The American Journal of Pathology, Vol. 183, No. 4, October 2013

ASIP

2013

AJP

CME Program

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Breast Cancer Theme Issue

REVIEWBreast Cancer Metastasis

Issues for the Personalization of Its Prevention andTreatmentNatascia Marino,* Stephan Woditschka,* L. Tiffany Reed,* Joji Nakayama,* Musa Mayer,y Maria Wetzel,z and Patricia S. Steeg*

From the Women’s Cancers Section,* Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes ofHealth, Bethesda, Maryland; AdvancedBC.org,y New York, New York; and the Michigan Breast Cancer Coalition,z Baldwin, Michigan

CME Accreditation Statement: This activity (“ASIP 2013 AJP CME Program in Pathogenesis”) has been planned and implemented in accordance with the Essential Areas andpolicies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and theAmerican Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.

The ASCP designates this journal-based CME activity (“ASIP 2013 AJP CME Program in Pathogenesis”) for a maximum of 48 AMA PRA Category 1 Credit(s)�. Physicians shouldonly claim credit commensurate with the extent of their participation in the activity.

CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.

Accepted for publicationJune 24, 2013.

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Address correspondence toNatascia Marino, Ph.D.,Women’s Cancers Section,Laboratory of Molecular Phar-macology, Center for CancerResearch, National CancerInstitute, Building 37, Room1126, 37 Convent Dr.,Bethesda, MD 20892. E-mail:marinon@mail.nih.gov.

opyright ª 2013 American Society for Inve

ublished by Elsevier Inc. All rights reserved

ttp://dx.doi.org/10.1016/j.ajpath.2013.06.012

Despite important progress in adjuvant and neoadjuvant therapies, metastatic disease often develops inbreast cancer patients and remains the leading cause of their deaths. For patientswith establishedmetastaticdisease, therapy is palliative, with few breaks and with mounting adverse effects. Many have hypothesizedthat a personalized or precision approach (the terms are used interchangeably) to cancer therapy, in whichtreatment is based on the individual characteristics of each patient, will provide better outcomes. Here, wediscuss the molecular basis of breast cancer metastasis and the challenges in personalization of treatment.The instability of metastatic tumors remains a leading obstacle to personalization, because information froma patient’s primary tumor may not accurately reflect the metastasis, and one metastasis may vary fromanother. Furthermore, the variable presence of tumor subpopulations, such as stem cells and dormant cells,may increase the complexity of the targeted treatments needed. Although molecular signatures and circu-lating biomarkers have been identified inbreast cancer, there is lackof validatedpredictivemolecularmarkersto optimize treatment choices for either prevention or treatment of metastatic disease. Finally, to maximizethe information that can be obtained, increased attention to clinical trial design in themetastasis preventivesetting is needed. (Am J Pathol 2013, 183: 1084e1095; http://dx.doi.org/10.1016/j.ajpath.2013.06.012)

Supported by the Intramural Program of the National Cancer Institute.This article is dedicated to the memory of Maria Wetzel, who died of

breast cancer in May 2013.Current address of J.N., Department of Biological Sciences, National

University of Singapore, Singapore.This article is part of a review series on the molecular pathogenesis of

breast cancer.

Although personalizing the treatment of breast and othercancers is a promising goal, individualizing treatments willrequire a wealth of newmolecular data and therapeutic options.Much of the recent progress has been in personalizing treatmentof early breast cancer. These efforts havemovedbeyondalreadyestablished therapies for patients with estrogen receptorepositive (ERþ) and/or progesterone receptorepositive (PRþ)and HER2-overexpressing (HER2þ) disease, primarily usingmultigene assays that are prognostic for risk of recurrence andpredictive for response to cytotoxic chemotherapy.

stigative Pathology.

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Personalized medicine for metastatic disease presentsgreater hurdles, however. The complexity, heterogeneity, andgenomic instability of metastatic breast cancer cells make

Table 1 Hallmarks of Metastasis and Their Implications for Personalized Medicine

Hallmark Implications for Personalized Medicine

Heterogeneity between primary tumors and metastases,and among metastases

Therapy based on primary tumor characteristics may not be effective

Redundancy of mechanistic pathways Need for combination therapiesVariable dormancy Clinical trials must address delayed relapsesContributions of cancer-initiating cells Incorporation of therapies that target stem cells

Challenges in Breast Cancer Metastasis

their evaluation and therapy a challenging process (Table 1).Thus, although this review sheds light on potentially impor-tant aspects of personalized medicine in metastatic breastcancer, the potential remains to be realized.

Metastasis has been described mechanistically as themigration of tumor cells from the primary tumor, followed byintravasation, survival, extravasation of the circulatorysystem, and progressive colonization of a distant site.1e3 Thismechanistic description does not capture other equally validcharacteristics, however. In a second definition, that ofparallel progression, the defining feature is tumor cellgenomic instability, promoting selection for characteristicsthat enable invasion and distant organ colonization.4 Fromthis perspective, it is not the steps in metastasis that arecritical, but rather the instability that fuels the process. In yetanother definition, metastasis is described in terms of seedand soil.5 Tumor cells (seeds) spread widely through thebody, but grow only in supportive locations (congenial soil).Thus the various microenvironments (soils) of metastasescontribute to the observed heterogeneity. Layered over thefundamentals of the metastatic process is acquired or innateresistance to therapies. Because only 5% of breast cancerpatients have stage IV disease at initial diagnosis (http://www.seer.cancer.gov/statfacts/html/breast.html; accessedFebruary 1, 2013), in the majority of metastatic patients themetastatic disease develops after hormone therapy, chemo-therapy, or biologics have been used in adjuvant treatment.

