accruing patients, with thegoal of randomizing 4,000 patients, and aims to answer thesamequestionas theTAILORx trialwith regard to thebenefit ofchemotherapy in patients with low to intermediate RS butspecifically for node-positive patients.

In line with aims at further personalizing oncologiccare, more precise prognostication is needed tomeetthe individual needs of patients. Gene expressionassays have the potential to fill the gap where clin-icopathologic criteria fall short.

TheMINDACT trial is another large randomizedclinical trialthat has incorporated MammaPrint into the design. The trialevaluates the clinical utility of MammaPrint (genomic factors[G]) in combinationwithclinicopathologic risk factorsassessedby Adjuvant! Online (clinical factors [C]). The study incorpo-rates early stage breast cancer cases that are LN2 or thathave one to three positive lymph nodes. The design is

complex and not only looks at MammaPrint utility but alsocompares adjuvant chemotherapy and adjuvant endocrineregimens. In order to evaluate MammaPrint utility, patientswho have discordant G and C results are randomized toadjuvant chemotherapy or no chemotherapy. Recently, thepilot phase, which comprised 800 patients, was published.In this first initial cohort of patients, 27% had discordant G andC risks and approximately half received chemotherapy. Thepilot phase demonstrated that the study is feasible, and theoutcomes are expected to be quite informative [47].

Last, the OPTIMA trial is a randomized phase III study thatwill compare outcomes of test-directed treatment with acontrol arm of chemotherapy plus endocrine therapy forpN12 or pT3N0 ER1, HER22 breast cancers. In addition toclinical outcomes, the study will evaluate cost-effectivenessand health resource utilization related to multigene assays forearly stage breast cancer in the U.K. [48].

DISCUSSIONAlthough the vast majority of stage III HR1, HER22 patientswill never develop recurrent disease, the fear of recurrenceoften drives patients and providers to choose adjuvantchemotherapy. The majority of patients receive no benefitfrom this treatment; however, this practice continues becausecurrent prognostic markers fail to identify which patientswithin this groupareatgreatest risk. In linewithaimsat furtherpersonalizing oncologic care, more precise prognosticationis needed to meet the individual needs of patients. Geneexpression assays have the potential to fill the gap whereclinicopathologic criteria fall short. Figure 3 shows a proposedalgorithm for clinical decision making using available multi-gene expression assays.

Figure 1. Numberof patients per intrinsic subtype and their risk categories as determinedby the70-geneprofile andRS (21-geneprofile)derived from microarray data.Within each intrinsic subtype, risk categories per patient are similar between assays, although completeconcordance is seen only for the basal-like group. Adapted from data in Table 2 of Fan et al. [36].

Abbreviation: RS, recurrence score.



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Figure 2. Overviewof clinical trials that have incorporated currently available assays into their designs to answer furtherquestions aboutthe assays utility.

Abbreviations: CT, chemotherapy; HR, hormone receptor; IC, informed consent; PIS, patient information sheet; R-C, chemotherapyrandomizationofanthracycline-basedCT (FECDforN1docetaxel/capecitabine);R-E,endocrine treatmentrandomization, letrozolevs.tamoxifen followed by letrozole; R-T, treatment decision randomization based on genomic vs. clinical prognosis; RS, recurrence score.



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We have reviewed the four main gene expression assaysused in the U.S. to evaluate risk of recurrence in early stagebreast cancer patients with HR1, LN2 disease. The assaysreviewed in this article each have their own individual ad-vantages. Oncotype DX has perhaps the strongest clinicalevidence, with 13 retrospective studies, totaling .4,000patients, suggesting it is clinically useful. The assay has beenshown to be prognostic, and, to some degree, it may predictchemotherapy response.

PAM50, thenewcomer to thegroup, hasbeenvalidated inlarge cohorts (.1,000 patients).The bulk of the data supportits prognostic value in patients with early stage, ER1/HER22breast cancer. Emerging, albeit limited, data showed thatPAM50 may also be predictive of response to neoadjuvantchemotherapy, althoughmore studies are needed to validateits predictive role in this setting [49]. One of the potentialadvantages of the PAM50 assay is that it can be performedand reported by hospital and commercial laboratories.Although reproducibility and technical expertise withina laboratory certified under the Clinical Laboratory Im-provement Amendments of 1988 is necessary, the ability tooffer testing within ones own institution has multiple benefitsincluding personalized overview of testing, cost control, likelyfaster turn-around time, andanopportunity for institutional datacollection. It should be noted that the FDA cleared only theProsigna ROR score for clinical use; therefore, reports fromcommercial laboratories may not include the intrinsic subtypeinformation.

