TOP ARTICLES SUPPLEMENT - Future Medicine€¦ · Specifically in the field of BC research, *omics technologies are currently applied to understand the molecular mechanisms, to

Breast Cancer TOP ARTICLES SUPPLEMENT

CONTENTSREVIEW: Potential biomarker panels in overall breast cancer management: advancements by multilevel diagnostics Personalized Medicine Vol. 13 Issue 5

REVIEW: Management of women with a hereditary predisposition for breast cancer Future Oncology Vol. 12 Issue 19

EDITORIAL: Intratumor and circulating clonal heterogeneity shape the basis of precision breast cancer therapy Future Oncology (Epub ahead of print)

REVIEW: Restoring anti-oncodriver Th1 responses with dendritic cell vaccines in HER2/neu-positive breast cancer: progress and potential Immunotherapy Vol. 8 Issue 10

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469Per. Med. (2016) 13(5), 469–484 ISSN 1741-0541

part of

Review

10.2217/pme-2016-0020 © 2016 Future Medicine Ltd

Per. Med.

Review 2016/08/2913

5

2016

Breast cancer (BC) prevalence has reached an epidemic scale with half a million deaths annually. Current deficits in BC management include predictive and preventive approaches, optimized screening programs, individualized patient profiling, highly sensitive detection technologies for more precise diagnostics and therapy monitoring, individualized prediction and effective treatment of BC metastatic disease. To advance BC management, paradigm shift from delayed to predictive, preventive and personalized medical services is essential. Corresponding step forwards requires innovative multilevel diagnostics procuring specific panels of validated biomarkers. Here, we discuss current instrumental advancements including genomics, proteomics, epigenetics, miRNA, metabolomics, circulating tumor cells and cancer stem cells with a focus on biomarker discovery and multilevel diagnostic panels. A list of the recommended biomarker candidates is provided.

First draft submitted: 25 February 2016; Accepted for publication: 16 May 2016; Published online: 27 July 2016

Keywords: breast cancer • cancer stem cells • circulating tumor cells • omics • personalized medicine • predictive diagnostics • prognosis • stratification • targeted therapy • validated biomarker

Introduction: deficits in breast cancer management & expectations by multilevel diagnosticsBreast cancer (BC) has been recognized as becoming epidemic in the 21st century [1] with around half a million deaths and about 1.7 million new cases annually [2]. Although BC mortality rates have slightly decreased over last decades, the incidence rates con-tinue to escalate [3]. BC is a multifactorial disease with highly heterogeneous risk fac-tors, origin, individual phenotypes and consequently, different molecular profiles, sensitivity to therapeutic approaches and fre-quently unpredictable clinical outcomes [4]. Current deficits in BC management include suboptimal screening programs exhibiting a number of cases with both false-positive and -negative diagnosis [5], an evident lack of screening programs focused specifically

on BC predisposition and initiation in young subpopulations, individualized patient profil-ing for more precise diagnosis and treatments tailored to the person that altogether result in enormous socioeconomic burden [1,6]. In par-ticular, the overall annual costs for BC man-agement across Europe are reported as being at the level of €15.0 billion [7]. Furthermore, study by Meneses et al. has underlined that the BC-linked economic burden is in liaison with decreased motivation and productivity at work, days missed from work, change in partner’s income, financial adversity and an overall poorer quality of life [8].

Currently, there is an evident lack of arti-cles providing evidence and summarizing biomarker panels for multilevel diagnostic approaches, which are particularly promising to optimize the overall BC management [1,6]. Potential complementarity and integration of

Potential biomarker panels in overall breast cancer management: advancements by multilevel diagnostics

Shantanu Girotra1, Kristina Yeghiazaryan1 & Olga Golubnitschaja*,1

1Department of Radiology, University of

Bonn, Germany

*Author for correspondence:

[email protected]

For reprint orders, please contact: [email protected]

470 Per. Med. (2016) 13(5) future science group

Review Girotra, Yeghiazaryan & Golubnitschaja

several detection technologies at molecular and cellu-lar levels in use or under development into innovative approaches by multilevel diagnostics is considered in this paper. Biomarker panels are overviewed as pro-vided by individual technologies, namely genomics, transcriptomics, proteomics, metabolomics as well as detection and analysis of circulating tumor cells (CTCs) and cancer stem cells (CSCs). The crucial role of integrative bioinformatics in development and suc-cessful clinical application of innovative approaches by multilevel diagnostics is discussed.

Crucial role of *omics in BC diagnostics & treatment*omics technologies encompass the utilization of detection methodology focused on complex molecular profiling. Most frequently considered complementary approaches are genomics, transcriptomics, proteomics, metabolomics and lipidomics. Specifically in the field of BC research, *omics technologies are currently applied to understand the molecular mechanisms, to analyze contributing molecular pathways [9], BC pre-disposition and phenotyping [10–12], heterogeneity of BC and BC metastatic disease (BCMD) [13], therapy targeting [14], drug resistance [15] and others [16–18].

Genomic analysis in BCCancer genomics has become a vital tool in cancer management. It employs different genomic approaches such as the next-generation sequencing platform, genome testing and genome wide association stud-ies [19–25]. Clinical implementation of genome testing is well justified, for example, for adjuvant chemotherapy application [26]. Next-generation sequencing platform provides multitude of genomic data including proba-bilistic approach for cancer predisposition, discovery of cancer-related genes/pathways and structural DNA modification (mutations, changes of corresponding gene copy number and chromosomal rearrangements), analysis of intratumoral heterogeneity and drug resis-tance [19]. Engaging the currently available genomic tools manifests a potential for better diagnosis and prognosis of BC [20–24]. Table 1 provides information about powerful biomarker candidates detectable by genomics.

Complementary to this subchapter considering BC relevant DNA structure modifications such as poly-morphisms, mutations, gene copy number, the follow-up subchapters are dedicated to the next levels of bio-marker detection, namely the gene regulation-specific patterns.

Table 1. Predictive and prognostic biomarkers detectable by genomics.

Biological material Biomarker (DNA template)

Comments Useful for Ref.

Peripheral blood lymphocytes

BRCA1 mutations (genomic DNA)

Higher in TNBC compared with non-TNBC; worsen OS in carriers compared with noncarriers

BC predictive diagnostics [10,27–28]

Primary invasive breast carcinomas tumor samples (human)

TP53 mutations Poor prognosis in ER+ BC therapy monitoring/prognostic

[29]

Peripheral blood samples MTHFR C677T polymorphism (genomic DNA)

Significantly associated with BC risk, BC development

BC predisposition/predictive diagnostics

[30–36]

Peripheral blood samples hOGG1 polymorphism c.977C>G (genomic DNA)

Relevance for development of p53+ TNBC phenotype in premenopausal patients

BC therapy monitoring/prognostic

[37]

Whole blood samples SNP rs17506395 in p63 TT genotype: poor prognosis in BC in Han Chinese population

BC therapy monitoring/ prognostic

[38]

Blood samples SNP rs28366003 in MT2A (genomic DNA)

Risk of BC in Polish population

BC predictive diagnostics [39]

Tissue samples (human) and peripheral blood samples

SNP rs17849079 in PIK3CA (genomic DNA)

Prevalent in BC cases compared with controls in Arab population

BC predictive diagnostics [40]

TNBC tissue samples (human)

High EGFR copy number Associated with poor outcome in TNBC phenotype


[41]

BC: Breast cancer; OS: Overall survival; rs: Reference SNP ID; TNBC: Triple-negative breast cancer.

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Biomarker panels in breast cancer management Review

Gene regulation level comprises a number of complementary approaches & allows for more precise diagnosis & targeted treatments in BCAbove-described BC-relevant alterations in DNA structure are the basic informational level con-sidering genetic predisposition to pathology and pharmacogenetic characteristics relevant for the drug application.

The following paragraphs are focused on more com-prehensive level of ‘post-genomic’ gene regulation to be essentially considered for individual patient profiles estimating synergistic effects of all risk factors includ-ing internal and external stress factors, lifestyle, diet, behavior-related biomarker patterns, among others.

Gene patterns differently regulated in BCThe data summarized below furnish information about biomarker-candidate elements in regard to molecular patterns linked to gene regulation (gene amplification/overexpression), which is specifically altered in BC. Table 2 outlines a particular importance of specific gene expression signature for BC and BCMD prognosis. Hence, a recurrence of triple-negative breast cancer (TNBC) can be predicted [42].

Epigenetic patterns specific for BCIn contrast to the basic DNA information analyzed by genomics, epigenomics analyses DNA patterns that are relevant for the specific gene activation such as DNA methylation status [48,49]. A role of epigenetic deregula-

tion (specifically altered epigenetic patterns) has been suggested for BC pathology based on comprehensive analysis by ‘epigenome-wide maps’ [50]. An impor-tance of DNA methylation in BC patient stratifica-tion, prediction and prognosis is evidence-based [51–53]. Furthermore, a biomarker panel is proposed specifi-cally for BC progression, which comprises the follow-ing genes: BRCA1, DAPK1, MSH2, CDKN2A, PGR, PRKCDBP and RANKL [52]. The functional role of above-mentioned genes includes tumor suppression, regulation of gene transcription, proapoptotic activ-ity, kinase inhibition and cell survival regulation. The other panel which is indicated to have prognostic value comprises BC-specific epigenetic patterns of the follow-ing 13 genes: CST6, DBC1, EGFR, GREM1, GSTP1, IGFBP3, PDGFRB, PPM1E, SFRP1, SFRP2, SOX17, TNFRSF10D and WRN [53]. Additionally, the same study demonstrated that the luminal type B tumors have higher average methylation status compared with basal-like tumors of the 48 selected genes [53] that are indicative for more precise BC phenotyping and stratified treatments. This gene panel was selected by previously performed genome-wide methylation studies, due to its relevance in BC. Epigenetic altera-tions in patterns of histones are shown to be involved in BC pathological mechanisms [54–56] being relevant for BCMD, patient stratification and related progno-sis [57–60]. Table 3 provides summarizing information to the most potent biomarker candidates. Corresponding predictive and/or prognostic value is notified.

Table 2. Predictive and prognostic biomarkers patterns differentially regulated in breast cancer.

Biological material Biomarker (DNA template)

Comments Useful for Ref.

Tissue samples (human), BC cell line (human)

PAK1 Amplification is associated with TamR

BC therapy monitoring/prognostic and optimal BC therapy algorithms/predictive

[43,44]

Tissue samples (mice), cell line (human) and BC tissue samples (human)

Nanog Altered expression contributes to BCMD, associated with poor prognosis

BC therapy monitoring/prognostic and BCMD therapy monitoring/prognostic

[45,46]

Tissue samples (human) Oct-4 and Nanog Overexpression promotes EMT process, contributing to BCMD

BC therapy monitoring/prognostic and BCMD therapy monitoring/prognostic

[46]

Normalized gene expression data and frozen-archived tumor material from early-stage BC patients

41-gene signature Predict DMFS and OS in node-negative BC

BC therapy monitoring/prognostic, BCMD therapy monitoring/prognostic

[47]

Tissue samples (human) 7-gene signature Predict distant recurrence in TNBC phenotype undergone adjuvant chemotherapy


[42]

BC: Breast cancer; BCMD: Breast cancer metastatic disease; DMFS: Distance metastasis-free survival; EMT: Epithelial mesenchymal transition; OS: Overall survival; TamR: Tamoxifen resistance; TNBC: Triple-negative breast cancer.



The role of specific miRNA patterns is reported for BC & BCMDmiRNAs are small (18–25 nucleotides), noncoding and evolutionary conserved RNAs discovered originally in Caenorhabditis elegans [77,78]. miRNAs function by neg-atively regulating expression of human genes affecting a number of biological processes such as cell development, differentiation, proliferation and apoptosis [78,79].

miRNA molecules have a crucial role in tumorigen-esis, which is complex, yet not entirely understood. Several miRNA species are reported as associated with metastatic disease in a spectrum of cancer types, for example, lymph node metastasis at early stages of squamous cell carcinoma [80], bone metastases spread by prostate cancer [81], gastric cancer development [82], colorectal cancer development and progression [83].

The role of miRNAs in BC and metastatic disease is reported with estimated high diagnostic and prognos-tic values [84–87]. Hence, miRNA are shown to regu-late signaling pathways implicated in BC development and progression [88]. Further supporting this, interplay between chemokine (C–C motif) ligand 18 (CCL18), a promoter of BC metastasis and miR progression

has been illustrated [88]. This study indicates miR-98 and miR-27b as downregulated by CCL18, which in turn creates a feedback loop that renders the CCL18-activated-N-Ras/ERK/PI3K/NFκB/Lin28b signaling pathway responsible for epithelial mesenchymal tran-sition characteristic for metastatic activity [88]. Con-sidering technological progress behind the issue, next-generation sequencing is used for detecting miRNA patterns specific for BC and their profiling in this patient cohort now [89,90]. Advanced technologies that utilize a profiling of circulating miRNAs (ct-miR) in blood may lead to a sufficient progress in BC diagnostics, due to minimally invasive and simplified sampling of blood, serum/plasma and urine [91,92]. Contextually, biobank-ing plays an important role in the field of miRNA research and prospective studies, which may lead to novel biomarker panels [93]. In order to bring miRNAs ‘from bench to bedside’, following deficits should be resolved: creation of robust prospective clinical trials, standardization of the analytical approach, detection of the exact origin of circulating miRNAs (tumor vs by-products of normal or dead cells) and validation of the disease-specific signature/miRNA patterns [94].

Table 3. Predictive and prognostic biomarkers patterns detected at epigenetic level in breast cancer.

Biological material Biomarker Comments Useful for Ref.