Another hallmark of metastasis is the redundancy ofpathways that mediate the process or its component steps.Genes promoting breast cancer metastasis abound,including ERBB2 (alias HER2, NEU), CTNNB1, KRAS,PI3KCA (alias PI3K), EGFR, MYC, TWIST1, SNAI1 (aliasSNAIL), SNAI2, MET, and ID1.6 Some genes are involvedin tumor cell survival and colonization in the metastaticsite in a generalized manner, including PTGS2, EREG,MMP1, LOX, ANGPTL4, and CCL5. Other genes, such asPTHLH (alias PTHRP), IL11, CSF2RB, IL6, and TNF(previously TNFA) function in a more organ-specificmanner.6 Several of these pathways in genetically engi-neered mouse models exhibit oncogene addiction, with theablation of the expression of a single gene causing tumorregression.7 Does this portend better responses to pathwayinhibitors? Most pathways are only partial contributors tothe metastatic process, meaning that their inhibition wouldhave at-best partial effects and could be overcome by othercontributory pathways. Additional pathways suppressmetastasis, either by inhibiting tumorigenesis8 or by

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specifically suppressing the metastatic process, the latterdefined as metastasis suppressor genes.9,10 Successfulpersonalized medicine approaches will have to deal withthe instability, complexity, and multifactorial nature of themetastatic process.

Research into the metastatic process is relevant topersonalizing both the adjuvant and metastatic clinicalsettings. In the adjuvant setting, micrometastases arethought to be present, and systemic therapy is adminis-tered to prevent their outgrowth. In the metastatic setting,treatment aims to shrink lesions that have completed themetastatic process and to prevent the outgrowth of furthermetastases. Both preclinical and clinical data suggest thata given drug may not be equally effective in both settings.

Factors Important to Personalizing Therapy forPreventing and Treating Metastatic Disease

Semipersonalized Medicine

There appear to be degrees of personalized medicine. Semi-personalized medicine is based on the identification of largegroups of patients with certain tumor characteristics that candirect a given patient to corresponding specific types oftherapy. True personalized medicine would be based on anindividual patient’s tumor, directing to a tailored therapymaximized for effectiveness for that one patient in particular.Semipersonalized medicine has already generated effectivetherapies for groups of patients and thus can provide a basis forpersonalized approaches. Here, we address three examples:HER2-directed therapies, anti-estrogenic therapies, andbisphosphonate and antibody therapies.

HER2-Directed TherapiesThe tyrosine kinase receptor proto-oncogene c-ErbB-2(hereafter referred to by the familiar alias HER2) is overex-pressed or amplified in approximately 25% of breast cancers,and is a significant prognostic marker of shorter relapse-freeand overall survival.11 HER2 is a transmembrane tyrosinekinase receptor and a member of the EGFR family, whichalso includes HER1 (EGFR), HER3, and HER4. Addition-ally, HER2 can interact reversibly with ligand-activatedfamily members to form active heterodimers, leading tophosphorylation of intracellular tyrosine residues. This acti-vation recruits cytoplasmatic signal transducers such asSTAT, p85-PI3K, PLC-g, and Src. Two of the main

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downstream pathways activated by HER2 are the MAPK andPI3KeAKT pathways promoting cell survival, cell prolif-eration, and migration.12

HER2þ breast cancer patients derive significant benefitfrom HER2-targeted therapy, such as the humanizedmonoclonal antibody trastuzumab combined with chemo-therapy in the adjuvant and metastatic settings.13 Althoughthe benefit of trastuzumab-based therapy is undeniable,approximately 50% of HER2-overexpressing breast cancersdo not respond to trastuzumab,13 suggesting the need forgreater precision. Lapatinib, a small-molecule inhibitor ofHER2 and EGFR, has shown efficacy in the metastaticsetting after relapse on trastuzumab-based therapy.14 Severalnewer therapies aimed at the HER superfamily (EGFR andHER2 to HER4) have been approved or are in late devel-opment; these include pertuzumab15 and trastuzumabemtansine (T-DM1).16 These vary in potency and specifictarget within the superfamily.

In animal models, overexpression of HER2 promotesmetastasis to lymphnode, lung, bone, andbrain.17e19StudiesofHER2 promotion of metastasis have identified several path-ways, including a bidirectional interaction with the TGF-b/Smad pathway,20 an increase in expression and stability of thehoming chemokine receptor CXCR4,21 an activation of Srcwith consequent phosphorylation of FAKtyr861 and activationof p120/Rac1/Cdc42,22 and an increase in angiogenesisthrough up-regulation of VEGF23 and angiopoietin-2.24 It willbe of interest to determinewhether inhibitors of these pathways,in combination with HER2 therapy, provide a better degree ofmetastasis prevention or shrinkage of established lesions.

