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Received: 25 October 2013/Accepted: 27 December 2013/Published online: 5 January 2014 Springer Science+Business Media New York 2014AbstractTriple-negative breast cancer (TNBC) is anKeywordsTriple negative Breast Future Diagnosis aggressive subtype comprising about 1020 % of breastcancer patients with an overall poor prognosis. Recently, itwas found to be a heterogeneous disease that has beenclassied into six subtypes based on molecular signature.In preclinical trials, these subtypes have different activesignaling pathways with variable response to chemother-apy. To improve treatment outcome of TNBC, therapyshould be tailored according to the active driving signalingaberration. Molecular testing represents the optimal way tostratify patients, but it has some difculties to be imple-mented in routine clinical practice. This article provides anassumption for stepped diagnostic algorithm of TNBCbased on immunohistochemistry markers in addition to asuggested tailored therapeutic strategy for advanced TNBCbased on the driving aberrations. Furthermore, most TNBCpatients develop early relapse despite adjuvant chemo-therapy. We provide a design for future adjuvant therapyfor the disease. This design is based on targeting proposedactive pathways in breast cancer stem cells responsible forregenerating the tumor and disease relapse. Finally, weprovide a proposed design for future clinical trials inTNBC to allow for investigation of different medications inthis heterogeneous disease based on upfront patient strati-cation and then allocation to the suitable treatment arms.

S. Elsamany (&) S. AbdullahMedical Oncology Department, Oncology Centre, KingAbdullah Medical City, 2677 Al-Mashaeer District,Makkah 57657, Saudi Arabiae-mail: samanyshereef@yahoo.com

S. ElsamanyMedical Oncology, Mansoura University, Mansoura, EgyptManagement

Introduction

Triple-negative breast cancer (TNBC) constitutes approxi-mately 1020 % of breast cancer patients and represents anaggressive subtype with poor overall prognosis [1]. TNBCpatients have a higher rate of early recurrence and distantmetastasistobrainandlungscomparedtootherbreastcancersubtypes [1]. Chemotherapy is the only systemic therapycurrently available for TNBC; however, most patients withTNBC relapse within 12 years and\30 % of patients sur-vive 5 years despite adjuvant chemotherapy [2].Despite initial high response rate to chemotherapy in themetastatic setting, TNBC patients develop rapid diseaseprogression resulting in a shorter overall survival comparedto ER? breast cancer [3].The treatment options for TNBC after chemotherapyfailure are limited. Overall, treatment of patients withTNBC has been challenging due to the heterogeneity of thedisease and the absence of well-dened molecular targets[4]. Although several small molecule inhibitors andmonoclonal antibodies have been tested in clinical trials inunselected TNBC patients, none has entered clinicalpractice due to limited efcacy [4]. Given so, improvingtherapeutic outcome of TNBC patients requires betterunderstanding of its molecular basis to identify moleculardrivers that can be therapeutically targeted [5].In this article, we will review recent data about themolecular basis of TNBC, with a particular emphasis onpotential targets of novel therapies. In addition, we willprovide assumptions of future diagnostic and therapeuticapproaches of this disease.

123Med Oncol (2014) 31:834DOI 10.1007/s12032-013-0834-y

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Triple-negative breast cancer: future prospects in diagnosisand management

Shereef Elsamany Sakher Abdullahchemotherapy [5]. However, data from ER-positive breastcancer revealed that single-agent PI3K inhibition leads tofeedback activation of ER pathway, while combined inhi-bition of both PI3K and ER may be more effective thaninhibition of either pathway alone [9]. This displays thevalue of combining PI3K inhibitors with other agents tooptimize therapeutic efcacy.

Promising synergism of PARP inhibitors and PI3Kinhibitors

Several PARP inhibitors are being tested in clinical trialssuch as olaparib (AZD2281), which has been shown to besafe and effective in BRCA-related cancers. Meanwhile, thebenet of iniparib in phase II trial was not conrmed in thesubsequent phase III trial [10].Emerging preclinical data demonstrates synergisticactivity when PARP inhibitors are combined with PI3Kinhibitors in cell lines with active PI3K pathway [11].PARP enzymatic activity is necessary for the repair ofsingle-strand breaks (SSBs) through the base excisionrepair (BER) pathway, while BRCA1 and BRCA2 proteinsare essential components for repair of the double-strandbreaks (DSBs) through homologous recombination (HR)[12]. When PARP is inhibited, unrepaired SSBs can betransformed to DSBs that, in BRCA-decient cells, can nolonger be repaired by HR, resulting in lethal DNA damage[13].PI3K pathway is required to maintain and stabilizeBRCA1/2 protein levels. In this way, PI3K inhibitionimpairs BRCA1/2 protein levels, which sensitizes BRCA-procient TNBC to PARP inhibition as demonstrated incell lines and animal models [11]. In addition, in an animalmodel of BRCA1-decient breast cancer, combined ther-apy of the PARP inhibitor olaparib with the PI3K inhibitorNVP-BKM120 caused a 14-fold delay in tumor growthcompared with therapy with either agent alone [14].