Tissue samples, peripheral blood

BRCA1 Altered promoter methylation in TNBC phenotype

BCMD predisposition/predictive diagnostics

[61–64]

Tissue samples, BC cell lines (human)

CXCL12 and CXCR4 Patients with altered CXCL12 methylation and unmethylated CXCR4 have shorter overall and disease-free survival


[65,66]

Primary tumors, lymph node metastases, plasma and blood cells

CXCL12 and ADAM23 Hypermethylation associated with advanced stage and LNM


[67]

Tissue samples, plasma, blood cells, BC cell line (human)

ADAM23 Increased promoter methylation observed in ER+ and associated to advanced grade, poor prognosis, role in metastasis in other studies


[67–69]

BC cell lines (human), tissue samples

CXCL12 and ESR1 Hypermethylation associated with advanced stage and metastasis


[70]

FFPE tumor samples BRCA1, DAPK1, MSH2, CDKN2A, PGR, PRKCDBP, RANKL

Promoter hypermethylation associated with tumor progression


[52]

Serum samples ITIH5, DKK3 and RASSF1A (tumor-suppressor genes)

Hypermethylation in samples of BC


[71]

Peripheral blood, cell culture (human), plasma samples, tissue samples

Global DNA methylation (genomic DNA)

Overall DNA hypomethylation in BC

BC predictive diagnostics

[72–76]

Corresponding methylation status and potential diagnostic value are notified.BC: Breast cancer; BCMD: Breast cancer metastatic disease; FFPE: Formalin-fixed paraffin-embedded tissue; LNM: Lymph node metastases; TNBC: Triple-negative breast cancer.


Biomarker panels in breast cancer management ReviewTa

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BC: Breast cancer; BCMD: Breast cancer metastatic disease; FFPE: Formalin-fixed paraffin-embedded tissue; TamR: Tamoxifen resistance; TNBC: Triple-negative breast cancer.



miRNA patterns relevant for BC are summarized in Table 4. Corresponding diagnostic and prognostic value is notified.

Proteomic patterns specific for BC predisposition, development & progressionClinical differential proteomics as the promising tool for

BC risk assessment

A new integrative concept for better BC management has been recommended in context of molecular patient profiling utilizing clinical differential proteomics [6]. This approach emphasizes the crucial importance of

multilevel diagnostics and application of technologies complementary to the protein level of detection. Con-sequently, the overall task includes multimodal diag-nostic approaches, disease-specific biomarker-patterns, individual patient profiles, creation of medical records and treatments tailored to the person [6]. Creation of the patient-specific ‘molecular portrait’ is an essential part of the diagnostic strategy. Furthermore, noninva-sive analytical tools utilizing blood samples for clinical proteomics and complementary analytical technolo-gies are well justified in case of BC, due to a direct relevance of molecular patterns in blood for BC pre-

Box 1. Systematic overview of the functional group panel; detection tool – clinical differential proteomics, diagnostic purpose – breast cancer risk assessment; the source of biological material – circulating leukocytes.

Functional group• Microfilamental network-associated and cytoskeletal-assembly proteins• Cell motility, migration and adhesion• Nucleoside/nucleotide turnover and metabolism• Protein metabolism (regulatory protein-synthesis and protein-modification enzymes, chaperons)• Energy metabolism• Vitamin metabolism• Mitochondrial proteins• Channels, membrane-architecture and intercellular-junction proteins• Antioxidant defense/red-ox control• Detoxification proteins• Stress-response/-protection-related protein• Cell-cycle machinery proteins• Heat-shock proteins• Apoptosis-related proteins/protection against apoptosis• Tissue remodeling enzymes• Extra-cellular transport and carrier-proteins• Signal-transduction proteins/signaling pathways• Longevity-/aging-related proteins• Inflammation-related/anti-inflammatory proteins• (Breast) cancer-related inhibitor/promoter• Cancer invasion and regulator of metastases formation.

Data taken from [6].

Table 5. Biomarker candidates nominated for specific diagnostic purposes.


BC cell line (human) PFN1 Downregulated in BCMD BC therapy monitoring/prognostic; BCMD therapy monitoring/prognostic

[127–130]

BC cell line (human) Annexin A6 Increased expression in BC BC therapy monitoring/prognostic [131]

FFPE tumor samples Vimentin Upregulated in TNBC BC therapy monitoring/prognostic [132]

Surgical specimen, blood samples, FFPE samples

MMP-9 Overexpressed in BC and BCMD

BC therapy monitoring/prognostic; BCMD therapy monitoring/prognostic

[133–135]

Frozen primary tumor samples

11-protein signature Predict metastasis in TNBC BC therapy monitoring/prognostic; BCMD therapy monitoring/prognostic

[136]

FFPE samples 10-protein expression panel

Stratify aggressive phenotype in familial BC

BC predisposition; BC therapy monitoring/prognostic

[137]

BC: Breast cancer; BCMD: Breast cancer metastatic disease; FFPE: Formalin-fixed paraffin-embedded tissues; TNBC: Triple-negative breast cancer.



disposition, development and progression. Therefore, analysis of molecular patterns in circulating leucocytes as the minimally invasive diagnostic tool and construc-tion of diagnostic windows based on individual patient profiling for efficient BC risk assessment is highly rec-ommended [6]; the approach that is further demon-strated as clinically useful [126]. Consequently, a panel of functional groups of proteins involved in BC-related molecular mechanisms is provided (see Box 1) [6].

Furthermore, Table 5 summarizes protein bio-markers demonstrated as specific for BC at the level of predisposition to BCMD and diagnostically useful for individual BC phenotypes. Alterations specific for BC are notified and diagnostic/prognostic utility is described, respectively.

Metabolomics demonstrates a high potential of clinical utility in BC managementWell-detectable metabolic differences have been described as specific for BC and metastatic dis-ease [138–141]. Consequently, metabolomics profiling is considered as being a powerful methodology for pre-diction and prognosis in BC development and progres-sion [142,143]. Metabolomics is the technological tool analyzing health and disease stage-specific metabolite

profiles in biological samples such as body fluids and tissue homogenates. The approach entails utilizing qualification and quantification of biomarkers in indi-vidual and comparative metabolic patterns. The most clinically useful approaches result in metabolite profil-ing and metabolic fingerprinting. Metabolite profiling is currently utilized for diagnostics of a whole spectrum of cancers [144]. Thereby, patient stratification is pos-sible regarding cancer predisposition and cancer sub-type followed by more precise prognosis and targeted therapy approaches [145–148]. Metabolomics employs highly sensitive technologies based on NMR spec-troscopy and mass spectrometry. The methodology is well standardized with an excellent level of results’ reproducibility that is decisive for its successful clinical application. Table 6 summarizes metabolic biomarkers specific for BC diagnosis and prognosis. According to currently published reports, following metabolic path-ways are certainly involved in BC pathology: gluco-neogenesis, tricarboxylic acid cycle, pentose phosphate pathway [149], serine/glycine pathway [140], glutamine and beta-alanine metabolism [146]. Many others are currently under consideration that further increases the potential of clinical utility for metabolomics in the overall BC management.

Table 6. Metabolites and metabolic pathways involved in breast cancer pathology.


Serum Low levels of histidine, higher levels of glucose and lipids

Predict disease relapse in early stage of BC


[138]

Urine, peripheral venous blood sample

PGE2 Increased levels in BC associated with increased BC risk


[142–143,150]

Tissue samples, cell lines (human)

Glutamine metabolism Enhanced in ER- and TNBC phenotypes and is associated with reduced patient survival in HER2+ phenotype


[141,145–148]

Cell lines, tissue samples Glycine/serine metabolism

Pathway highly activated in BC BC therapy monitoring/prognostic

[139–140,151]

Tissue samples Glycine High levels in HER2+ phenotype BC therapy monitoring/prognostic

[141]

BC: Breast cancer; TNBC: Triple-negative breast cancer.

Table 7. Circulating tumor cells in breast cancer prediction and prognosis.


Peripheral whole blood sample

CTC enumeration Predict treatment outcome in BCMD

BC therapy monitoring/prognostic; BCMD therapy monitoring/prognostic

[168]

Cell lines (human) and peripheral blood sample

PLS3 High expression in CTC undergoing EMT

BC therapy monitoring/prognostic [170]

Blood sample CD133-expressing CTCs

More frequent in luminal tumors

BC therapy monitoring/prognostic [172]

BC: Breast cancer; BCMD: Breast cancer metastatic disease; CTC: Circulating tumor cell; EMT: Epithelial mesenchymal transition.



Cellular levelCirculating tumor cellsCTCs, dislocated from a cancerous tissue via blood flow, demonstrate high metastatic potential nesting distanced organs and developing secondary tumors of particular aggressive phenotypes. Clinical interest in the CTCs research field is permanently increasing, due to two main reasons: first, CTC can be detected by minimally invasive approach in peripheral blood and second, a lot of diagnostic information can be received for more precise prediction and prognosis [152]. Informa-tion provided by CTCs has been recently demonstrated to have an independent prognostic value in metastatic disease [153], in monitoring treatment response [154,155], efficacy of chemotherapy [156] and prediction of recur-rence [157]. Despite low density of CTC in circulating blood flow, innovative detection technologies allow for an accurate tracing of CTC and their representative isolation from patients’ blood samples [158,159]. Clini-cal application of CTC is considered for a spectrum of cancer types [160–166]. BC is demonstrated as spread-ing the highest number of CTC in blood flow [152] fol-lowed by high CTC level in prostate and other cancer

types [167]. Contextually, this approach is well promis-ing in diagnosis and prognosis of breast and prostate cancer metastatic disease. Hence, a prospective study on 393 metastatic BC patients revealed that serial CTC enumeration and related parameters such as changes in CTCs over a course of treatment are helpful for effi-cient monitoring of the disease progression and treat-ments tailored to the patient [168]. Furthermore, recent studies on Plastin3, a novel biomarker for CTCs detec-tion [169], highlight that, first, it is of great clinical util-ity in BC regarding the risk of recurrence and second, it is a marker for poor prognosis especially relevant for TNBC cases [170]. CTCs could also play an important role in the detection of BC at early stages and associated pathology development [171]. Table 7 summarizes poten-tial CTC-related biomarker candidates useful for BC and BCMD prognosis and therapy monitoring.

Cancer stem cellsCSCs are also known as tumor-initiating cells. CSC populations are highly heterogeneous with a particular potential for reproduction and generation of cells dif-ferentiated into cancer phenotypes [173,174]. It has been

Table 8. Cancer stem cells in breast cancer therapy monitoring and prognosis.


Tissue samples [171,172], cell lines (human [172])

CD44+/CD24-/low Marker for basal-like tumors, associated with more aggressive phenotype


[190,191]

Tissue samples [172,173] and cell line (human [172])

ALDH1+ Marker for basal-like and HER2+ overexpressing tumors


[191,192]

BC: Breast cancer.

Figure 1. Potential biomarker panels in breast cancer management. BC: Breast cancer; BCMD: Breast cancer metastatic disease.

Technology Biological material

Application

Genomics• Tissue samples• Blood samples• BC cell lines

• BC predisposition/predictive diagnostics• BC therapy monitoring/prognostic• BCMD therapy monitoring/prognostic

• BC predictive diagnostics• BC therapy monitoring/prognostic• BCMD therapy monitoring/prognostic• BCMD predisposition/predictive diagnostics

• Tissue samples• Blood samples• Serum samples• BC cell lines

Epigenetics



suggested that bulk of BC tumor consists of differenti-ated CSC [175–179]. Furthermore, CSCs strongly modu-late (increase) tumor drug resistance [180–182]. Conse-quently, current CSC-related studies are dedicated to innovative therapy approaches targeted specifically to the CSC populations [183–185].

Breast cancer stem cells are known to be highly tumor-igenic. They are characterized by increased chemoresis-tance compared with the differentiated BC cells [181,182]. They are involved in local invasion and migration being responsible for metastatic activity [186], aggressive phe-notype in HER2-overexpressing breast cancer stem cells [187] and altered drug resistance [188]. The most common phenotypes used for identification of CSC in BC are CD44+/CD24low/- and elevated ALDH [189]. Spe-cifically in BC, well-detailed characterization of CSCs may result in promising adjuvant therapies tailored to stratified BC subtypes and individual CSC populations. CSC biomarker patterns specific for BC are summarized in Table 8. Corresponding clinical utility is noted.

ConclusionBreast cancer is a multifactorial disease with highly het-erogeneous risk factors, origin, individual phenotypes, and consequently, different molecular profiles, sensitivity to therapeutic approaches, and frequently unpredictable clinical outcomes. Breast cancer metastatic disease is the major cause of about a half of million deaths annually registered for the BC patient cohort worldwide. There are evident deficits in the overall BC management. Con-sequently, a paradigm change towards predictive, pre-ventive and personalized medicine (PPPM) in the overall breast cancer management is essential and urgent. For the most effective implementation of advanced PPPM services in the field of breast cancer, an innovative approach of multilevel diagnostics is recommended.

Future perspectiveBC is a multifactorial disease with highly heteroge-neous risk factors, origin, individual phenotypes and consequently, different molecular profiles, sensitivity

Executive summary

Deficits in breast cancer management & expectations by multilevel diagnostics• Current deficits in breast cancer (BC) management include suboptimal screening programs demonstrating

a number of cases with both false-positive and -negative diagnosis, an evident lack of screening programs focused specifically on BC predisposition and initiation in young subpopulations, individualized patient profiling for more precise diagnosis and treatments tailored to the person that altogether result in enormous socioeconomic burden.

• Furthermore, there is an evident lack of articles providing evidence and summarizing biomarker panels for multilevel diagnostic approaches, which are particularly promising to optimize the overall BC management.

• The crucial role of integrative bioinformatics in development and successful clinical application of innovative approaches by multilevel diagnostics is discussed.

Crucial role of *omics in BC diagnostics & treatment• Next-generation sequencing platform provides multitude of genomic data including probabilistic approach

for cancer predisposition, discovery of cancer-related genes/pathways and structural DNA modification (mutations, changes of corresponding gene copy number and chromosomal rearrangements), analysis of intratumoral heterogeneity and drug resistance.

• Gene regulation level comprises a number of complementary approaches and allows for more precise diagnosis and targeted treatments in BC.

• Epigenetic deregulation in BC pathology has been detected by ‘epigenome-wide maps’; an importance of DNA methylation in BC patient stratification, prediction and prognosis is evidence-based.

• The role of miRNAs in BC and metastatic disease is reported with estimated high diagnostic and prognostic values. However, in order to bring miRNAs ‘from bench to bedside’, following deficits should be resolved: creation of robust prospective clinical trials, standardization of the analytical approach, detection of the exact origin of circulating miRNAs (tumor vs by-products of normal or dead cells) and validation of the disease-specific signature/miRNA-patterns.