Anti-Estrogenic TherapiesAt diagnosis, 75% of breast tumors are ERþ and can poten-tially respond to tamoxifen, aromatase inhibitors, or otherhormonal therapies.25,26 ERþ tumors tend to metastasize tothe bone,27 and often metastasize late.28 A recent long-termfollow-up study after 5 years of tamoxifen therapy showedthat metastatic relapses continue over the next 10 years andhave not leveled off at that time point, suggesting a contin-uous break from dormancy.29

Estrogen effects are mediated by two specific nuclearreceptors, estrogen receptor a (ER-a) and estrogen receptorb (ER-b). ER-a is expressed in breast and is associated withincreased proliferation and metastasis. On binding to theligand, ER regulates the transcription of target genes. ERcan also form multiprotein complexes with membrane-related factors such as Src, G-proteins, RTK, and PELP1.30

Downstream of ER lies the activation of Src, Ras, andMAPK signaling to promote cell proliferation, PI3KeAktto induce survival and invasion, and the Rho familyGTPases Rac and Cdc42 to promote cell migration, inva-sion of the extracellular matrix, and metastasis.31 Crosstalkbetween ER signaling and growth factor pathways iscorrelated with both cancer progression and resistance tohormonal therapy.32 A link between ER signaling and theepithelialemesenchymal transition has been described.33

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Finally, estrogen affects the cytokine milieu in the cancermicroenvironment.34

The wide range of endocrine therapy options provides anopportunity to select the optimal sequence and combinationof therapeutic agents after recurrence or relapse. Currentstrategies focus on combinations with growth factor and PI3kinase pathway targeting agents, such as gefitinib and ever-olimus.35,36 Combinations with other pathway inhibitors mayhold promise. The potential of gene signatures for individu-alizing therapy in the ERþ setting is under testing in theTAILORx trial. The purpose of this randomized phase IIItrial is to identify the best individual therapy for node-negative, ERþ breast cancer patients, classifying them byusing the 21-gene signature Oncotype DX.37 If validated, thissignature could provide patients with an individualized esti-mate of therapeutic benefit.

Bisphosphonates and RANKL AntibodyBreast cancer patients with bone metastases have distincttherapeutic options, including bisphosphonates and (morerecently) denosumab, typically in combination with endo-crine or HER2-directed therapies. The bone metastaticprocess has been described as a vicious cycle.38 Alterationsin the bone microenvironment are the initiators of this cycle;major changes include hypoxia, acidic pH, and increasedlevels of extracellular calcium and growth factors.39 Theosteolytic vicious cycle is characterized as bone lysis withconcurrent infiltration of metastatic tumor cells. TGF-b andparathyroid hormone-related protein (PTHrP) both play animportant role in osteolysis. PTHrP, produced by tumorcells, activates osteoblasts and osteoclasts via the RANKligand (RANKL) pathway, resulting in bone resorption.Bone resorption releases growth factors, ionized calcium,and ionized phosphate. Release of ionized calcium results inan elevation of circulating PTHrP, increasing the propensityfor osteolysis by osteoclasts and thus promoting the viciouscycle. Release of growth factors from the bone matrix, suchas TGF-b, in turn activates tumor cells, contributing to thecycle.39 Denosumab is a human monoclonal antibodyagainst RANKL. In a phase III trial, denosumab reduced therisk of developing multiple skeletal-related events (time tofirst and subsequent events) by 23%, compared with thebisphosphonate zoledronic acid.40

It must be noted that one consequence of semi-personalized medicine is that some patients will be over-treated, because not all members of the large groups onwhich semipersonalized treatment is based will benefit alike.

Factors That We Ignore at Our Peril

Metastatic disease is largely incurable because of the veryfactors that define it (Figure 1). As precision treatmentapproaches develop in the adjuvant and metastatic settings,consideration must be given in trial design to these factors:heterogeneity, genomic instability, sites of metastasis, tumorsubpopulations, and microenvironmental influences.

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Intratumor

heterogeneity

Primary tumor –

metastasis

heterogeneity

Metastatic biopsy

limitations

Lack of drugs

targeting newly

identified pathways

Pathway

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Heterogeneous

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cells)

Tumor

microenvironmental

influence

Lack of specific

clinical trials

Personalized

Therapy

Lack of predictive

biomarkers

Tumor

resistance (de

novo, acquired)

Figure 1 The complex of challenges in person-alizing therapy in breast cancer metastasis. Metas-tasis is an intricate disease state, and many aspectsof its biology and origin remain poorly understood.Thus, the complexity of the disease (with its geneticinstability, tumor heterogeneity, and dormancyphenotype) limits accurate diagnosis and tailoringof treatments for patients. The lack of validatedpredictive biomarkers able to determine treatmentchoice and scarcity of metastasis-specific clinicaltrials constitutes a further limitation for imple-mentation of personalizedmedicine in breast cancermetastasis.