Recent view of androgen receptor-positive TNBC

The luminal androgen receptor (LAR) subtype was iden-tied in 11 % of TNBC by gene analysis as reported byLehmann et al [5]; however, androgen receptor (AR) wasfound to be expressed in one-third of TNBC via immu-nohistochemistry [15]. This subtype showed enhancedsteroid hormone signaling and androgen receptor mRNAwas detected at an average of ninefold higher than otherTNBC subtypes [16]. Interestingly, LAR subtype belongsto either luminal A or luminal B intrinsic subtype despitebeing negative for ER expression [16].This subtype showed high response rate to anti-andro-gens in preclinical trials [5]. Furthermore, the anti-Subtype

Basal-like 1

Basal-like 2

Immunomodulatory

Mesenchymal

Mesenchymal stemlike

Luminal androgenreceptorGene expression prole

High expression of genes involved in cell cycleprogression, cell division and DNA damageresponse pathwaysHigh expression of genes involved in cell cycleprogression, cell division and growth factorsignalingHigh expression of genes involved in immuneprocessesHigh expression of genes involved in motilityand extracellular matrixHigh expression of genes involved in motility,extracellular matrix and growth factorsignalingHigh expression of genes involved inhormonally regulated pathways834 Page 2 of 7

Table 1 Subtypes of TNBC based on gene expression prolingMed Oncol (2014) 31:834

have enhanced response to PI3K inhibitors compared toMolecular classication of TNBC

TNBC has been recently classied into six subtypes on thebasis of gene analysis (Table 1) [5]. These subtypes haveprognostic signicance with the basal-like subtypes havingthe worst clinical outcome [4].

Basal-like subtypes

Basal-like 1 (BL-1) and basal-like 2 (BL-2) are the mostfrequent TNBC subtypes (47 %) [5]. They are character-ized by high proliferation rate with an average Ki-67staining of 70 % in addition to high expression of genesinvolved in cell cycle and cell division [5]. They have highsensitivity to chemotherapy, especially the antimitoticagents as demonstrated by high rate of pathological com-plete response (pCR) with neoadjuvant taxan-based che-motherapy (63 % in BL-1 and BL-2 subtypes compared to1431 % in other TNBC subtypes) [6].Interestingly, the majority of studies evaluating neoad-juvant chemotherapy have identied high Ki-67 level as apredictive factor of pCR [7]. In the study of Fasching et al.[7] involving TNBC patients, pCR was 57 % in patientswith Ki-67 [35 % compared to 4 % in those with Ki-67\35 %. Interestingly, Adamo and Anders [8] suggestedthat TNBC patients can be divided into two subgroups withdifferential response and prognosis after preoperativechemotherapy based on Ki-67 level.

Mesenchymal and mesenchymal stem-like subtypes

Mesenchymal (M) and mesenchymal stem-like (MSL)subtypes have low level of proliferation, but they areenriched with phosphatidylinositol-3-kinase (PI3K) andWnt pathways activity. In preclinical trials, these subtypes

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Fig. 1 Wnt/b-catenin signaling in TNBC. a In the absence of Wntbinding to LRP5/6 and FZD, APC binds to axin and GSK3. Thiscomplex promotes phosphorylation of b-catenin via CK1. Phosphor-ylated b-catenin is then degraded by proteasome system. b Wntproteins bind to LRP5/6 and FZD forming a complex that prevents

androgen bicalutamide is currently being evaluated in aphase II study in AR-positive ER-/PR-negative metastaticbreast cancer patients [15].Meanwhile, PI3K pathway activation is involved in theresistance to anti-androgens as demonstrated in animalmodels of AR-positive TNBC treated with bicalutamide[17]. In addition, synergistic activity between PI3K inhib-itors and anti-androgens has been shown in cell lines andexperimental animals [17]. These preclinical data providethe rationale for future clinical trials, evaluating the com-bination of AR antagonists with PI3K inhibitors in AR-positive TNBC patients.