• Creation of the patient specific ‘molecular portrait’ is an essential part of the diagnostic strategy: differential proteomics is the promising tool for that.

• Metabolomics demonstrates a high potential of clinical utility in BC management.Cellular level• Clinical interest in the circulating tumor cells is permanently increasing due to two main reasons: first,

circulating tumor cells can be detected by minimally invasive approach in peripheral blood, and second, a lot of diagnostic information can be received for more precise prediction and prognosis.

• Breast cancer stem cells (BCSCs) are highly tumorigenic being responsible for metastatic activity. Individual characterization of BCSCs may result in promising adjuvant therapies tailored to stratified BC subtypes and individual BCSC populations.



to therapeutic approaches and frequently unpredictable clinical outcomes. Currently, there are evident deficits in the overall BC management such as suboptimal screening programs differentiating early versus late adulthood and the elderly, lack of the targeted preven-tion specifically in teenager age and young subpopula-tions, patient stratification for more precise diagnosis and treatments tailored to the person. Over the next 5–10 years, the overall BC management will remark-ably evolve utilizing the paradigm change from reac-tive to PPPM. The most effective implementation of advanced PPPM services includes:

• Innovative approaches of multilevel diagnostics considered in this paper – see Figure 1;

• Application of individualized patient profiles [6,126,193].

• Creation of optimized screening programs [6,126];

• Development of novel biomarker panels for BC therapeutics [194];

• Application of multidrug treatments tailored to the person [195].

Financial & competing interests disclosureThe authors have no relevant affiliations or financial in-

volvement with any organization or entity with a financial

interest in or financial conflict with the subject matter or

materials discussed in the manuscript. This includes employ-

ment, consultancies, honoraria, stock ownership or options,

expert testimony, grants or patents received or pending, or

royalties.

No writing assistance was utilized in the production of this

manuscript.

ReferencesPapers of special note have been highlighted as: •ofinterest;••ofconsiderableinterest

1 Golubnitschaja O, Debald M, Yeghiazaryan K et al. Breast cancer epidemic in the early 21st century: evaluation of risk factors, cumulative questionnaires and recommendations for preventive measures. Tumor Biol. Doi:10.1007/s13277-016-5168-x (2016) (In press).

•• Demonstratinganurgentnecessityforparadigmshiftintheoverallbreastcancermanagementfromreactivetopredictive,preventiveandpersonalizedmedicalservices,summarizingall-knownbreastcancer-relatedriskfactorsandprovidingrecommendationsforinnovativemultileveldiagnosticapproaches.

2 Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-tieulent J, Jemal A. Global cancer statistics, 2012. CA Cancer J. Clin. 65(2), 87–108 (2015).

3 Ekwueme DU, Guy GP, Rim SH et al. Health and economic impact of breast cancer mortality in young women, 1970–2008. Am. J. Prev. Med. 46(1), 71–79 (2014).

4 Reis-Filho JS, Pusztai L. Gene expression profiling in breast cancer: classification, prognostication, and prediction. Lancet 378(9805), 1812–1823 (2011).

5 Marmot MG, Altman DG, Cameron DA, Dewar JA, Thompson SG, Wilcox M. The benefits and harms of breast cancer screening: an independent review. Br. J. Cancer 108(11), 2205–2240 (2013).

6 Golubnitschaja O, Yeghiazaryan K, Costigliola V et al. Risk assessment, disease prevention and personalised treatments in breast cancer: is clinically qualified integrative approach in the horizon? EPMA J. 4(1), 6 (2013).

•• SummarizingproteinbiomarkerpanelsspecificforbreastcancerdevelopmentandprogressionandprovidingcompleteinformationaboutfunctionalgroupsinvolvedinBCpathology;thepaperusefulforcreationofindividualizedpatientprofiles,targetedpredictionandpreventionofbreastcancer.

7 Luengo-fernandez R, Leal J, Gray A, Sullivan R. Economic burden of cancer across the European Union: a population-based cost analysis. Lancet Oncol. 14(12), 1165–1174 (2013).

8 Meneses K, Azuero A, Hassey L, Mcnees P, Pisu M. Does economic burden influence quality of life in breast cancer survivors? Gynecol. Oncol. 124(3), 437–443 (2012).

9 Wang L, Xiao Y, Ping Y et al. Integrating multi-omics for uncovering the architecture of cross-talking pathways in breast cancer. PLoS ONE 9(8), e104282 (2014).

• Demonstratinghighlevelofclinicalutilityformultidimensional(multiomics)datatoidentifythecancer-relatedpathwaysandtoimprovetheprecisionofdiagnosis.

10 Tun NM, Villani G, Ong K, Yoe L, Bo ZM. Risk of having BRCA1 mutation in high-risk women with triple-negative breast cancer: a meta-analysis. Clin. Genet. 85(1), 43–48 (2014).

11 Garrisi VM, Tommasi S, Facchiano A et al. Proteomic profile in familial breast cancer patients. Clin. Biochem. 46(3), 259–265 (2013).

12 Rhee JK, Kim K, Chae H et al. Integrated analysis of genome-wide DNA methylation and gene expression profiles in molecular subtypes of breast cancer. Nucleic Acids Res. 41(18), 8464–8474 (2013).

13 Xu H, Eirew P, Mullaly SC, Aparicio S. The omics of triple-negative breast cancers. Clin. Chem. 60(1), 122–133 (2014).

14 Muellner MK, Mair B, Ibrahim Y et al. Targeting a cell state common to triple-negative breast cancers. Mol. Syst. Biol. 11(1), 789 (2015).

15 Chen S. An “omics” approach to determine the mechanisms of acquired aromatase inhibitor resistance. OMICS 15(6), 347–352 (2011).

16 Kim D, Li R, Dudek SM, Ritchie MD. Predicting censored survival data based on the interactions between meta-dimensional omics data in breast cancer. J. Biomed. Inform. 56, 220–228 (2015).

17 Pauling JK, Christensen AG, Batra R et al. Elucidation of epithelial–mesenchymal transition-related pathways in a



triple-negative breast cancer cell line model by multi-omics interactome analysis. Integr. Biol. (Camb.) 6(11), 1058–1068 (2014).

18 Ternès N, Arnedos M, Koscielny S, Michiels S, Lanoy E. Statistical methods applied to omics data: predicting response to neoadjuvant therapy in breast cancer. Curr. Opin. Oncol. 26(6), 576–583 (2014).

19 Desmedt C, Voet T, Sotiriou C, Campbell PJ. Next-generation sequencing in breast cancer: first take home messages. Curr. Opin. Oncol. 24(6), 597–604 (2012).

• Summarizingtheroleofnext-generationsequencinginbreastcancerdiagnostics,providinginformationfornoveltargetsfortreatmentsanddiseasemonitoringtailoredtotheperson.

20 Ahsan H, Halpern J, Kibriya MG et al. A genome-wide association study of early-onset breast cancer identifies PFKM as a novel breast cancer gene and supports a common genetic spectrum for breast cancer at any age. Cancer Epidemiol. Biomarkers Prev. 23(4), 658–669 (2014).

21 Lindström S, Thompson DJ, Paterson AD et al. Genome-wide association study identifies multiple loci associated with both mammographic density and breast cancer risk. Nat. Commun. 5, 5303 (2014).

22 Michailidou K, Beesley J, Lindstrom S et al. Genome-wide association analysis of more than 120,000 individuals identifies 15 new susceptibility loci for breast cancer. Nat. Genet. 47(4), 373–380 (2015).

23 Hsiung CN, Chu HW, Huang YL et al. Functional variants at the 21q22.3 locus involved in breast cancer progression identified by screening of genome-wide estrogen response elements. Breast Cancer Res. 16(5), 455 (2014).

24 Purrington KS, Slager S, Eccles D et al. Genome-wide association study identifies 25 known breast cancer susceptibility loci as risk factors for triple-negative breast cancer. Carcinogenesis 35(5), 1012–1019 (2014).

25 Rich TA, Woodson AH, Litton J, Arun B. Hereditary breast cancer syndromes and genetic testing. J. Surg. Oncol. 111(1), 66–80 (2015).

26 Abu-khalf M, Pusztai L. Influence of genomics on adjuvant treatments for pre-invasive and invasive breast cancer. Breast 22(Suppl. 2), S83–S87 (2013).

27 Fostira F, Tsitlaidou M, Papadimitriou C et al. Prevalence of BRCA1 mutations among 403 women with triple-negative breast cancer: implications for genetic screening selection criteria: a Hellenic Cooperative Oncology Group Study. Breast Cancer Res. Treat. 134(1), 353–362 (2012).

28 Zhong Q, Peng HL, Zhao X, Zhang L, Hwang WT. Effects of BRCA1- and BRCA2-related mutations on ovarian and breast cancer survival: a meta-analysis. Clin. Cancer Res. 21(1), 211–220 (2015).

29 Silwal-pandit L, Vollan HK, Chin SF et al. TP53 mutation spectrum in breast cancer is subtype specific and has distinct prognostic relevance. Clin. Cancer Res. 20(13), 3569–3580 (2014).

30 He JM, Pu YD, Wu YJ et al. Association between dietary intake of folate and MTHFR and MTR genotype with risk of breast cancer. Genet. Mol. Res. 13(4), 8925–8931 (2014).

31 Liang H, Yan Y, Li T et al. Methylenetetrahydrofolate reductase polymorphisms and breast cancer risk in Chinese population: a meta-analysis of 22 case–control studies. Tumour Biol. 35(2), 1695–1701 (2014).

32 Wang ZG, Cui W, Yang LF, Zhu YQ, Wei WH. Association of dietary intake of folate and MTHFR genotype with breast cancer risk. Genet. Mol. Res. 13(3), 5446–5451 (2014).

33 Li K, Li W, Dong X. Association of 677 C>T (rs1801133) and 1298 A>C (rs1801131) polymorphisms in the MTHFR gene and breast cancer susceptibility: a meta-analysis based on 57 individual studies. PLoS ONE 9(6), e71290 (2014).

34 Diakite B, Tazzite A, Hamzi K, Jouhadi H, Nadifi S. Methylenetetrahydrofolate reductase C677T polymorphism and breast cancer risk in Moroccan women. Afr. Health Sci. 12(2), 204–209 (2012).

35 Awwad N, Yousef AM, Abuhaliema A, Abdalla I, Yousef M. Relationship between genetic polymorphisms in MTHFR (C677T, A1298C and their Haplotypes) and the incidence of breast cancer among Jordanian females – case–control study. Asian Pac. J. Cancer Prev. 16(12), 5007–5011 (2015).

36 Qi X, Ma X, Yang X et al. Methylenetetrahydrofolate reductase polymorphisms and breast cancer risk: a meta-analysis from 41 studies with 16,480 cases and 22,388 controls. Breast Cancer Res. Treat. 123(2), 499–506 (2010).

37 Xie H, Xia K, Rong H, Chen X. Genetic polymorphism in hOGG1 is associated with triple-negative breast cancer risk in Chinese Han women. Breast 22(5), 707–712 (2013).

38 Zhang N, Huo Q, Wang X et al. A genetic variant in p63 (rs17506395) is associated with breast cancer susceptibility and prognosis. Gene 535(2), 170–176 (2014).

39 Krześlak A, Forma E, Jóźwiak P et al. Metallothionein 2A genetic polymorphisms and risk of ductal breast cancer. Clin. Exp. Med. 14(1), 107–113 (2014).

40 Karakas B, Colak D, Kaya N et al. Prevalence of PIK3CA mutations and the SNP rs17849079 in Arab breast cancer patients. Cancer Biol. Ther. 14(10), 888–896 (2013).

41 Park HS, Jang MH, Kim EJ et al. High EGFR gene copy number predicts poor outcome in triple-negative breast cancer. Mod. Pathol. 27(9), 1212–1222 (2014).

42 Park YH, Jung HH, Do IG et al. A seven-gene signature can predict distant recurrence in patients with triple-negative breast cancers who receive adjuvant chemotherapy following surgery. Int. J. Cancer 136(8), 1976–1984 (2015).

43 Holm C, Rayala S, Jirström K, Stål O, Kumar R, Landberg G. Association between Pak1 expression and subcellular localization and tamoxifen resistance in breast cancer patients. J. Natl Cancer Inst. 98(10), 671–680 (2006).

44 Bostner J, Ahnström waltersson M, Fornander T, Skoog L, Nordenskjöld B, Stål O. Amplification of CCND1 and PAK1 as predictors of recurrence and tamoxifen resistance in postmenopausal breast cancer. Oncogene 26(49), 6997–7005 (2007).

45 Lu X, Mazur SJ, Lin T, Appella E, Xu Y. The pluripotency factor nanog promotes breast cancer tumorigenesis and metastasis. Oncogene 33(20), 2655–2664 (2014).

46 Wang D, Lu P, Zhang H et al. Oct-4 and Nanog promote the epithelial-mesenchymal transition of breast cancer stem



cells and are associated with poor prognosis in breast cancer patients. Oncotarget 5(21), 10803–10815 (2014).

47 Yin ZQ, Liu JJ, Xu YC et al. A 41-gene signature derived from breast cancer stem cells as a predictor of survival. J. Exp. Clin. Cancer Res. 33(1), 49 (2014).

48 Meldi KM, Figueroa ME. Epigenetic deregulation in myeloid malignancies. Transl. Res. 165(1), 102–114 (2015).

49 Jovanovic J, RØnneberg JA, Tost J, Kristensen V. The epigenetics of breast cancer. Mol. Oncol. 4(3), 242–254 (2010).

50 Locke WJ, Zotenko E, Stirzaker C et al. Coordinated epigenetic remodelling of transcriptional networks occurs during early breast carcinogenesis. Clin. Epigenetics 7(1), 52 (2015).

•• Summarizingepigeneticnetworksanddemonstratingbiologicalvalueandpotentialclinicalutilityofepigenome-widemapsforearlybreastcarcinogenesis.

51 Győrffy B, Bottai G, Fleischer T et al. Aberrant DNA methylation impacts gene expression and prognosis in breast cancer subtypes. Int. J. Cancer 138(1), 87–97 (2016).

52 Li Y, Melnikov AA, Levenson V et al. A seven-gene CpG-island methylation panel predicts breast cancer progression. BMC Cancer 15, 417 (2015).