Challenges in Breast Cancer Metastasis

HeterogeneityRecent research has demonstrated heterogeneity in primarytumors and corresponding breast cancer metastases at themorphological, molecular, and genomic levels, and suchheterogeneity may be a significant determinant of anticancertherapy response.41 Studies evaluating the traditionalmarkers HER2, ER, and PR indicate 5% to 22%, 13% to33%, and 31% to 32% discordance, respectively, betweenprimary tumor and distant metastases.42e44 A retrospectivestudy of 255 patients with matched primary breast cancerand liver metastatic samples available for evaluation re-ported discordance of 14.5%, 48.6%, and 13.9% for ER,PR, and HER2, respectively.45 Interestingly, the authorsreported the influence of receptor status discordance on thetherapeutic regimen: based on the metastatic biopsy, treat-ment was changed for 12.1% of the patients. Larger studieswill be needed to fully assess the frequency and potentialbenefits of altering therapy because of discordance of tumorcharacteristics in metastatic sites.

For other therapeutic targets, discordance between primarytumors and metastases reigns. Akcakanat et al46 determinedwhether primary tumors differed from their metastases intheir expression of p-Akt and p-4E-BP1, components of thetherapeutically relevant PI3K pathway. They observed poorconcordance between immunohistochemical levels of p-Aktand p-4E-BP1 expression in primary tumors and metastases.Wu et al47 observed extensive heterogeneity betweenprimary breast carcinomas and their paired metastases, aswell as amongmultiple metastatic breast carcinomas from thesame patient. They observed down-regulation of ER and PRand overexpression of COX-2, MET, EGFR, and mesothelinin metastatic versus primary lesions, and they concluded thattherapeutic targets identified in the primary breast carcinoma,or even in some metastatic breast carcinomas, might notreflect targets present in all metastatic sites.47 DNA methyl-ation of five marker genes [CCND2, RARB, TWIST1,SCGB3A1 (alias HIN1), and RASSF1] has been reported to

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be discordant between primary tumor and metastasis, andbetween metastases in a warm autopsy study.48

Mutational profiling of breast cancer metastases and eithermatched or unmatched primary tumors has recently identifieda number of genetic alterations that, although not unique tometastases, occur more frequently in secondary lesions. Forexample, mutations in the tumor-suppressor gene TP53,which occur in roughly a quarter of primary breast carci-nomas, were found at higher frequency (87%) in a series of 23brain metastases of breast cancer, with a striking preponder-ance of complex TP53 mutations, such as frameshift, splice,and nonsense mutations, as well as in-frame insertions anddeletions.49 Increased amplification of MYC in systemicmetastases compared with primary breast tumors has beendescribed.50 In brain metastases, the tumor-suppressor PTENwas more frequently mutated or lost because of allelicimbalance, compared with primary breast tumors.51

Recently, advanced next-generation sequencing tech-niques have been used to interrogatewhole cancer genomes atthe single-nucleotide level and have distinguished betweenmutations in breast cancer metastases that are present in rarecell populations of the primary tumor and to those arising denovo during metastatic progression. In the brain-metastaticprogression of a basal-like breast cancer, most mutationswere shared between the primary tumor and the metastasis,although a significant enrichment of missense mutations inNRK (a JNK activating protein kinase), PTPRJ (a proteintyrosine phosphatase), and WWRTR1 (a modulator ofmesenchymal stem cell differentiation) was observed in themetastatic lesion.52 In contrast, the majority of mutationspresent in a metastatic lobular breast cancer specimen werenot present in the primary tumor, suggesting substantialgenetic evolution during the metastatic process.53

These data highlight two concerning findings. First, wecannot accurately predict the molecular profile of metastaticdisease by profiling the primary tumor. Second, onemetastasis may be distinct from another within the same

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individual. Effective personalized medicine will have toaccount not only for a person’s individual primary tumordata, but also for variances within and between metastases.Moreover, we still lack drugs targeting many of the newlyidentified pathways that are altered in metastatic lesions.The creation of new therapies based on detailed molecularprofiling remains challenging.

Genomic InstabilityThe instability of metastatic breast cancer cells likely drivesheterogeneity. Comparative genomic hybridization has beenused as a tool to identify large scale genomic instability andidentified two classes of breast cancer genomic structure:monogenomic, with one major clonal subpopulation withhigh chromosomal stability, and polygenomic, with multipleclonal subpopulations. Polygenomic clones may haveshared or segregated anatomical distributions. Disparateclonal evolution may explain molecular discordance at thetime of disease relapse.54 Comparison of the primary tumorand concurrent lymph node infiltrates using comparativegenomic hybridization revealed extensive clonal genomicheterogeneity, indicating that the evolution of tumors canbegin early.55 In addition, adjuvant therapy may contributeto biological discordance by clonal selection pressure,exemplified by the evolution of ER� subpopulations afterendocrine therapy for predominantly ERþ disease.

Failure in the DNA break repair system, mitotic chro-mosome transmission, or the spindle mitotic checkpoint cancause the chromosomal lesions that are hallmarks ofgenomic instability. Consequently, high mutation rates andchromosomal rearrangements (deletions, duplications, andamplifications) may drive tumor progression by disruptionof tumor-suppressor genes, formation of fusion proteins,constitutive activation of enzymes, or amplification ofoncogenes.56 Whole-genome sequencing analyses of breastcancers revealed unexpectedly high levels of somaticmutations,57 which may confer a selective advantage on thetumor cells and thus promote their clonal expansion.