Wnt/b-catenin pathway in TNBC

Wnt/b-catenin signaling pathway is preferentially activatedin TNBC [18], especially in BL2 and mesenchymal sub-types [5]. Wnt proteins bind to the lipoprotein receptor-related protein 5/6 (LRP5/6) and frizzled (FZD) trans-membrane receptor proteins, to form a complex thatMed Oncol (2014) 31:834Page 3 of 7 834axin, CK1 and GSK3 from inducing b-catenin degradation. Free b-catenin is translocated to the nucleus to bind with TCF transcriptionfactor. The TCFb-catenin complex activates Wnt target genes. APCadenomatous polyposis coli, GSK3 glycogen synthase kinase-3, CK1casein kinase-1

prevents b-catenin degradation, which allows its translo-cation to the nucleus to enhance cell proliferation [18](Fig. 1).Dysfunction of the Wnt ligands at the cell surface leadsto aberrant activation of Wnt/b-catenin signaling thatdrives breast carcinogenesis [19]. Among the 10 FZDproteins, FZD7, overexpressed in 67 % of TNBC, is likelythe most important one involved in breast cancer tumori-genesis [19]. Furthermore, downregulation of FZD7 orLRP5/6 suppressed cell growth and tumor transformationin TNBC cell lines [20].Porcupine is an acyltransferase enzyme required for thefunction and secretion of Wnt ligands. The porcupineinhibitor LGK974 is a Wnt inhibitor under development forthe treatment of cancers driven by the Wnt pathway in aWnt ligand-dependent manner [21]. In preclinical evalua-tion, LGK974 attenuated tumor growth and induced tumorregression as a single agent as well as in combination withpaclitaxel in a human primary breast cancer model [22].LGK974 is currently evaluated in a phase I trial in patientswith advanced malignancies, including malignant

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about possible combinations of Wnt, CAV1 and S6K positivities. *3Chemotherapy may be considered given that no other options forthose patients

these genes are either targets for dasatinib or substrates forSrc kinases. Interestingly, expression of these genes wasparticularlyobservedinTNBCcelllines [26].Finnetal[24]also reported an association between CAV1 (Caveolin 1)mRNA expression and sensitivity to dasatinib. Similarly,Tryfonopoulos et al [25] detected an association betweendasatinib sensitivity and high protein level of CAV1.Despite promising activity in TNBC cell lines, the Srcinhibitor dasatinib had disappointing results in clinicalstudies involving unselected TNBC patients. In a phase IItrial including patients with advanced TNBC, dasatinibdisplayed modest activity (5 % partial response, 10 %disease control) [4]. So, proper use of Src inhibitors inTNBC requires selecting patients who are more likely tobenet from this therapy.

Tailored therapy for advanced TNBC

Evidence from preclinical trials illustrates that there isdifferential response to therapeutic agents in TNBC834 Page 4 of 7Med Oncol (2014) 31:834Fig. 2 Proposed future tailored treatment algorithm of advancedTNBC. PI3KI, PI3K inhibitor; PARPI, PARP inhibitor. *1 No dataabout possible simultaneous Wnt?ve/CAV?ve disease. *2 No data

melanoma, pancreatic cancer and TNBC (www.clinicaltrials.gov).

New insights on Src pathway in TNBC

Src is a non-receptor tyrosine kinase involved in celladhesion and motility [23]. Preclinical studies in breastcancer cell lines displayed that compared to other subtypes,TNBC is uniquely sensitive to growth inhibition by the Srcinhibitor, dasatinib, and synergistic activity with chemo-therapy has been shown in TNBC cell lines [24, 25]. Themesenchymal-like subtypes, being enriched in cell motilitygenes, are the most common subtypes with high Srcactivity and were found to be more sensitive to Srcinhibitors in preclinical trials [4].Src expression is more frequent in TNBC; however, thelevel of Src expression is not predictive of dasatinib sensi-tivity [25]. Huang et al [26] identied a six-gene panel thatcould predict sensitivity to dasatinib. These genes includedEPHA2, CAV1, CAV2, ANXA1, PTRF and IGFBP2. All