53 Li Z, Guo X, Wu Y et al. Methylation profiling of 48 candidate genes in tumor and matched normal tissues from breast cancer patients. Breast Cancer Res. Treat. 149(3), 767–779 (2015).

54 Müller BM, Jana L, Kasajima A et al. Differential expression of histone deacetylases HDAC1, 2 and 3 in human breast cancer – overexpression of HDAC2 and HDAC3 is associated with clinicopathological indicators of disease progression. BMC Cancer 13(1), 215 (2013).

55 Seo J, Min SK, Park HR et al. Expression of histone deacetylases HDAC1, HDAC2, HDAC3, and HDAC6 in invasive ductal carcinomas of the breast. J. Breast Cancer 17(4), 323–331 (2014).

56 Park SY, Jun JA, Jeong KJ et al. Histone deacetylases 1, 6 and 8 are critical for invasion in breast cancer. Oncol. Rep. 25(6), 1677–1681 (2011).

57 Yokoyama Y, Matsumoto A, Hieda M et al. Loss of histone H4K20 trimethylation predicts poor prognosis in breast cancer and is associated with invasive activity. Breast Cancer Res. 16(3), R66 (2014).

58 Healey MA, Hu R, Beck AH et al. Association of H3K9me3 and H3K27me3 repressive histone marks with breast cancer subtypes in the Nurses’ Health Study. Breast Cancer Res. Treat. 147(3), 639–651 (2014).

59 Zhang L, Deng L, Chen F et al. Inhibition of histone H3K79 methylation selectively inhibits proliferation, self-renewal and metastatic potential of breast cancer. Oncotarget 5(21), 10665–10677 (2014).

60 Feng Q, Zhang Z, Shea MJ et al. An epigenomic approach to therapy for tamoxifen-resistant breast cancer. Cell Res. 24(7), 809–819 (2014).

61 Timms KM, Abkevich V, Hughes E et al. Association of BRCA1/2 defects with genomic scores predictive of DNA damage repair deficiency among breast cancer subtypes. Breast Cancer Res. 16(6), 475 (2014).

62 Gupta S, Jaworska-Bieniek K, Narod SA, Lubinski J, Wojdacz TK, Jakubowska A. Methylation of the BRCA1 promoter in peripheral blood DNA is associated with triple-negative and medullary breast cancer. Breast Cancer Res. Treat. 148(3), 615–622 (2014).

63 Xu Y, Diao L, Chen Y et al. Promoter methylation of BRCA1 in triple-negative breast cancer predicts sensitivity to adjuvant chemotherapy. Ann. Oncol. 24(6), 1498–1505 (2013).

64 Yamashita N, Tokunaga E, Kitao H et al. Epigenetic inactivation of BRCA1 through promoter hypermethylation and its clinical importance in triple-negative breast cancer. Clin. Breast Cancer 15(6), 498–504 (2015).

65 Ramos EA, Grochoski M, Braun-Prado K et al. Epigenetic changes of CXCR4 and its ligand CXCL12 as prognostic factors for sporadic breast cancer. PLoS ONE 6(12), e29461 (2011).

66 Wendt MK, Cooper AN, Dwinell MB. Epigenetic silencing of CXCL12 increases the metastatic potential of mammary carcinoma cells. Oncogene 27(10), 1461–1471 (2008).

67 Fridrichova I, Smolkova B, Kajabova V et al. CXCL12 and ADAM23 hypermethylation are associated with advanced breast cancers. Transl. Res. 165(6), 717–730 (2015).

68 Verbisck NV, Costa ET, Costa FF et al. ADAM23 negatively modulates alpha(v)beta(3) integrin activation during metastasis. Cancer Res. 69(13), 5546–5552 (2009).

69 Costa FF, Verbisck NV, Salim AC et al. Epigenetic silencing of the adhesion molecule ADAM23 is highly frequent in breast tumors. Oncogene 23(7), 1481–8 (2004).

70 Ramos EA, Camargo AA, Braun K et al. Simultaneous CXCL12 and ESR1 CpG island hypermethylation correlates with poor prognosis in sporadic breast cancer. BMC Cancer 10(1), 23 (2010).

71 Kloten V, Becker B, Winner K et al. Promoter hypermethylation of the tumor-suppressor genes ITIH5, DKK3, and RASSF1A as novel biomarkers for blood-based breast cancer screening. Breast Cancer Res. 15(1), R4 (2013).

72 Kuchiba A, Iwasaki M, Ono H et al. Global methylation levels in peripheral blood leukocyte DNA by LUMA and breast cancer: a case–control study in Japanese women. Br. J. Cancer 110(11), 2765–2771 (2014).

73 Hon GC, Hawkins RD, Caballero OL et al. Global DNA hypomethylation coupled to repressive chromatin domain formation and gene silencing in breast cancer. Genome Res. 22(2), 246–258 (2012).

74 Choi JY, James SR, Link PA et al. Association between global DNA hypomethylation in leukocytes and risk of breast cancer. Carcinogenesis 30(11), 1889–1897 (2009).

75 Legendre C, Gooden GC, Johnson K, Martinez RA, Liang WS, Salhia B. Whole-genome bisulfite sequencing of cell-free DNA identifies signature associated with metastatic breast cancer. Clin. Epigenetics 7(1), 100 (2015).

76 Bernardino J, Roux C, Almeida A et al. DNA hypomethylation in breast cancer: an independent parameter of tumor progression? Cancer Genet Cytogenet. 97(2), 83–89 (1997).

77 Krishnan P, Ghosh S, Wang B et al. Next generation sequencing profiling identifies miR-574-3p and miR-660-5p



as potential novel prognostic markers for breast cancer. BMC Genomics 16(1), 735 (2015).

78 Mcguire A, Brown JA, Kerin MJ. Metastatic breast cancer: the potential of miRNA for diagnosis and treatment monitoring. Cancer Metastasis Rev. 34(1), 145–155 (2015).

79 Takahashi RU, Miyazaki H, Ochiya T. The roles of microRNAs in breast cancer. Cancers (Basel). 7(2), 598–616 (2015).

•• SummarizingtheroleofmiRNApanelinbreastcancerdiagnosisandtreatmentconsideringthemechanismsoftumorinitiation,promotionofbreastcancermetastaticdiseaseanddrugresistanceaswellastargetingnewtherapies.

80 Chen J, Yao D, Li Y et al. Serum microRNA expression levels can predict lymph node metastasis in patients with early-stage cervical squamous cell carcinoma. Int. J. Mol. Med. 32(3), 557–567 (2013).

81 Zhang HL, Qin XJ, Cao DL et al. An elevated serum miR-141 level in patients with bone-metastatic prostate cancer is correlated with more bone lesions. Asian J. Androl. 15(2), 231–235 (2013).

82 Kim SY, Jeon TY, Choi CI et al. Validation of circulating miRNA biomarkers for predicting lymph node metastasis in gastric cancer. J. Mol. Diagn. 15(5), 661–669 (2013).

83 Toiyama Y, Hur K, Tanaka K et al. Serum miR-200c is a novel prognostic and metastasis-predictive biomarker in patients with colorectal cancer. Ann. Surg. 259(4), 735–743 (2014).

84 Ma L, Teruya-feldstein J, Weinberg RA. Tumour invasion and metastasis initiated by microRNA-10b in breast cancer. Nature 449(7163), 682–688 (2007).

85 Cizeron-clairac G, Lallemand F, Vacher S, Lidereau R, Bieche I, Callens C. MiR-190b, the highest up-regulated miRNA in ERα-positive compared with ERα-negative breast tumors, a new biomarker in breast cancers? BMC Cancer 15, 499 (2015).

86 Niu J, Xue A, Chi Y et al. Induction of miRNA-181a by genotoxic treatments promotes chemotherapeutic resistance and metastasis in breast cancer. Oncogene 35(10), 1302–1313 (2016).

87 Kodahl AR, Lyng MB, Binder H et al. Novel circulating microRNA signature as a potential non-invasive multi-marker test in ER-positive early-stage breast cancer: a case control study. Mol. Oncol. 8(5), 874–883 (2014).

88 Lin X, Chen L, Yao Y et al. CCL18-mediated down-regulation of miR98 and miR27b promotes breast cancer metastasis. Oncotarget 6(24), 20485–20499 (2015).

89 Krishnan P, Ghosh S, Wang B et al. Next generation sequencing profiling identifies miR-574-3p and miR-660-5p as potential novel prognostic markers for breast cancer. BMC Genomics 16(1), 735 (2015).

90 Wu Q, Lu Z, Li H, Lu J, Guo L, Ge Q. Next-generation sequencing of microRNAs for breast cancer detection. J. Biomed. Biotechnol. 2011, 597145 (2011).

91 Erbes T, Hirschfeld M, Rücker G et al. Feasibility of urinary microRNA detection in breast cancer patients and its potential as an innovative non-invasive biomarker. BMC Cancer 15, 193 (2015).

92 Mishra S, Srivastava AK, Suman S, Kumar V, Shukla Y. Circulating miRNAs revealed as surrogate molecular signatures for the early detection of breast cancer. Cancer Lett. 369(1), 67–75 (2015).

93 Kinkorová J. Biobanks in the era of personalized medicine: objectives, challenges, and innovation. EPMA J. 7, 1 (2015).

94 Christodoulatos GS, Dalamaga M. Micro-RNAs as clinical biomarkers and therapeutic targets in breast cancer: quo vadis? World J. Clin. Oncol. 5(2), 71–81 (2014).

95 Fkih M’hamed I, Privat M, Ponelle F, Penault-llorca F, Kenani A, Bignon YJ. Identification of miR-10b, miR-26a, miR-146a and miR-153 as potential triple-negative breast cancer biomarkers. Cell. Oncol. (Dordr.). 38(6), 433–442 (2015).

96 Taylor MA, Sossey-Alaoui K, Thompson CL, Danielpour D, Schiemann WP. TGF-β upregulates miR-181a expression to promote breast cancer metastasis. J. Clin. Invest. 123(1), 150–163 (2013).

97 Guo LJ, Zhang QY. Decreased serum miR-181a is a potential new tool for breast cancer screening. Int. J. Mol. Med. 30(3), 680–686 (2012).

98 Miller TE, Ghoshal K, Ramaswamy B et al. MicroRNA-221/222 confers tamoxifen resistance in breast cancer by targeting p27Kip1. J. Biol. Chem. 283(44), 29897–29903 (2008).

99 Wei Y, Lai X, Yu S et al. Exosomal miR-221/222 enhances tamoxifen resistance in recipient ER-positive breast cancer cells. Breast Cancer Res. Treat. 147(2), 423–431 (2014).

100 Adachi R, Horiuchi S, Sakurazawa Y, Hasegawa T, Sato K, Sakamaki T. ErbB2 down-regulates microRNA-205 in breast cancer. Biochem. Biophys. Res. Commun. 411(4), 804–808 (2011).

101 Bockhorn J, Yee K, Chang YF et al. MicroRNA-30c targets cytoskeleton genes involved in breast cancer cell invasion. Breast Cancer Res. Treat. 137(2), 373–382 (2013).

102 Dobson JR, Taipaleenmäki H, Hu YJ et al. hsa-mir-30c promotes the invasive phenotype of metastatic breast cancer cells by targeting NOV/CCN3. Cancer Cell Int. 14(1), 73 (2014).

103 Ebrahimi F, Gopalan V, Smith RA, Lam AK. miR-126 in human cancers: clinical roles and current perspectives. Exp. Mol. Pathol. 96(1), 98–107 (2014).

104 Wang CZ, Yuan P, Li Y. MiR-126 regulated breast cancer cell invasion by targeting ADAM9. Int. J. Clin. Exp. Pathol. 8(6), 6547–6553 (2015).

105 Zhang Y, Yang P, Sun T et al. miR-126 and miR-126* repress recruitment of mesenchymal stem cells and inflammatory monocytes to inhibit breast cancer metastasis. Nat. Cell. Biol. 15(3), 284–294 (2013).

106 Shinden Y, Iguchi T, Akiyoshi S et al. miR-29b is an indicator of prognosis in breast cancer patients. Mol. Clin. Oncol. 3(4), 919–923 (2015).

107 Song B, Wang C, Liu J et al. MicroRNA-21 regulates breast cancer invasion partly by targeting tissue inhibitor of metalloproteinase 3 expression. J. Exp. Clin. Cancer Res. 29(1), 29 (2010).

108 Wang Y, Zhang Y, Pan C, Ma F, Zhang S. Prediction of poor prognosis in breast cancer patients based on microRNA-21



expression: a meta-analysis. PLoS ONE 10(2), e0118647 (2010).

109 Pan F, Mao H, Deng L, Li G, Geng P. Prognostic and clinicopathological significance of microRNA-21 overexpression in breast cancer: a meta-analysis. Int. J. Clin. Exp. Pathol. 7(9), 5622–5633 (2014).

110 Si ML, Zhu S, Wu H, Lu Z, Wu F, Mo YY. miR-21-mediated tumor growth. Oncogene 26(19), 2799–2803 (2007).

111 Si H, Sun X, Chen Y et al. Circulating microRNA-92a and microRNA-21 as novel minimally invasive biomarkers for primary breast cancer. J. Cancer Res. Clin. Oncol. 139(2), 223–229 (2013).

112 Qian B, Katsaros D, Lu L et al. High miR-21 expression in breast cancer associated with poor disease-free survival in early stage disease and high TGF-beta1. Breast Cancer Res. Treat. 117(1), 131–140 (2009).

113 Zeng H, Fang C, Nam S, Cai Q, Long X. The clinicopathological significance of microRNA-155 in breast cancer: a meta-analysis. Biomed. Res. Int. 2014, 724209 (2014).

114 Kong W, He L, Richards EJ et al. Upregulation of miRNA-155 promotes tumour angiogenesis by targeting VHL and is associated with poor prognosis and triple-negative breast cancer. Oncogene 33(6), 679–689 (2014).

115 Zheng SR, Guo GL, Zhang W et al. Clinical significance of miR-155 expression in breast cancer and effects of miR-155 ASO on cell viability and apoptosis. Oncol. Rep. 27(4), 1149–1155 (2012).