Sites of MetastasisThe extent to which metastases are site specific remainspoorly understood. Certainly the vicious cycle has beenestablished as being important to bone metastasis.39 How-ever, other pathways also contribute to bone metastasis, andPTHrP, involved in the vicious cycle for bone, has beenimplicated in metastasis to other sites.58 Specific therapies areavailable for patients with bone metastases, and these need tobe factored in for a personalized regimen. Other pathwayshave been reported to mediate lung59 and liver metastasis.60

Our research group and others have investigated brain-permeable compounds that prevent the formation of brainmetastases or could potentially prevent the development ofadditional brain metastases in patients with a limited numberof lesions. Lapatinib is the only traditional breast cancer drug,of 18 drugs tested, that has brain metastasis preventiveactivity in the HER2þ setting.61,62 In the model used,

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however, HER3 activation was lacking in the tumor cells;other models with activated HER3 signaling may show moreresistance. Personalization of these trends would require theidentification of those patients with HER2þ tumors at highestrisk for development of brain metastases, as well as anunderstanding of the complex signaling pathways involved.A prospective study showed that 37.3% of all metastaticbreast cancer patients with HER2þ tumors developed brainmetastases over 7.1 to 13.3months of follow-up,63 but furtherpersonalization of this trend is needed. Several attempts havebeen reported in preliminary form, but none have beenprospectively confirmed.64 Other drugs that can partiallyprevent the formation of brain metastases in preclinicalmodels but are not part of the breast cancer armamentariuminclude pazopanib,65 a Plk1 inhibitor,66 vorinostat,67 andTPI-287.68 Guidance is needed to identify which patientswould benefit from these nontraditional therapies.

Tumor SubpopulationsIn addition to molecular heterogeneity, subpopulations oftumor cells with distinct functional capabilities may exist,and these would need to be factored in for any successfulpersonalized treatment.Tumor stem cells or cancer-initiating cells may be one

subpopulation. Stem cells are defined as cells that have theability to perpetuate themselves through self-renewal and togenerate mature cells of a particular tissue through differenti-ation.69 A small subpopulation of tumor cells characterizedby CD44þCD24�/low was enriched in stem cell-like proper-ties.70 In addition to the CD44þCD24�/low subpopulation,aldehyde dehydrogenase 1 [encoded byALDH1A1 (previouslyALDH1)] was reported to identify stem cell-like properties onbreast cancer cells. Both aldehyde dehydrogenase and CD44/CD24 expression have been linked to aggressive metastaticbehavior.71Metastasis is an inefficient process, and very few ofthe cells released from a primary tumor can reinitiate tumorgrowth at distant sites.72 The formation of metastases has beenthought to result from the dissemination of cancer cells pos-sessing stem cell-like properties and their proliferation atdistant sites. Stem cell-like properties have been proposed toconfer not only metastatic potential but also chemotherapyresistance.73 Recently, a plasticity model was proposed byPolyak, Weinberg, and colleagues.74 They demonstrated inmammalian breast cells that undifferentiated CD44�/low-

CD24þ

epithelial cells can revert to a stem-like state(CD44þCD24�/low), driven by the epithelialemesenchymaltransition. To our knowledge, it remains uncertain whethertumor stem cells also evolve in the metastatic process, whichwould determine whether the stem cell subpopulation from theprimary tumor resection is adequate.Another tumor subpopulation that we ignore at our peril

is dormant tumor cells. For some breast cancer patients,metastasis occurs soon after a primary tumor develops,whereas for other patients metastases emerge years or evendecades after initial treatment. Tumor cells that remainlatent for a prolonged period of time are termed dormant.

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Challenges in Breast Cancer Metastasis

Dormancy itself can be heterogeneous, reflecting a balanceof proliferation and apoptosis, cell cycle quiescence, and/orantiangiogenic mechanisms.75,76 An experimental studydemonstrated doxorubicin resistance in dormant breastcancer cells,77 which suggests that these cells can surviveinitial chemotherapy and awaken later.

To address the chemoresistance of dormant tumor cells,novel therapeutic strategies based on mechanistic pathwaysmediating dormancy are needed. Several clues haveemerged from the preclinical literature. When HEp3 cellswere xenografted into the immunodeficient chick embryo,dormancy was accompanied by a balance between reducedactivation of ERK proliferative MAP kinase and elevatedactivation of p38 stress MAP kinase; this balance wasregulated by the urokinase receptor and by interactions withfibronectin.78 A similar inverse balance between ERK andp38 was observed in other models.79 The data suggest thatp38-activating drugs or ERK inhibitors hold benefit. Thelysophosphatidic acid receptor 1 (LPA-1) inhibitor Debio0719 significantly decreased metastatic progression in twotriple-negative breast cancer model systems using themurine mammary carcinoma cell line 4T1 and human breastcancer cell line MDA-MB-231. The inhibitor preventedthese cells from proliferating in distant organs such as liverand lung, as evidenced by reduced Ki-67 staining; cancercells in distant organs in the inhibitor-treated mice showeda reduction in ERK activation and an increase in p38 acti-vation.79 In a three-dimensional culture system, inhibition ofintegrin b1 or MLCK prevented transition from a quiescentto a proliferative state,80 which has been reported also inother models.81 These studies suggest that integrin b1inhibitors may control dormancy.