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subtypes depending on the active signaling pathway [5].Currently, many trials in TNBC are ongoing (clinical tri-als.gov), but unfortunately, most of these trials involveunselected patients and are not directed by predictivebiomarkers, which make their success, in a heterogeneousdisease with different driving molecular pathways, extre-mely doubtful.In view of this, TNBC can be considered an excellentexample of the value of tailored therapy. A more logicapproach should consider upfront stratication of TNBCpatients based on genetic or surrogate immunohistochem-istry (IHC) biomarkers that reect various subtypes anddriving pathways to allow treatment to be personalizedaccording to the intrinsic tumor signature. Ideally,stratifying patients based on genetic testing represents theoptimal approach, but the cost, complexity and requirementof facilities and trained personnel for genetic testing maymake classifying patients based on IHC surrogate markersa more attractive practical approach.The following is a suggested stepped testing algorithmfor advanced TNBC patients that may allow for propertreatment selection.1.Testing for Ki-67, CAV1 and Wnt proteins expressionvia IHC:Fig. 4 Proposed design forfuture clinical trials in TNBCMed Oncol (2014) 31:834

Fig. 3 Proposed futureadjuvant therapy of TNBC.PI3KI, PI3K inhibitor; PARPI,PARP inhibitorPage 5 of 7 834Testing for Wnt pathway proteins: FZD7 and LRP5/6 are selected being the most commonly overex-pressed in TNBC.

123Testing for CAV1 protein level: as a marker ofdasatinib sensitivity.2.In Ki-67\35 %: testing for AR and PI3K activity:AR testing: via IHC.PI3K testing: through IHC testing for S6 kinase(S6K), the downstream signaling protein of PI3Kpathway, as a surrogate marker [27].It is to be noted that Ki-67[35 % is selected as surro-gate marker for BL1 and BL2 subtypes based on averageKi-67 of 70 % in these subtypes [5] and high response rateto chemotherapy in patients with Ki-67[35 % [7]. Testingfor AR and PI3K activity is suggested to be reserved forpatients with Ki-67\35 % given the low proliferation ratein LAR and mesenchymal-like subtypes.Next, based on the above panel of predictive markers,the following diagram illustrates proposed future tailoredtreatment algorithm for advanced TNBC (Fig. 2).

Proposed design for future adjuvant therapy

As stated above, most patients with TNBC relapse within12 years despite adjuvant chemotherapy [2], whichhighlights the need for additional therapy in early stageTNBC to prevent rapid relapse. According to cancer stemcells (CSCs) theory, these cells are intrinsically resistant tochemotherapy and could later regenerate the tumorresulting in relapse [28]. Therefore, drugs that inhibit keyactive pathways in CSCs, such as PI3K and Wnt pathways,may suppress their tumor initiating property, which mayrepresent promising therapeutic strategies in the adjuvantsetting of TNBC [29].Constitutive activation of the Wnt/b-catenin signalingpathway is essential for maintenance, clonogenicity ofCSCs and, most importantly, Wnt/b-catenin signalingconfers resistance of CSCs to radiation and chemotherapy[30]. Noteworthy, Wnt pathway inhibition can controlCSCs. For example, salinomycin, through induction ofLRP6 degradation, was found to be a selective breast CSCsinhibitor [30]. This suggests that targeting Wnt/b-cateninpathway can control breast CSCs which may optimize thetherapy of TNBC [31].PI3K/Akt/mTOR signaling pathway is critical for CSCBased on the above, the following therapeutic strategy issuggested for early stage TNBC after nishing adjuvantchemotherapy (Fig. 3).Proposed design of future clinical trials in TNBCOf course, these proposed approaches of management ofearly and advanced TNBC should be evaluated in well-planned clinical trials. To facilitate patients recruitmentand conduction of trials in this disease with variable sub-types where a plenty of drugs need to be investigated,upfront testing of patients for predictive markers should beconsidered. Multiple treatment arms should be availableand patients can be allocated to the suitable therapy basedon their testing results (Fig. 4).

Conclusion

Treatment outcome of TNBC is still unsatisfactory even inthe early stage of the disease. TNBC should be viewed as agroup of different diseases that have similar phenotype butdifferent genotypes with variable response to chemother-apy. Recent data revealed different molecular subtypes ofthe disease with different driving molecular pathways andprovided insights about potential therapeutic targets. Thisconcept raises the real need for personalized therapies ofTNBC to improve the treatment outcome of this disease.Several questions are still to be answered. The propersequence of therapies, possible combinations, duration oftherapy and validation of predictive markers are open eldsfor future research. In addition, possible exclusion ofadjuvant chemotherapy needs to be assessed in TNBCsubtypes with low proliferation where chemotherapy haslittle effect and other therapies may be more benecial.AcknowledgmentsThe authors would like to thank Dr. Mian Us-man Farooq for his technical support in the preparation of the guresof the manuscript.The authors declared that they have no conictConict of interestof interest.

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