116 Erbes T, Hirschfeld M, Rücker G et al. Feasibility of urinary microRNA detection in breast cancer patients and its potential as an innovative non-invasive biomarker. BMC Cancer 15, 193 (2015).

117 Shaker O, Maher M, Nassar Y, Morcos G, Gad Z. Role of microRNAs -29b-2, -155, -197 and -205 as diagnostic biomarkers in serum of breast cancer females. Gene 560(1), 77–82 (2015).

118 Zhang CM, Zhao J, Deng HY. MiR-155 promotes proliferation of human breast cancer MCF-7 cells through targeting tumor protein 53-induced nuclear protein 1. J. Biomed. Sci. 20(1), 79 (2013).

119 Mattiske S, Suetani RJ, Neilsen PM, Callen DF. The oncogenic role of miR-155 in breast cancer. Cancer Epidemiol. Biomarkers Prev. 21(8), 1236–1243 (2012).

120 Liu J, Mao Q, Liu Y, Hao X, Zhang S, Zhang J. Analysis of miR-205 and miR-155 expression in the blood of breast cancer patients. Chin. J. Cancer Res. 25(1), 46–54 (2013).

121 Jung EJ, Santarpia L, Kim J et al. Plasma microRNA 210 levels correlate with sensitivity to trastuzumab and tumor presence in breast cancer patients. Cancer 118(10), 2603–2614 (2012).

122 Rothé F, Ignatiadis M, Chaboteaux C et al. Global microRNA expression profiling identifies miR-210 associated with tumor proliferation, invasion and poor clinical outcome in breast cancer. PLoS ONE 6(6), e20980 (2011).

123 Toyama T, Kondo N, Endo Y et al. High expression of microRNA-210 is an independent factor indicating a poor

prognosis in Japanese triple-negative breast cancer patients. Jpn J. Clin. Oncol. 42(4), 256–263 (2012).

124 Hong L, Yang J, Han Y, Lu Q, Cao J, Syed L. High expression of miR-210 predicts poor survival in patients with breast cancer: a meta-analysis. Gene 507(2), 135–138 (2012).

125 Pérez-Rivas LG, Jerez JM, Carmona R et al. A microRNA signature associated with early recurrence in breast cancer. PLoS ONE 9(3), e91884 (2014).

126 Debald M, Yeghiazaryan K, Cebioglu M, Kuhn W, Schild HH, Golubnitschaja O. ‘Suspect molecular signature’ in blood as the indicator of undiagnosed breast cancer, cancer risk and targeted prevention. EPMA J. 4(1), 22 (2013).

•• Demonstratingclinicalutilityofmultileveldiagnosticsandprovidingrecommendationsforimplementationofbiomarkerpanelsbyinnovativeminimal-invasivebloodanalysis.

127 Coumans JV, Gau D, Poljak A, Wasinger V, Roy P, Moens PD. Profilin-1 overexpression in MDA-MB-231 breast cancer cells is associated with alterations in proteomics biomarkers of cell proliferation, survival, and motility as revealed by global proteomics analyses. OMICS 18(12), 778–791 (2014).

128 Ding Z, Joy M, Bhargava R et al. Profilin-1 downregulation has contrasting effects on early vs late steps of breast cancer metastasis. Oncogene 33(16), 2065–2074 (2014).

129 Zou L, Ding Z, Roy P. Profilin-1 overexpression inhibits proliferation of MDA-MB-231 breast cancer cells partly through p27kip1 upregulation. J. Cell. Physiol. 223(3), 623–629 (2010).

130 Bae YH, Ding Z, Zou L, Wells A, Gertler F, Roy P. Loss of profilin-1 expression enhances breast cancer cell motility by Ena/VASP proteins. J. Cell. Physiol. 219(2), 354–364 (2009).

131 Sakwe AM, Koumangoye R, Guillory B, Ochieng J. Annexin A6 contributes to the invasiveness of breast carcinoma cells by influencing the organization and localization of functional focal adhesions. Exp. Cell. Res. 317(6), 823–837 (2011).

132 Karihtala P, Auvinen P, Kauppila S, Haapasaari KM, Jukkola-vuorinen A, Soini Y. Vimentin, zeb1 and Sip1 are up-regulated in triple-negative and basal-like breast cancers: association with an aggressive tumour phenotype. Breast Cancer Res. Treat. 138(1), 81–90 (2013).

133 Wu QW, Yang QM, Huang YF et al. Expression and clinical significance of matrix metalloproteinase-9 in lymphatic invasiveness and metastasis of breast cancer. PLoS ONE 9(5), e97804 (2014).

134 Heo DS, Choi H, Yeom MY, Song BJ, Oh SJ. Serum levels of matrix metalloproteinase-9 predict lymph node metastasis in breast cancer patients. Oncol. Rep. 31(4), 1567–1572 (2014).

135 Yousef EM, Tahir MR, St-Pierre Y, Gaboury LA. MMP-9 expression varies according to molecular subtypes of breast cancer. BMC Cancer 14(1), 609 (2014).

136 Liu NQ, Stingl C, Look MP et al. Comparative proteome analysis revealing an 11-protein signature for aggressive triple-



negative breast cancer. J. Natl Cancer Inst. 106(2), djt376 (2014).

137 Schirosi L, De Summa S, Tommasi S et al. Immunoprofile from tissue microarrays to stratify familial breast cancer patients. Oncotarget 6(29), 27865–27879 (2015).

138 Tenori L, Oakman C, Morris PG et al. Serum metabolomic profiles evaluated after surgery may identify patients with oestrogen receptor negative early breast cancer at increased risk of disease recurrence. Results from a retrospective study. Mol. Oncol. 9(1), 128–139 (2015).

139 Kim SK, Jung WH, Koo JS. Differential expression of enzymes associated with serine/glycine metabolism in different breast cancer subtypes. PLoS ONE 9(6), e101004 (2014).

140 Antonov A, Agostini M, Morello M, Minieri M, Melino G, Amelio I. Bioinformatics analysis of the serine and glycine pathway in cancer cells. Oncotarget 5(22), 11004–11013 (2014).

141 Cao MD, Lamichhane S, Lundgren S et al. Metabolic characterization of triple negative breast cancer. BMC Cancer 14(1), 941 (2014).

142 Cui Y, Shu XO, Gao YT et al. Urinary prostaglandin E2 metabolite and breast cancer risk. Cancer Epidemiol. Biomarkers Prev. 23(12), 2866–2873 (2014).

143 Fawzy MS, Aly NM, Shalaby SM, El-Sawy WH, Abdul-Maksoud RS. Cyclooxygenase-2 169C>G and 8473T>C gene polymorphisms and prostaglandin E2 level in breast cancer: a case–control study. Gene 527(2), 601–605 (2013).

144 Budhu A, Terunuma A, Zhang G, Hussain SP, Ambs S, Wang XW. Metabolic profiles are principally different between cancers of the liver, pancreas and breast. Int. J. Biol. Sci. 10(9), 966–972 (2014).

145 Budczies J, Pfitzner BM, Györffy B et al. Glutamate enrichment as new diagnostic opportunity in breast cancer. Int. J. Cancer 136(7), 1619–1628 (2015).

146 Budczies J, Brockmöller SF, Müller BM et al. Comparative metabolomics of estrogen receptor positive and estrogen receptor negative breast cancer: alterations in glutamine and beta-alanine metabolism. J. Proteomics 94, 279–288 (2013).

147 Van geldermalsen M, Wang Q, Nagarajah R et al. ASCT2/SLC1A5 controls glutamine uptake and tumour growth in triple-negative basal-like breast cancer. Oncogene doi:10.1038/onc.2015.381 (2015) (Epub ahead of print).

148 McGuirk S, Gravel SP, Deblois G et al. PGC-1α supports glutamine metabolism in breast cancer. Cancer Metab. 1(1), 22 (2013).

149 Drabovich AP, Pavlou MP, Dimitromanolakis A, Diamandis EP. Quantitative analysis of energy metabolic pathways in MCF-7 breast cancer cells by selected reaction monitoring assay. Mol. Cell. Proteomics 11(8), 422–434 (2012).

150 Reader J, Holt D, Fulton A. Prostaglandin E2 EP receptors as therapeutic targets in breast cancer. Cancer Metastasis Rev. 30(3–4), 449–463 (2011).

151 Yin K. Positive correlation between expression level of mitochondrial serine hydroxymethyltransferase and breast cancer grade. Onco Targets Ther. 8, 1069–1074 (2015).

152 Pesta M, Kulda V, Narsanska A, Fichtl J, Topolcan O. May CTC technologies promote better cancer management? EPMA J. 6(1), 1 (2015).

153 Wallwiener M, Hartkopf AD, Baccelli I et al. The prognostic impact of circulating tumor cells in subtypes of metastatic breast cancer. Breast Cancer Res. Treat. 137(2), 503–510 (2013).

154 Pearl ML, Dong H, Tulley S et al. Treatment monitoring of patients with epithelial ovarian cancer using invasive circulating tumor cells (iCTCs). Gynecol. Oncol. 137(2), 229–238 (2015).

155 Torphy RJ, Tignanelli CJ, Kamande JW et al. Circulating tumor cells as a biomarker of response to treatment in patient-derived xenograft mouse models of pancreatic adenocarcinoma. PLoS ONE 9(2), e89474 (2014).

156 Hartkopf AD, Wagner P, Wallwiener D, Fehm T, Rothmund R. Changing levels of circulating tumor cells in monitoring chemotherapy response in patients with metastatic breast cancer. Anticancer Res. 31(3), 979–984 (2011).

157 Tinhofer I, Konschak R, Stromberger C et al. Detection of circulating tumor cells for prediction of recurrence after adjuvant chemoradiation in locally advanced squamous cell carcinoma of the head and neck. Ann. Oncol. 25(10), 2042–2047 (2014).

158 Beije N, Jager A, Sleijfer S. Circulating tumor cell enumeration by the CellSearch system: the clinician’s guide to breast cancer treatment? Cancer Treat. Rev. 41(2), 144–150 (2015).

159 Wang HY, Ahn S, Kim S et al. Detection of circulating tumor cells in patients with breast cancer using the quantitative RT-PCR assay for monitoring of therapy efficacy. Exp. Mol. Pathol. 97(3), 445–452 (2014).

160 Galletti G, Portella L, Tagawa ST, Kirby BJ, Giannakakou P, Nanus DM. Circulating tumor cells in prostate cancer diagnosis and monitoring: an appraisal of clinical potential. Mol. Diagn. Ther. 18(4), 389–402 (2014).

161 Resel Folkersma L, San José Manso L, Galante Romo I, Moreno Sierra J, Olivier Gómez C. Prognostic significance of circulating tumor cell count in patients with metastatic hormone-sensitive prostate cancer. Urology 80(6), 1328–1332 (2012).

162 Tognela A, Spring KJ, Becker T et al. Predictive and prognostic value of circulating tumor cell detection in lung cancer: a clinician’s perspective. Crit. Rev. Oncol. Hematol. 93(2), 90–102 (2015).

163 Fu L, Liu F, Fu H et al. Circulating tumor cells correlate with recurrence in stage III small-cell lung cancer after systemic chemoradiotherapy and prophylactic cranial irradiation. Jpn J. Clin. Oncol. 44(10), 948–955 (2014).

164 Tsujiura M, Ichikawa D, Konishi H, Komatsu S, Shiozaki A, Otsuji E. Liquid biopsy of gastric cancer patients: circulating tumor cells and cell-free nucleic acids. World J. Gastroenterol. 20(12), 3265–3286 (2014).

165 Toyoshima K, Hayashi A, Kashiwagi M et al. Analysis of circulating tumor cells derived from advanced gastric cancer. Int. J. Cancer 137(4), 991–998 (2015).

166 Uenosono Y, Arigami T, Kozono T et al. Clinical significance of circulating tumor cells in peripheral blood from patients with gastric cancer. Cancer 119(22), 3984–3991 (2013).



167 Allard WJ, Matera J, Miller MC et al. Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant diseases. Clin. Cancer Res. 10(20), 6897–6904 (2004).

168 Wallwiener M, Riethdorf S, Hartkopf AD et al. Serial enumeration of circulating tumor cells predicts treatment response and prognosis in metastatic breast cancer: a prospective study in 393 patients. BMC Cancer 14(1), 512 (2014).

169 Yokobori T, Iinuma H, Shimamura T et al. Plastin3 is a novel marker for circulating tumor cells undergoing the epithelial-mesenchymal transition and is associated with colorectal cancer prognosis. Cancer Res. 73(7), 2059–2069 (2013).

170 Ueo H, Sugimachi K, Gorges TM et al. Circulating tumour cell-derived plastin3 is a novel marker for predicting long-term prognosis in patients with breast cancer. Br. J. Cancer 112(9), 1519–1526 (2015).

171 Maltoni R, Fici P, Amadori D et al. Circulating tumor cells in early breast cancer: a connection with vascular invasion. Cancer Lett. 367(1), 43–48 (2015).

172 Nadal R, Ortega FG, Salido M et al. CD133 expression in circulating tumor cells from breast cancer patients: potential role in resistance to chemotherapy. Int. J. Cancer 133(10), 2398–2407 (2013).

173 Clarke MF, Dick JE, Dirks PB et al. Cancer stem cells--perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res. 66(19), 9339–9344 (2006).

174 Mannello F. Understanding breast cancer stem cell heterogeneity: time to move on to a new research paradigm. BMC Med. 11(1), 169 (2013).

175 Al-hajj M, Becker MW, Wicha M, Weissman I, Clarke MF. Therapeutic implications of cancer stem cells. Curr. Opin. Genet. Dev. 14(1), 43–47 (2004).

176 Wicha MS. Cancer stem cells and metastasis: lethal seeds. Clin. Cancer Res. 12(19), 5606–7 (2006).

177 Visvader JE, Lindeman GJ. Cancer stem cells: current status and evolving complexities. Cell Stem Cell 10(6), 717–728 (2012).

178 Shackleton M, Quintana E, Fearon ER, Morrison SJ. Heterogeneity in cancer: cancer stem cells versus clonal evolution. Cell 138(5), 822–829 (2009).