Regardless of personalized medicine approaches, clinicaltrial designs to validate dormancy-maintenance regimenswill need new designs and endpoints. We currently have novalidated mechanism to determine whether a patient harborsdormant tumor cells, nor for evaluating the potential of anysuch cells for awakening or their molecular characteristics.Disseminated tumor cells in the bone marrow have beenproposed as a potential reservoir for dormant tumor cells tonest, and their presence is prognostic.82 However, we do notknow what proportion of tumor cells reside in bone marrowversus in the secondary organs, and the invasiveness of bonemarrow biopsies precludes their routine use.

Microenvironmental InfluencesA major contributor to tumor metastasis is the tumormicroenvironment, which includes fibroblasts, vasculature,immune and inflammatory cells, and extracellular matrix.Microenvironments (also known as niches) participate inreciprocal interactions between tumor cells and theirsurroundings.83 Microenvironments are not static. They aremodified by tumor cells and infiltrated by immune andother circulating cells, a state often referred to as reactive; inturn, the reactive microenvironment can fuel tumorprogression. To date, a complete portrait of the metastatic

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microenvironment by organ site, through time, is lackingeven in experimental models. Layered onto this lack of basicinformation is limited understanding of the role that themicroenvironment plays in chemotherapeutic resistance.83

Simplistically, aspects of the microenvironment havebeen targeted for interruption (eg, angiogenesis). Againsimplistically, this complex process has been reduced to theangiogenesis promoter VEGF (targeted by bevacizumab) ora handful of angiogenesis receptors such as VEGFRs andPDGFRs (targeted by sorafenib, pazopanib, and otherkinase inhibitors). Although the U.S. Food and DrugAdministration (FDA) has approved bevacizumab forcertain metastatic cancer indications, conditional approvalin metastatic breast cancer was withdrawn over efficacyand toxicity concerns (http://www.cancer.gov/cancertopics/druginfo/fda-bevacizumab, last accessed February 1, 2013).In terms of efficacy, there is much that we do not understand.For example, the reliance of metastasis on angiogenesis (asopposed to co-option of the existing vasculature or othermechanisms) is debated,84 an effect of angiogenesis onhypoxia-induced tumor progression has been reported,85 andother angiogenic factors and receptors are known to exist.84

Such detailed knowledge of microenvironmental evolutionwill be critical to identifying newer and more effectivemetastasis preventive and therapeutic strategies and to theireventual personalization.

Oligometastatic or Limited Metastatic Disease?

The clinical state of oligometastatic disease, as described in1995 byHellman andWeichselbaum,86 refers to a subgroup ofcancer patients with a limited metastatic burden. In general,only one organ is involved, and with only a single detectablemetastatic lesion. Does the oligometastatic state benefit fromdistinct treatments, and would its personalization be different?Given their relatively limited nature, oligometastases could beremoved by local therapy, such as surgical excision orstereotactic radiotherapy. In some cases, local therapy is fol-lowed by a systemic treatment similar to adjuvant therapy, toreduce possible subclinical systemic spreading of the meta-static disease. For a subgroup of oligometastatic breast cancerpatients, the multidisciplinary approach may be curative. Theselection of a subgroup of oligometastatic patients that maybenefit from aggressive local treatment, currently based ona long disease-free interval, young age, good performancestatus, negative HER2 status, and a low number of involvedsites,87 can be refined to more personalized levels.

Approaches to Personalized Medicine forMetastatic Breast Cancer

Can Prognostic and Predictive Signatures GuideIndividualized Therapy?

In addition to standard histopathological tools, a number ofmultigene expression signatures have been identified for

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estimating the natural history of the disease. Although thereis generally little overlap among the genes in these signa-tures, most prognostic signatures are related to tumorproliferation phenotypes. The two most commonly consid-ered platforms, MammaPrint (Agendia, Amsterdam,Netherlands; Irvine, CA) and Oncotype DX (GenomicHealth, Redwood City, CA), for example, have only onegene in common.88,89 Immunohistochemical detection ofKi-67 is a relatively inexpensive, well-established prolifer-ation marker in breast cancer with demonstrated predictivepower.90 The predictive ability of these platforms may beuseful for personalization of therapy to prevent metastasis.

Another relevant question is whether signatures basedon characteristics other than proliferation (eg, dormancy,stemness, or metastatic colonization potential) may holdgreater potential for personalizing the prevention of metas-tasis. A starting point would be to determine what tumorcohorts with what follow-up data are needed, to be collectedas a centralized resource for validation of potential signatures.