This skepticism regarding the clinical utility of molecularsubtypes was reiterated in a publication from the Annals ofOncology thatevaluated themedical usefulnessof the intrinsicsubtypes as defined by PAM50 compared with IHC-classifiedbreast cancer subtypes (HER2 and HR status) [50]. An expertpanel, the IMPAKT task force, composed of pathologists,clinicians, biostatisticians, and scientists, reviewed literatureto determine evidence for the use of PAM50 molecular

subtypes in clinical practice. After a thorough review of theevidence, the group determined that data were insufficientto support the clinical validity and utility of PAM50 molecu-lar subtypes. They recommended against using the intrinsicsubtypes to guide treatment decisions, in part because theremay be some discrepancy between intrinsic subtypes definedby PAM50 compared with IHC-defined classes, which wereconsidered to be more strongly validated tests. These rec-ommendations fall in line with the FDA recommendations foruse of this assay. However, the IMPAKT task force did notaddress the utility of the PAM50 ROR score, which shouldbe considered separately and which we feel has been wellestablished based on the available data.

Looking at all four of the assays reviewed in this paper,we return to the study by Fan et al., which was the only oneto evaluate all of these assays together in a single investiga-tion [39]. Fan et al. used the gene sets from each assay(MammaPrint, Oncotype DX, PAM50, and the two-gene ratiofrom the BCI) to compare predicted outcomes for an inde-pendent set of breast cancers. It is important to note thatthe gene set for the PAM50 ROR was not compared with theotherassays in this studybut rather the intrinsic subtypes fromPAM50 were used. The study concluded that the gene sets asa whole identified similar groups of high-risk and low-riskcancers, although100%concordancewasnot seen.The largestdiscrepancy was within the luminal A group, in which somecases identified as low risk by the MammaPrint, or 70-gene,assay were classified as intermediate risk by the OncotypeDX, or 21-gene, assay. The purpose of this study was not todetermine which assay was most predictive of outcomes butrather to evaluate the inherent concordance between genesets. Although interesting in terms of biological principles,underscoring the concept that the assays are ultimatelymeasuring the same tumor properties, the study does notdistinguish one assay from another in terms of usefulness forclinical practice.

Perhaps themostuseful study fordirecting clinical decisionmaking, in our opinion, was the head-to-head comparison ofPAM50 and Oncotype DX [36]. In this investigation, PAM50providedmore prognostic information thanOncotypeDXwithregard to likelihood ratios and distant recurrence rates overamedianof10yearsof follow-up.Comparison indices revealedthat the PAM50 ROR outperformed the Oncotype DX RS forevery tumorsubtype. Inaddition,hazard ratios for the low-andhigh-risk groups categorizedbyRORwere improved comparedwith the low- and high-risk groups identified by the RS. Al-though this is only a single study, it provides high-quality datawith which to compare the two tests.

That being said, a major drawback of all of the gene ex-pression assays reviewed is that HER21 and LN1 patientswere included in the testcohorts. By including thesehigher riskindividuals, the recurrence scores may have less inherentclinical utility because they are currently only being applied toHR1, HER22, LN2 disease. Including HER21 and LN1 high-risk patients in the test cohort may bias the analysis andcompromise the ability to determinewhether the assays havejust as strong predictive potential in a population with lowerrisk overall. A study that excludes such patients wouldprovide more clinically relevant information. These studiesare upcoming.

Figure 3. Suggested schema for clinical decision making.



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Including HER21 and LN1 high-risk patients in thetest cohortmay bias the analysis and compromise theability to determine whether the assays have just asstrong predictive potential in a populationwith lowerrisk overall.

The prohibitive cost of genomic studies is another obstacleassociated with these tests. Although clearly dependent onthe health care system of the country and on individual healthcare plans, the valueofgene expression assays is still controlled,for the most part, by individual biotechnology companies.Because the tests are not considered entirely routine, as ofyet, there is an additional price tag beyond standard tumorprocessing. We hope that, given the abundance of availabletests, competition will eventually drive down the cost of ge-nomic testing.

We still have room to improve on our prognosticationefforts. With recent studies revealing the utility of immune-related biomarkers for breast cancer, perhaps, in the nearfuture, levels of tumor-infiltrating lymphocytes or interleukinexpression will be incorporated into standard prognostictesting [51]. For now, the currently available assays provide at

least more information than that provided by traditional IHC.They have been shown to provide reproducible results usingroutinely stored tumor specimens. More important, exploita-tion of the tumor genetic milieu has proven to be an essentialtool for better understanding of tumor biology. As such,the assays enhance our ability to study the nature of eachindividual tumor; given this information, they serve as a guidefor physicians caring for intermediate-risk early stage breastcancer patients. The practice of medicine is both science andart, and ultimately decisions regarding adjuvant chemother-apywill involve amyriad of factors including but not limited togenomic assays.

AUTHOR CONTRIBUTIONSConception/Design: Christina Adaniel, Francisco J. EstevaProvision of study material or patients: Adriana HeguyCollection and/or assembly of data: Christina Adaniel, Komal JhaveriData analysis and interpretation: Adriana Heguy, Francisco J. EstevaManuscriptwriting:ChristinaAdaniel, Komal Jhaveri, AdrianaHeguy, Francisco

J. EstevaFinal approval ofmanuscript: Christina Adaniel, Komal Jhaveri, Adriana Heguy,

Francisco J. Esteva

DISCLOSURESThe authors indicated no financial relationships.

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