179 Dick JE. Stem cell concepts renew cancer research. Blood 112(13), 4793–4807 (2008).

180 Chen X, Zhang J, Zhang Z, Li H, Cheng W, Liu J. Cancer stem cells, epithelial-mesenchymal transition, and drug resistance in high-grade ovarian serous carcinoma. Hum. Pathol. 44(11), 2373–2384 (2013).

181 Li X, Lewis MT, Huang J et al. Intrinsic resistance of Wigenic breast cancer cells to chemotherapy. J. Natl Cancer Inst. 100(9), 672–679 (2008).

182 Chuthapisith S, Eremin J, El-Sheemey M, Eremin O. Breast cancer chemoresistance: emerging importance of cancer stem cells. Surg. Oncol. 19(1), 27–32 (2010).

183 Wang N, Wang Z, Peng C et al. Dietary compound isoliquiritigenin targets GRP78 to chemosensitize breast cancer stem cells via β-catenin/ABCG2 signaling. Carcinogenesis 35(11), 2544–2554 (2014).

184 Wang Z, Wang N, Li W et al. Caveolin-1 mediates chemoresistance in breast cancer stem cells via β-catenin/ABCG2 signaling pathway. Carcinogenesis 35(10), 2346–2356 (2014).

185 Saha S, Mukherjee S, Mazumdar M et al. Mithramycin A sensitizes therapy-resistant breast cancer stem cells toward genotoxic drug doxorubicin. Transl. Res. 165(5), 558–577 (2015).

186 Bozorgi A, Khazaei M, Khazaei MR. New findings on breast cancer stem cells: a review. J. Breast Cancer 18(4), 303–312 (2015).

187 Duru N, Fan M, Candas D et al. HER2-associated radioresistance of breast cancer stem cells isolated from HER2-negative breast cancer cells. Clin. Cancer Res. 18(24), 6634–6647 (2012).

188 Peitzsch C, Kurth I, Kunz-Schughart L, Baumann M, Dubrovska A. Discovery of the cancer stem cell related determinants of radioresistance. Radiother. Oncol. 108(3), 378–387 (2013).

189 Guo W. Concise review: breast cancer stem cells: regulatory networks, stem cell niches, and disease relevance. Stem Cells Transl. Med. 3(8), 942–948 (2014).

190 Camerlingo R, Ferraro GA, De Francesco F et al. The role of CD44+/CD24-/low biomarker for screening, diagnosis and monitoring of breast cancer. Oncol. Rep. 31(3), 1127–1132 (2014).

191 Ricardo S, Vieira AF, Gerhard R et al. Breast cancer stem cell markers CD44, CD24 and ALDH1: expression distribution within intrinsic molecular subtype. J. Clin. Pathol. 64(11), 937–946 (2011).

192 Kang EJ, Jung H, Woo OH et al. Association of aldehyde dehydrogenase 1 expression and biologically aggressive features in breast cancer. Neoplasma 61(3), 352–362 (2014).

193 Yeghiazaryan K, Cebioglu M, Braun M, Kuhn W, Schild HH, Golubnitschaja O. Noninvasive subcellular imaging in breast cancer risk assessment: construction of diagnostic windows. Pers. Med. 8(3), 321–330 (2011).

•• Presentinginnovativetechnologyforbreastcancerriskassessmentandprovidingrecommendationsforclinicalapplication.

194 Grech G, Zhan X, Yoo BC et al. EPMA position paper in cancer: current overview and future perspectives. EPMA J. 6(1), 9 (2015).

195 Golubnitschaja O, Costigliola V. General report & recommendations in predictive, preventive and personalised medicine 2012: white paper of the European Association for Predictive, Preventive and Personalised Medicine. EPMA J. 3(1), 14 (2012).

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REVIEW

Management of women with a hereditary predisposition for breast cancer

Ismail Jatoi*,1 & John R Benson2

1Division of Surgical Oncology & Endocrine Surgery, University of Texas Health Science Center, San Antonio, TX, USA 2Addenbrookes Hospital, Cambridge University, Cambridge, UK

*Author for correspondence: [email protected]

Women with a hereditary breast cancer predisposition have three management options: screening, chemoprevention (risk-reducing medication) and risk-reducing surgery. However, no randomized trials have addressed the effect of these strategies in mutation carriers. In the general population, randomized trials failed to demonstrate a benefit for screening in premenopausal women. Moreover, although chemoprevention reduces breast cancer incidence in high-risk populations, this benefit is potentially confined to estrogen receptor-positive tumors. Finally, observational studies suggest that prophylactic mastectomy and even prophylactic salpingo-ophorectomy reduces breast cancer risk in BRCA mutation carriers, but there are systematic biases associated with such studies. Therefore, women with a hereditary predisposition for breast cancer should be informed of the three risk-reducing strategies, and that their benefits are not fully understood.

First submitted: 11 April 2016; Accepted for publication: 14 June 2016; Published online: 7 July 2016

KEYWORDS • breast cancer • chemoprevention • genetic testing • hereditary predisposition • prophylactic surgery • screening

Hereditary predisposition indicates innately increased susceptibility to development of a disease based on genetic makeup and in particular specific germline mutations passed from one generation to another [1]. These genetic changes are not necessarily causative and phenotypic manifestations of cancer are variable. In recent years there has been an upsurge of interest in identifying women with genetic predisposition and this has been facilitated by enhanced understanding of the genetic basis for breast cancer susceptibility derived from genome-wide linkage analysis and mutational screening [2]. The focus has shifted from high-penetrance genes to intermediate- and low-penetrance mutations that confer lower levels of risk individually but collectively are more significant [3]. There has been frenzied public interest in genetic testing and risk reduction strategies following revelation in the New York Times in May 2013 that the famed actress Angelina Jolie had chosen to undergo bilateral prophylactic mastectomy (BPM) due to carriage of a BRCA1 gene mutation [4]. Genetic counseling and testing for breast cancer predisposition has been widely implemented in many coun-tries and the number of women seeking gene testing continues to rise. Nonetheless, despite these advances in genetics, approximately 30% of the familial breast cancer risk remains unaccounted for by mutations in currently known genes [5].

Risk factors for breast cancerThe three major risk factors for development of breast cancer are gender, age and family history/genetic susceptibility [6,7]. Gender is the single greatest risk factor with breast cancer being


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100-fold more common in women than men [7,8]. Thus approximately 12.7% of all women in the USA will be diagnosed with breast cancer (one in eight) compared with a figure of only 0.1% for men [7]. The second most important risk factor is age, with disease incidence rising dramatically during the fourth and fifth decades but less so after age 50 years (Clemmenson’s hook) and evidence of incidence leveling off after the age of 75–80 years [8]. This effect of ageing is illus-trated by comparisons of estimated risk levels for younger versus older women. Thus for a 30-year-old woman, the risk of developing breast cancer during the next 10 years of life is 0.44% (one in 227) but the corresponding risk for a 70-year-old woman is 3.82% (one in 26) – a tenfold difference [8]. Finally, family history and genetic predisposition are associated with variable levels of risk depending on intensity of familial cluster-ing (numbers of first-degree relatives with early onset/bilateral breast cancer) and identification of specific gene mutations [9].

The etiology of breast cancer is multifactorial and the vast majority of cases are nonhereditary (sporadic) with no identifiable predisposing gene mutation [10]. Nonetheless, environmental and lifestyle factors are important determinants of risk which can potentially be modified to reduce overall risk for development of breast cancer [11]. Thus, maintaining a healthy weight, undertaking regular exercise, limiting alcohol intake, avoid-ing/cessation of smoking and participation in regular screening may potentially help minimize risk in those individuals with a genetic predispo-sition [12]. There is evidence that environmental and life-style factors can modify risk in muta-tion carriers; for example, a recent population-based study in Israel found that breast cancer risk amongst BRCA1 or BRCA2 mutation carri-ers was much higher for women born after 1958 when compared with those born prior to this date [13]. Changes in lifestyle are could poten-tially be responsible for these differences between birth cohorts. It is tempting to speculate that contemporary levels of obesity have increased breast cancer among mutation carriers. However, it is also important to note that this study relied on hospital records to discern whether family members were diagnosed with breast or ovar-ian cancer in cohorts born before 1958 and after 1958. Thus, it is also possible that the higher incidence rates in the later cohort might be due to differences in screening and diagnostic methods between these two cohorts.

●● High-risk predisposing genesBRCA1 & BRCA2Although many individuals may possess a rela-tive with breast cancer and therefore by defini-tion have a positive family history for the disease, only about 10% of all breast cancers are currently attributable to germline mutations [2]. Of par-ticular relevance are the high-penetrance muta-tions, and these have an autosomal dominant pattern of inheritance consistent with vertical transmission of high-risk genetic predisposition from one generation to another [9]. The precise level of risk and the probability of harboring a mutation in a recognized high-penetrance gene is determined by family history of breast (and some other) cancers [2]. A method termed link-age analysis was previously used to calculate life-time risk for development of breast cancer based on information from family cancer history such as the number of affected first and second degree relatives with breast/ovarian cancer, age of onset and bilateral breast cancer [14]. Hereditary pre-disposition occurs in only about 10% of all cases, and is conferred by cancer susceptibil-ity genes that conform to Knudson’s model or ‘two-hit hypothesis’ [15,16]. Approximately half of mutations in hereditary breast cancer involve the breast cancer genes BRCA1 and BRCA2 [2]. These are large tumor suppressor genes located on chromosomes 17q21 and 13q12 respectively and mutated in approximately 1/300 individu-als [17]. Among BRCA1 mutation carriers, the average cumulative risks for breast and ovarian cancer by 80 years of age are about 67 and 45% respectively, while for BRCA2 mutation carri-ers, the average cumulative risks for breast and ovarian cancer are 66 and 12%, respectively [2]. However, there is considerable variation in reported magnitude of risk for breast and ovar-ian cancer among BRCA1 and BRCA2 mutation carriers with risk partly dependent on precise location of the mutation within the gene [18]. Initial estimates of risk were based on studies of selected high-risk families whilst more recent estimates are derived from population-based studies and these are generally associated with lower levels of risk.

At the cellular level, the effects of BRCA1 and BRCA2 are recessive and both copies of an allele must be lost or mutated for cancer to develop. Individuals with a germline mutation in these genes have a dominantly inherited susceptibil-ity and the second hit occurs in the somatic copy. Over 75% of breast cancers that develop

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in BRCA1 mutation carriers are estrogen recep-tor (ER)-negative, and 69% are triple-negative (ER-negative, progesterone receptor-negative and EGFR2-negative) [2]. By contrast, 77% of breast cancers in BRCA2 mutation carriers are ER-positive, and only 16% are triple-negative [2]. Women with triple-negative breast cancer under the age of 50 years have a much greater likelihood of harboring a BRCA1 gene muta-tion, potentially justifying screening for BRCA mutations [19].

It should also be noted that after an initial diagnosis of unilateral breast cancer, BRCA1 and BRCA2 mutation carriers have an elevated risk of developing contralateral breast cancer and this has therapeutic implications [20]. For these patients, bilateral mastectomy should be considered as an option [20].

Other high-risk genesBesides BRCA1 and BRCA2 mutations, there are several other gene mutations that dramati-cally increase the risk of developing breast cancer (high-penetrance mutations). These include mutations in the STKII (Peutz-Jeghers syndrome), pTEN (Cowden’s syndrome), TP53 (Li-Fraumeni syndrome), CDH1 (diffuse heredi-tary gastric cancer syndrome) and PALB2 (part-ner and localizer of BRCA2) genes [9,21]. These mutations are generally associated with other malignancies and disease manifestations. Thus, the STK11 mutation is associated with gastro-intestinal polyposis and breast cancer, and the pTEN mutation with thyroid cancer and breast cancer. The TP3 mutation is associated with numerous other cancers such as sarcomas, brain tumors and gastrointestinal tumors in addition to breast cancer. The CDH1 mutation is linked with gastric cancer and invasive lobular breast cancer and the PALB2 mutation with breast can-cer. No further high-penetrance genes have been identified from genome-wide linkage studies and it seems unlikely that any other major genes in this class exist [22]. Mutations of TP53 are com-mon in many cancers and promote tumor growth by removing a ‘brake’ on cell proliferation [23]. Germline TP53 mutations are associated with a lifetime risk for development of breast cancer in excess of 50% [24]. The PALB2 gene encodes for a protein that interacts with the BRCA2 gene product to repair damaged DNA and maintain fidelity of DNA replication [21]. Mutations of the PALB2 gene are associated with a breast cancer risk of 35–40% by age 70 years which is slightly

lower than for BRCA2 mutations [21]. The pTEN gene is also involved in growth regulation and mutations confer a more variable level of risk ranging from 20 to 50% [25].

●● Low/intermediate-risk predisposing genesIn addition to the BRCA1 and BRCA2 muta-tions, the high-penetrant gene mutations dis-cussed above individually confer high lifetime risks (generally greater than 50%), but collec-tively these mutations are estimated to account for no more than 20–25% of the familial aggre-gation of breast cancer [26,27]. Much of the resid-ual unexplained familial cancer risk is likely to be due to low- and moderate-penetrance muta-tions [27]. Technological advances and mapping of single nucleotide polymorphisms that are common genetic variants has permitted compre-hensive analyses with identification of potential candidate genes such as CHEK2, ATM, FGFR and TNCR9 [28]. CHEK2 and ATM are con-sidered moderate-risk gene mutations whereas mutations in the FGFR and TNCR9 genes con-fer low risk. Protein-truncating mutations in CHEK2 confer a lifetime breast cancer risk of about 20–30% but probably contribute no more than 3% to familial relative risk [29].

Genetic testing & risk predictionBreast cancer risk assessments falls into two dis-tinct categories: assessment of the risk of car-rying a mutation that increases breast cancer risk, and assessment of breast cancer risk with or without such a mutation [30]. Genetic testing represents a great advance in provision of accu-rate risk quantitation. Once a mutation has been identified for a particular family, that individual has a very high chance of developing breast can-cer without some form of intervention. Herein lies a potential dilemma; when women consent to genetic testing, they must be counseled appro-priately and able to cope with the information gained from the test – whatever the result [31]. Women who wish to consider genetic testing should be referred to genetic counselors and a detailed family history obtained to determine whether genetic testing is warranted and which specific mutations should be sought. Counseling should take account of broader issues such as personal medical history as well as ethnicity and cultural influences. NICE has recently recom-mended that women undergo genetic testing when the chance of finding a high-penetrance mutation is 10% (reduced from the previous

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mandate of 20%) [32]. Most women overestimate their risk and genetic testing allows accurate risk assessment that more confidently informs any proposed management decisions [33].