Metastatic Biopsies

As noted above, a substantial number of patients havediscordant findings between matched biopsies of primarytumor and metastatic sites. Furthermore, one metastasis maydiffer from another within the same individual, raising thequestion of how many biopsies are needed. Biopsy of meta-static tissue could potentially improve outcome by identifyingwhat new genetic or molecular pathways are activated, leadingto altered and ideally more efficacious therapy. In 2011, theguidelines of the National Comprehensive Cancer Network(http://www.nccn.com/files/cancer-guidelines/breast/index.html#/1, last accessed February 1, 2013) indicated theimportance of biopsy to confirm recurrence, if possible, and itsnecessity if either HER2 or hormone receptor testing wasnegative or never performed. The extent to which theseguidelines are practiced is unknown. Issues include reim-bursement, the pain and morbidity for the patient, and tech-nical difficulties (especially for the biopsy of bonemetastases).

Botteri et al91 reported that biopsies of liver metastasiswere useful for confirmation or exclusion of advanceddisease and for reassessment of the biology of the metastaticdisease, and thus can contribute to defining a more effectivetreatment strategy, either by proposing new treatmentoptions or avoiding ineffective therapies. Indeed, theyobserved a positive effect of liver biopsy on survival inpatients with early metastases. Clearly, additional largerstudies will be needed to validate the technique.

Can CTCs Guide Personalized Therapy?

A minimally invasive tumor assessment would be preferableto an invasive biopsy. Serial reconfirmation of diseasebiocharacteristics before commencing new therapy and atrelapse could potentially personalize and optimize thera-peutic decisions. Detection and biocharacterization of

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circulating tumor cells (CTCs) in the peripheral blood ofpatients with advanced breast cancer could optimally serveas a real-time tumor biopsy.92 The cardinal feature not onlyof breast cancer metastases but of all cancer metastases isthe circulation of cancer cells from a primary tumor todistant organs such as lung, liver, bone, or brain.CTCs are rare events, occurring at rates as low as one cell

per 105 to 107 peripheral blood mononucleated cells. Theirdetection is complicated by significant leukocyte contami-nation.92 Recent advanced methodologies in the detectionand characterization of CTCs include microchips, filtration,microscopic approaches, highly sensitive quantitativeRT-PCR, the FDA-approved CellSearch system (JanssenDiagnostics, Raritan, NJ) system, or a combination ofmolecular and imaging methods.93 Detection based onEpCAM or cytokeratin expression on CTCs is potentiallycomplicated by the epithelialemesenchymal transition,where expression of these proteins is lost.94

Prospective studies have demonstrated that detection ofCTCs in metastatic breast cancer can successfully predictprogression-free survival and overall survival.95 TheGerman SUCCESS trial is the largest study (performed withthe CellSearch system) to evaluate the prognostic relevanceof CTCs in breast cancer patients in the adjuvant setting.96

In 2007, CTCs were cited for the first time in the Amer-ican Society of Clinical Oncology recommendations ontumor markers.97

Although CTCs are promising, several issues in the liter-ature need resolution before CTCs can be proposed as a part ofa personalized medicine regimen. The proportion of CTCsthat are metastatically competent (as opposed to shed tumorcells destined to die) is unclear. According to the experimentalliterature, the vast majority of tumor cells shed into thecirculation never form a metastatic lesion.72 Can therapeuticdecisions bemade on the characteristics of all CTCs obtained,or do we need to know which ones are metastaticallycompetent? In addition, it is known that the molecular char-acteristics of CTCs do not always match those of the primarytumor and that in the same blood sample, heterogeneous CTCsubpopulations with different hormone receptor and otherphenotypes coexist.98,99 Given our increasing ability toprofile CTCs not only for standard histopathological markersbut also for gene expression, mutations, and epigeneticalterations, the prognostic and predictive ability of thesesubpopulations of tumor cellsmust be confirmed. Thismay bebest accomplished by the collection of longitudinal banks ofCTCs with associated patient follow-up data to semi-personalized therapy, for use inmultiple molecular platforms.Finally, we note that CTCs are not obtained in all patients,even some with metastatic disease; necessarily, CTCs canprovide information only when they are available.

miRNAs

Other components of blood, such as standard cancer markersand microRNAs (miRNAs) may also have predictive ability.

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miRNAs, which are small noncoding RNA molecules con-sisting of approximately 22 nucleotides, have been identifiedin the serum of cancer patients.100 miRNAs are thought toregulate the expression of multiple genes, based on theirbinding sites, and are involved in different cellularprocesses, including apoptosis, differentiation, metabolism,and cancer.101 It will be of interest to determine whethera specific set of miRNAs control metastasis and/or therapyresistance pathways.

New Trial Designs Will Be Needed

Towhat extent is our failure to improve the lives of metastaticbreast cancer patients, or those at high risk of developingmetastases, a failure not of drug development, but of clinicaltrial design?

Even before personalized medicine is developed, newtrial designs will be needed to address the goal of metastasisprevention, both in the adjuvant setting and for preventionof additional metastases in the limited metastatic setting(secondary prevention). The preclinical literature aboundswith evidence that compounds currently in clinical trialsprevent the formation of metastases (they are given earlyand continuously, and fewer metastases develop at theendpoint).102,103 We have advocated for phase II random-ized primary and secondary metastasis prevention trials.104

In brief, for semipersonalized groups, patients couldreceive standard of care and be randomized to a metastasispreventive agent or placebo. The endpoint of interest wouldbe time to the development of a first metastasis, or time tothe development of a new metastasis. This type of trialdesign could validate drugs with efficacy to hold singletumor cells or micrometastases in check, but that cannotshrink an established lesion in standard phase II metastaticsetting trials. It is noteworthy that the primary preventionscheme dovetails with recent FDA guidance on neoadjuvantclinical trial designs.105 Thus, patients who do not achievea complete pathological response could be optimal candi-dates for primary metastasis prevention trials.

New trials will also be needed to address the inductionand breakdown of metastatic dormancy as this phenotype isdrugged.106 Long-term therapy may induce drug insensi-tivity (resistance) as well as prolonged toxicity.

Trials with other designs that may be particularly germaneto the personalization of treatment are underway. In theSAFIR-01 trial, coordinated by Dr. Fabrice André, high-throughput technologies (microarray gene expressionprofiling and comparative genomic hybridization array,OncoMap platform, and next-generation sequencing) areused to identify metastatic breast cancer patients whosetumor metastases present specific molecular alterations toadd a targeted regimen to the standard treatment. More than400 mutations are analyzed. Moreover, these technologiesdetermine whether such genomic alterations are single (ex-pected efficacy of a single agent) or multiple (rationale fora combination). This approach attempts to improve

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personalized medicine and to lower risk, compared witha single-biomarker trial. However, the high cost of the high-throughput technologies and the small size that a personal-ized approach can reach must be considered as limitations.107

The role of adaptive trial designs is being developed inthe I-SPY trial series, although in the neoadjuvant setting.Coordinated by Drs. Laura Esserman and Donald Berry, theI-SPY 2 trial uses biomarkers (HR status, HER2 status, andthe MammaPrint 70-gene signature status) to stratifypatients based on their predicted potential response totreatment, and evaluates phase II drugs in combination withstandard chemotherapy.108 An adaptive trial can use morethan one type of adaptation, such as stopping a treatmentearly, changing or dropping arms or doses, and changing theproportion of patients randomized to each arm. Thisapproach allows a rapid identification of effective newagents and drug combinations, as well as of the breastcancer subtypes that will benefit from the new therapy. It ishoped that this trial design will also reduce adverse effectsand spare patients from enrolling in trials from which theywill not benefit. How adaptive designs fit into the adjuvantand metastatic settings remains incompletely resolved.

Redundancy

The metastatic process is inherently redundant, in thatmultiple pathways can accomplish the same task. Forexample, tumor cell motility may be mediated by integrin,Rac1, Rho, MMP, FAK, multiple growth factors, and othersignaling pathways.1 Although each of these pathways ismechanistically validated, an inhibitor to one pathway likelyselects for the rare tumor cell that can use an alternativepathway for the same function. The same genomic insta-bility that fuels this adaptive response to the mechanisticrequirements of metastasis likely also promotes an adaptiveresponse for the development of resistance to chemother-apies and hormonal therapies. Personalized medicine mayactually be most effective when administered as a cocktailof therapies to combat both the complex molecular wiringand the predicted resistance mechanisms, similar to thecocktails effective for the treatment of AIDS.

Patient Advocate Perspective

The life expectancy formost individualswithmetastatic breastcancer is still less than 5 years. Although patients receivesemipersonalized treatment, the life-saving efficacy observedin the adjuvant setting is absent from the metastatic setting.Treatment of metastatic breast cancer is largely a hit-or-missproposition characterized by increasing resistance withsuccessive lines of therapy. Although some guidelines areavailable, they are basedbynecessity on consensus ofwhat hasbeen shown to be best for groups of patients in clinical trials.

Apart from the predictive value of HER2 and ER status ingoverning treatment choices, treatment of metastatic diseaseis almost wholly an empirical process, especially in the later

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stages of the disease, involving a series of trial-and-errorattempts to control the cancer. The lack of specific predic-tive biomarkers for treatment response means that weeks tomonths of toxic and expensive treatments are undergonebefore scans indicate whether a treatment is working.

We still understand little about why one treatment worksand another does not. A much more personalized approachwould include other factors than the ER and HER2expression of a tumor. Because treatment efficacy and bothde novo and acquired resistance may be affected by otherthan genomic factors, perhaps we need to use the termpersonalized medicine more inclusively.

Many relevant and urgent questions need answering.What drives the dormant cancer cells to grow again or sendoutgrowths from quiescent, stable lesions? Is it an individ-ual’s metabolism? Is it the microenvironment and stromasurrounding the cells? Why do some patients get only bonemetastases? Or only one lesion in one organ? It seems as ifsequencing the genome of one individual lesion may notanswer such questions.

If a more truly personalized approach comes withinfinancial and logistic reach, the hope is that it could greatlyboost not only the quantity but also the quality of lives. Thedream of living a relatively normal life span could becomea reality. The challenges, however, seem too steep, and formany reasons. How will researchers, each with tiny piecesof a giant puzzle, come together to make sense of theincreasing amounts of data? Who is going to develop drugsthat may have quite a limited market? Who will the payersbe? Who will benefit from the personalized genomicapproach? Finally, how will personalized medicine, andtreatment of metastatic breast cancer in particular, reachthose living in low-resource countries?

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