Three categories of mutation are recognized – positive (deleterious) pathogenic mutations, no mutations and variations of uncertain clinical significance. When a variation of uncertain clinical significance is found on genetic test-ing, the associated cancer risk is unknown and therefore default back to indirect risk assess-ment is necessary using models such as Claus, Boadicea and Tyrer-Cuzik [30]. Testing with low-penetrance gene panels can create poten-tial clinical dilemmas. How will results of these tests lead to a change in management? Is the information useful? An individual may have a very strong family history of breast cancer, yet the panel is negative. Genetic testing should be offered to the person affected first; if a mutation, for example, in CHEK2 is found in the mother but not the individual, is their particular risk the same as the general population or a higher level of risk (20–30%) based on family history of breast/ovarian cancer (and determined by other as yet nonidentified gene mutations). There are no trials of low-penetrance genes and patients should be warned that there is much uncer-tainty regarding the significance of any result. It remains unclear whether newer techniques for genetic testing improve risk prediction as com-pared with already existing methods based on family history and empiric risk assessment tools. Ultimately, genetic testing must yield clinically actionable results. The challenge at present is how to maximize the yield of clinically action-able results whilst minimizing results that may confuse patients with little chance of benefit.

Management options for women with high-penetrance mutationsOnce a gene mutation is identified in an asymp-tomatic woman, three management options should be discussed:

● Screening;

● Chemoprevention (risk-reducing medication);

● Risk-reducing surgery.

Management of these high-risk women can be challenging and sometimes a woman may choose a combination of more than one option depending on personal circumstances and esti-mated levels of risk. For instance, a BRCA1

mutation carrier may elect to undergo breast cancer screening until she has had her children and completed breastfeeding. She may subse-quently opt for risk-reducing surgery targeted at both breasts and ovary (bilateral mastectomy with immediate reconstruction and bilateral salpingo-oophorectomy).

●● ScreeningAlthough no randomized trials have specifically addressed the efficacy of breast cancer screening in mutation carriers, there are trials that have examined mortality benefits of screening in the general population [34]. At total of nine trials have examined the effectiveness of mammogra-phy screening and there are two trials that have examined the efficacy of screening breast self-examination (BSE). The two trials on screening BSE were undertaken in Shanghai, China and St Petersburg, Russia, and showed that screen-ing BSE had no benefit in reducing breast can-cer mortality [35,36]. Additionally, two trials in India are currently assessing the potential value of screening clinical breast examination, and mortality data are eagerly awaited from these trials [37,38].

Meta-analyses of mammography screening trials demonstrate reductions in breast cancer-specific mortality of about 20% for the inter-vention group, with much of the benefit con-fined to women aged 50–69 years of age at trial entry [34]. It should be noted that most muta-tion carriers seek screening prior to age 50 years, and the efficacy of mammography screening in this age group has not been conclusively dem-onstrated. Indeed, two mammography screen-ing trials (Canadian National Breast Screening Study 1 and the UK Age trial) were specifically designed to assess the efficacy of mammographic screening in women below aged 50 years and neither showed any benefit [39,40]. Moreover, improvements in therapy are likely diminishing both absolute and relative benefits of population-based mammography screening [41]. This is con-sistent with the observed trends over time in the mammography screening trials. The first rand-omized trial of breast cancer screening (Health Insurance Plan, NY, USA) was initiated in 1963 and demonstrated that mammography screen-ing could reduce breast cancer mortality by about 25–30% [42]. Of note, adjuvant systemic therapy was not generally available to patients during the era of the Health Insurance Plan trial since which time there has been an incremental

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decrease in mortality reductions attributed to mammographic screening. This has occurred against a background of advances in technology and prompts the question of why the benefits of screening are decreasing in the contemporary era and whether enhanced screening techniques offering greater sensitivity can ever yield any mortality gains [41]. In the three most recent mammography screening trials (Canadian National Breast Screening Study I and II and the UK Age trial), adjuvant systemic therapy was widely available to patients and these trials failed to show any benefit from mammography screening [39,40].

Mammography screening is associated with ionizing radiation and mutation carriers may lack ability to effectively repair damaged DNA resulting from radiation exposure [43]. There is a theoretical concern that mammography screen-ing may potentially increase breast cancer risk in mutation carriers and that modalities of screen-ing such as MRI may therefore be more appro-priate [43]. Screening breast MRI is twice more sensitive for cancer detection than screening mammography and is not associated with ion-izing radiation [44]. Moreover, in a prospective cohort study among women with an elevated familial risk of breast cancer, the addition of screening mammography did not increase breast cancer detection rates when compared with screening MRI alone and the latter may there-fore represent the optimum screening s trategy for mutation carriers [45].

●● Chemoprevention (risk-reducing medication)This is a potential means of reducing breast cancer risk in mutation carriers, but once again there is no randomized trial data that has spe-cifically addressed the efficacy of breast cancer risk-reducing medication in mutation carriers. Nonetheless, these trials of risk-reducing medica-tion (Table 1) have addressed the benefits of these medications in women at high risk for breast cancer amongst whom there would be some BRCA mutation carriers. Placebo-controlled trials have evaluated both selective ER modula-tors in pre- and post-menopausal women and aromatase inhibitors in postmenopausal women. Trials of tamoxifen have revealed a reduction of about 50% (ranging from 31 to 67%) in the cumulative incidence of both invasive and noninvasive breast cancer at a median follow-up ranging from 96 to 158 months [46–49]. It

has not been fully established if the benefit of risk-reducing medication is confined to lower-ing risk of only ER-positive breast cancers in mutation carriers [50]. Risk reductions in excess of 50% were noted for BRCA2 but not BRCA1 mutation carriers in the NSABP P-1 study which may relate to ER positivity of tumors in BRCA2 mutation carriers, but there were very few known BRCA mutation carriers in this study [51] The International Breast Intervention Study (IBIS)-1 likewise randomized high-risk pre- and post-menopausal women to either tamoxifen or pla-cebo with some women having used hormone replacement therapy and about one-third having undergone hysterectomy [52]. At 20 years follow-up breast cancer incidence was significantly lower in the intervention group (7.8%) compared with the control arm (12.3%) with a sustained benefit from tamoxifen beyond the initial 5-year pulse of treatment. An update of the head-to-head comparison of tamoxifen and another selective ER modulator (raloxifene) in the STAR trial confirms that there is some attrition of efficacy of raloxifene relative to tamoxifen with a sig-nificantly increased risk of invasive cancer in the raloxifene arm [53]. Aromatase inhibitors (alone or sequence before/after tamoxifen) have been shown to have marginally improved antitumor efficacy than tamoxifen alone and are associated with notably greater reductions in contralateral cancer – up to 70% compared with 50% for tamoxifen [54]. Although aromatase inhibitors are associated with a lower risk of thromboem-bolism compared with tamoxifen, there is an increased risk of fractures and arthralgias. The potential side effects of aromatase inhibitors has led to some caution in pursuance of risk-reducing strategies based on these agents [55]. Nonetheless, based on positive results from the MAP3 trial that examined the benefit of exemes-tane (25 mg daily for 5 years) versus placebo in a randomized comparison of 4560 high-risk postmenopausal women, current guidelines for use of risk-reducing medications incorporate an aromatase inhibitor option (see Table 1) [56]. Thus tamoxifen (20 mg per day orally for 5 years) should be discussed as an option for risk reduc-tion in pre- and postmenopausal women, whilst a similar recommendation applies to raloxifene (60 mg per day orally for 5 years) for postmen-opausal women only. Furthermore, American Society of Clinical Oncology (ASCO) guide-lines now acknowledge the aromatase inhibitor exemestane (25 mg per day orally for 5 years)

Future Oncol. (2016) 12(19)2282



as an additional option for breast cancer risk re duction in postmenopausal women.

Although clinical trials have shown these agents can reduce the risk of developing breast cancer, no mortality benefit has been conclu-sively demonstrated. Moreover, as previously indicated, there remains some concern that these agents might only be effective in the primary prevention of ER-positive breast cancer and are perhaps ineffective in prevention of ER-negative disease. Thus, in the context of BRCA mutation carriers, these agents might be recommended for patients with BRCA2 mutations, but not for those who carry a BRCA1 abnormality. As mentioned above, approximately 77% of breast cancers in the BRCA2 cohort are ER positive (comparable to the general population) while three quarters of breast cancers developing in BRCA1 mutation carriers are ER negative and 69% are triple-negative breast cancer.

●● SurgeryFor women with a genetic predisposition to breast cancer, risk-reducing surgery has been associated with the greatest potential benefit in terms of decreasing the chance of develop-ing breast cancer. Bilateral prophylactic surgery seems to reduce the expected risk of breast can-cer by more than 90% among carriers of a gene mutation [44]. In a seminal retrospective study of more than 600 women at moderate to high risk who had undergone BPM, the incidence of breast cancer was reduced by 89.5% (p < 0.001) [57]. An established Gail model for risk estimation was used to predict the number of breast cancer cases expected in these two groups without surgical intervention. This together with several smaller studies have validated BPM as a management option for women at increased risk for develop-ment of breast cancer [44]. However, there are no randomized trials that have specifically exam-ined the effectiveness of risk-reducing surgery in

mutation carriers. Nonetheless, both prospec-tive and retrospective cohort studies indicate that BPM is associated with reduced breast cancer risk of more than 90% in BRCA1 and BRCA2 mutation carriers [10]. Moreover, among those women with a BRCA gene predisposition, bilateral prophylactic salpingo-oophorectomy (BSO) was associated with ovarian and other BRCA-related adnexal cancer risk reductions of between 80 and 96% [58]. It should be noted that BSO (rather than bilateral oophorectomy alone) is the recommended surgical procedure for risk-reduction in BRCA1 and BRCA2 mutation car-riers because these women are also at increased risk for development of fallopian tube cancers.

Systematic biases potentially threaten the validity of observational studies and several of these have relevance to studies on risk-reducing surgery. These include performance bias, detec-tion bias and selection bias [10]. The first of these may result if the performance of a specific risk-reducing operation is not confirmed in an objective manner (i.e., from checking of medical or surgical records) and investigators are reliant instead on self reports. Attrition bias may result if follow-up of patients who undergo risk-reducing surgery is different from the control group [59]. For example, patients having risk-reducing sur-gery might be discharged from further follow-up while nonsurgical patients might continue with regular follow-up and monitoring. This could lead to under-reporting of malignancies in the risk-reducing surgery group. Detection bias may result if outcomes are not assessed consensually for both groups in the study [60]. For example, women who undergo risk-reducing mastectomy are generally not referred for any subsequent breast imaging whilst controls continue to be screened. In consequence, detection of higher rates of occult cancers would be expected in the control group. Finally, the baseline characteris-tics of women undergoing risk-reducing surgery

Table 1. Trials of chemopreventive agents.

Study Women (n) Median follow-up (months) Relative risk (95% CI)†

NSABP P-1 13,338 84 0.38 (95% CI: 0.28–0.5)Royal Marsden 2471 158 0.6 (95% CI: 0.43–0.86)IBIS-I 7139 96 0.66 (95% CI: 0.5–0.87)Italian 5408 132 0.77 (95% CI: 0.51–1.16)STAR 19,747 81 1.24 (95% CI: 1.05–1.47)IBIS-II‡ 3864 60 0.47 (95% CI: 0.5–0.87)MAP3‡ 4560 35 0.35 (95% CI: 0.18–0.70)†Estrogen receptor-positive breast cancer. ‡Postmenopausal women.

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may differ from those who do not have surgi-cal intervention, thus introducing an element of selection bias. For example, women having risk-reducing surgery might be of higher socio-economic status with better access to health-care that can potentially influence measured outcomes. Collectively, these various sources of bias may lead to over-estimation of the benefit of risk-reducing surgery.

It is claimed that BSO not only reduces ovar-ian cancer risk, but also decreases breast can-cer risk by 50% among mutation carriers when undertaken during the premenopausal years [61]. This effect is presumably due to estrogen depri-vation and epidemiological studies have shown that for unselected patients, ovarian ablation reduces the risk of breast cancer in premeno-pausal women by up to two-thirds. When risk-reducing surgery is combined (BPM and BSO) the net reduction in risk of developing breast cancer is 95%. Once again, these estimates of benefit should be interpreted with caution and recent analyses have suggested that much of the apparent benefit of BSO on breast cancer risk might be accounted for by selection bias rather than being a true reduction in risk [62]. However, others continue to argue that BSO does indeed have benefit in reducing breast cancer risk in BRCA mutation carriers [63]. Nonetheless, oophorectomy should not be recommended specifically to reduce breast cancer risk (in con-junction with surveillance imaging) and risk-reducing mastectomy should remain the opti-mal approach for minimizing risk of developing breast cancer in BRCA1 and BRCA2 mutation carriers.

Notwithstanding these limitations of observa-tional studies, it is highly unlikely that there will ever be randomized evidence available to assess the efficacy of risk-reducing surgery in muta-tion carriers. The decision to proceed with risk-reducing surgery is greatly influenced by an indi-vidual’s personal beliefs and values and a woman is unlikely to relinquish her right to self-destiny in this respect. Therefore future management strategies will be based on observational studies that have consistently shown huge benefits in terms of risk-reduction from surgical ablation.

Contralateral prophylactic mastectomy in mutation carriersSeveral of these issues relating to BPM are perti-nent to CPM in patients with a proven BRCA1/2 gene mutation. It is clear that the latter group

of patients have an elevated risk of developing breast cancer due to an inherent genetic pre-disposition present within all cells of the con-tralateral breast tissue. If a BRCA1 or BRCA2 mutation carrier without any personal history of breast cancer is considered eligible for BPM, then a CPM in the same individual must be eas-ier to justify on statistical probability for breast cancer development [10]. The risk of developing contralateral breast cancer in mutation carri-ers appears to be three- or four-times greater than that of average risk cohorts [64]. Moreover, a recent study from The Netherlands suggests that age at diagnosis of the first breast cancer is a significant predictor of of contralateral breast cancer risk in BRCA1 and BRCA2 mutation car-riers [65]. Those diagnosed with breast cancer before age 41 years had a 10-year contralateral breast cancer risk of 23.9% compared with 12.6% for those diagnosed 41–49 years of age. Thus mutation carriers who present with uni-lateral breast cancer should generally consider bilateral mastectomy with immediate breast reconstruction rather than breast conserving surgery or unilateral mastectomy, particularly if they are young.

There is a paucity of data specifically address-ing the efficacy of CPM in BRCA1 and BRCA2 mutation carriers, either in terms of risk reduc-tion or improved survival. Van Sprundel and colleagues examined the incidence of invasive contralateral breast cancer among a group of 148 women who were mutation carriers for BRCA1 and BRCA2 and had received treatment for early stage unilateral invasive breast cancer [66]. Just over half of these women underwent CPM whilst the others remained under intensive surveillance. At a median follow-up of 3.5 years, one patient had developed an invasive contralateral cancer in the CPM group while six were observed in the surveillance group (p < 0.001) [66]. Therefore CPM reduced the risk of contralateral breast cancer by more than 90% and this is of similar magnitude to the risk reduction associated with BPM. There was no impact of CPM on breast cancer specific survival with this limited period of follow-up.

There would appear to be a compelling argu-ment for offering CPM to mutation carriers who are diagnosed with unilateral breast can-cer [20,65]. Additional factors such as dense breast parenchyma and/or florid benign changes would strengthen this decision-making process in favor of prophylactic surgery.

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Surgical techniquesWhen contemplating risk-reducing mastectomy there are several technical points to consider. First, it is essential to ensure that breast imag-ing has been undertaken within the preced-ing 3 months to exclude any incidental breast cancer [67]. Bilateral diagnostic MRI is usually recommended for BRCA mutation carriers, but despite a normal MRI examination in these cir-cumstances, definitive histo-pathological exami-nation of the mastectomy specimen may reveal a small cancer and patients should be warned about this prior to surgery. Another issue per-tains to surgical staging of the axilla; if pre-oper-ative MRI shows normal axillary nodes with no parenchymal abnormality, the chance of finding tumor foci within the axillary nodes is very low and does not justify routine sentinel lymph node biopsy. The latter is not without risk and can increase the chance of infection post-operatively and lead to seroma formation and lymphedema in about 5% of patients [68].

Several types of mastectomy are utilized in the context of risk-reducing surgery for mutation carriers [10].

●● Subcutaneous mastectomyThis procedure is historical to some extent and although previously employed in the context of surgical prophylaxis is no longer recommended as a risk-reducing strategy for mutation carri-ers [10,69]. Subcutaneous mastectomy aims to remove all breast parenchymal tissue whilst preserving a thin sliver of breast tissue deep to the nipple–areola complex (NAC) in order to ensure viability of the latter. This operation is often undertaken in younger women with dense breast tissue that can be difficult to dissect off the under-surface of the NAC without compro-mise of vascular supply. The procedure can be done through an incision along the inframam-mary fold although this can present problems with accessing the superior limit of the breast tissue.

●● Skin-sparing mastectomyThis procedure refers to extirpation of the nip-ple–areola complex and breast tissue with preser-vation of the breast skin envelope [10]. It is feasible to dissect the skin and subcutaneous tissues from the breast parenchyma without risk of leaving remnant breast tissue and therefore skin-sparing mastectomy ensures almost complete removal of breast tissue while maintaining the benefits

of skin preservation for purposes of immediate reconstruction. Some surgeons consider this to be the optimal breast cancer risk-reducing strat-egy for mutation carriers. It potentially achieves the combined goals of maximal risk-reduction whilst ensuring excellent cosmetic outcomes (nipple reconstruction can be undertaken at a later stage).

●● Nipple-sparing mastectomyThis is the latest development in preservation of the breast skin envelope [10]. Unlike subcutane-ous mastectomy where breast tissue is deliber-ately retained deep to the NAC, nipple-sparing mastectomy – or total skin-sparing mastectomy – aims to remove almost all breast tissue in the vicinity of the nipple–areola complex. The main lactiferous ducts converge upon the nipple and breast tissue and are inextricably linked with the tissues of the nipple itself. The areola can be readily dissected off the underlying breast tissue but in younger women with dense breasts this can be technically challenging and sometimes a thin layer of breast tissue must be retained to ensure viability of the NAC. With a nipple-sparing mastectomy, excision of the retro-areolar tissue is a particularly important maneuver and a balance must be achieved between complete excision of the duct system and preservation of blood supply to the NAC. The ducts are usu-ally cored out of the nipple, although micro-anatomical studies suggest that breast tissue within the nipple contains no terminal duct lobular units. Nipple-sparing mastectomy is an appropriate option for risk-reduction but remains controversial for patients with estab-lished breast malignancy [70,71]. Patients should be offered the choice of nipple preservation and informed that there is no evidence of any sig-nificant attrition of risk-reduction consequent to retention of the NAC. However, patients should be warned about the possibility of nipple necro-sis and need for any subsequent debridement. These risk-reducing procedures are not followed by adjuvant treatments such as radiotherapy or chemotherapy that may compromise the blood supply to the NAC or increase the chance of infection.

●● Simple mastectomyThis procedure is appropriate for patients not seeking immediate breast reconstruction in order to avoid redundant flap tissue that would oth-erwise result from any skin-sparing mastectomy

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without restoration of the breast mound [10]. There is no evidence that more extensive extir-pation reduces the risk of any subsequent can-cer and simple mastectomy is only indicated for mutation carriers who decline immediate breast reconstruction.

Despite careful dissection within the onco-logical plane, some breast tissue will remain following either skin-sparing or nipple-sparing mastectomy and this is particularly likely at the periphery of the breast where access is more restricted with certain types of incision [72]. In theory, mutation carriers will continue to have a low risk of breast cancer development due to this residual tissue but there is no evidence at present that any form of radiological surveillance is indicated following risk-reducing surgery by a competent operator [2]. Many clinicians dis-charge mutation carriers from further follow-up after such surgery but others recommend screen-ing clinical breast examination every 6 months. Breast cancer developing in mutation carriers following mastectomy is likely to be manifest as a superficial lesion which is readily detectable on clinical examination. These follow-up visits provide an opportunity for continued dialog with patients and their families (some of whom may be scheduled for risk-reducing surgery th emselves at a future date).

ConclusionThe management of women with a hereditary predisposition for breast cancer will continue to be a focus of interest in the years ahead with increasing utilization of genetic testing ser-vices [73]. Women who harbor a mutation confer-ring elevated risk for breast cancer development have several options to potentially reduce that risk. Physicians and healthcare personnel should be appropriately trained and educated to discuss these options and guide the informed consent process. Surgeons must liaise appropriately with genetic counselors as well as medical oncologists and gynecologists to ensure patients receive com-prehensive assessment and a coordinated plan for any risk reduction. Further advances in technol-ogy and bioinformatics may assist in more accu-rate risk prediction and defining which patients with low-penetrance genes may benefit from interventions aimed at risk reduction.

Future perspectiveIn the years ahead, wider use of genetic testing will result in the identification of large numbers of women who have a hereditary predisposition for breast cancer. In this article, we have empha-sized the high-penetrance mutations (BRCA1, BRCA2, p53, pTEN, CDH1, STK11, PALB2), for which prophylactic surgery, breast MRI

EXECUTivE SUMMARY ● Three major risk factors for development of breast cancer are gender, age and family history/genetic susceptibility.

● There has been an upsurge of interest in identifying women with a genetic predisposition for breast cancer

● Only about 10% of all breast cancers are currently attributable to germline mutations.

● High-penetrance mutations that increase breast cancer risk include those in the BRCA1, BRCA2, STK11 (Peutz-Jeghers syndrome), pTEN (Cowden’s syndrome), p53 (Li-Fraumeni syndrome), CDH1 (diffuse hereditary gastric cancer syndrome) and PALB2 (partner and localizer of BRCA2) genes.

● NICE has recently recommended that women undergo genetic testing when the chance of finding a high-penetrance mutation is 10% or greater.

● Once a high-penetrance gene mutation has been identified in an asymptomatic woman, three risk-reducing strategies should be discussed: screening (to include breast MRI screening), chemoprevention and risk-reducing surgery. However, there are no randomized trials that have addressed the effect of these risk-reducing strategies in mutation carriers.

● Observational studies suggest that bilateral prophylactic mastectomy reduces breast cancer risk by more than 90% in mutation carriers.

● If unilateral breast cancer is diagnosed in a mutation carrier, bilateral mastectomy should be considered, because these patients have a three- or four-times greater risk of developing contralateral breast cancer than average risk cohorts.

● Following bilateral prophylactic mastectomy, mutation carries will continue to have a low risk of breast cancer development due to the presence of some residual tissue, but there is no evidence that any form of radiological surveillance is beneficial.

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ReferencesPapers of special note have been highlighted as: • of interest; •• of considerable interest

1 Thompson ER, Rowley SM, Li N et al. Panel testing for familial breast cancer: calibrating the tension between research and clinical care. J. Clin. Oncol. 34(13), 1455–1459 (2016).

2 Hartmann LC, Lindor NM. The role of risk-reducing surgery in hereditary breast and ovarian cancer. N. Engl. J. Med. 374(5), 454–468 (2016).

•• ExcellentreviewofthecharacteristicsofbreastandovariancancersassociatedwiththeBRCA1andBRCA2mutations,andalsoanexcellentoverviewoftheroleofprophylacticsurgeryinthepreventionofthosecancers.

3 Sheikh A, Hussain SA, Ghori Q et al. The spectrum of genetic mutations in breast cancer. Asian Pac. J. Cancer Prev. 16(6), 2177–2185 (2015).

4 Evans DG, Barwell J, Eccles DM et al. The Angelina Jolie effect: how high celebrity profile can have a major impact on provision of cancer related services. Breast Cancer Res. 16(5), 442 (2014).

5 Turnbull C, Rahman N. Genetic predisposition to breast cancer: past, present, and future. Annu. Rev. Genomics Hum. Genet. 9, 321–345 (2008).

6 Singletary SE. Rating the risk factors for breast cancer. Ann. Surg. 237(4), 474–482 (2003).

7 Anderson WF, Jatoi I, Tse J, Rosenberg PS. Male breast cancer: a population-based comparison with female breast cancer. J. Clin. Oncol. 28(2), 232–239 (2010).

8 Mcpherson K, Steel CM, Dixon JM. ABC of breast diseases. Breast cancer-epidemiology, risk factors, and genetics. BMJ 321(7261), 624–628 (2000).

9 Oseni T, Jatoi I. An overview of the role of prophylactic surgery in the management of individuals with a hereditary cancer predisposition. Surg. Clin. North Am. 88(4), 739–758, vi (2008).

10 Jatoi I, Benson JR, Liau SS et al. The role of surgery in cancer prevention. Curr. Probl. Surg. 47(10), 750–830 (2010).

•• Providesanoverviewoftheroleofsurgeryforthepreventionofbreast,ovary,colon,thyroidandgastriccancers,forindividualswithahereditarypredispositionforthosecancers.

11 Benson JR, Jatoi I, Keisch M, Esteva FJ, Makris A, Jordan VC. Early breast cancer. Lancet 373(9673), 1463–1479 (2009).

•• Overviewofimportantconceptspertainingtoepidemiology,localtherapytherapyandadjuvantsystemictherapyofearlybreastcancer.

12 Harvie M, Howell A, Evans DG. Can diet and lifestyle prevent breast cancer: what is the evidence? Am. Soc. Clin. Oncol. Educ. Book 2015, e66–73 (2015).

13 Gabai-Kapara E, Lahad A, Kaufman B et al. Population-based screening for breast and ovarian cancer risk due to BRCA1 and BRCA2. Proc. Natl Acad. Sci. USA 111(39), 14205–14210 (2014).

14 Narod SA, Amos C. Estimating the power of linkage analysis in hereditary breast cancer. Am. J. Hum. Genet. 46(2), 266–272 (1990).

15 Knudson AG Jr. Hereditary cancer, oncogenes, and antioncogenes. Cancer Res. 45(4), 1437–1443 (1985).

16 Knudson AG Jr. Genetics of human cancer. Annu. Rev. Genet. 20 231–251 (1986).

17 King MC, Levy-Lahad E, Lahad A. Population-based screening for BRCA1 and BRCA2: 2014 Lasker Award. JAMA 312(11), 1091–1092 (2014).

• Interestingeditorial,callingforpopulation-basedscreeningforBRCA1andBRCA2mutationcarriers.

18 Rebbeck TR, Mitra N, Wan F et al. Association of type and location of BRCA1 and BRCA2 mutations with risk of breast and ovarian cancer. JAMA 313(13), 1347–1361 (2015).

19 Hutchinson L. Screening: BRCA testing in women younger than 50 with triple-negative breast cancer is cost effective. Nat. Rev. Clin. Oncol. 7(11), 611 (2010).

20 Jatoi I. Options in breast cancer local therapy: who gets what? World J. Surg. 36(7), 1498–1502 (2012).

21 Antoniou AC, Casadei S, Heikkinen T et al. Breast-cancer risk in families with mutations in PALB2. N. Engl. J. Med. 371(6), 497–506 (2014).

22 Mavaddat N, Antoniou AC, Easton DF, Garcia-Closas M. Genetic susceptibility to breast cancer. Mol. Oncol. 4(3), 174–191 (2010).

23 Lane DP, Hupp TR. Drug discovery and p53. Drug Discov. Today 8(8), 347–355 (2003).

24 Bougeard G, Renaux-Petel M, Flaman JM et al. Revisiting Li-Fraumeni syndrome from TP53 mutation carriers. J. Clin. Oncol. 33(21), 2345–2352 (2015).

25 Nelen MR, Padberg GW, Peeters EA et al. Localization of the gene for Cowden disease to chromosome 10q22–23. Nat. Genet. 13(1), 114–116 (1996).

26 Thompson D, Easton D. The genetic epidemiology of breast cancer genes. J. Mammary Gland Biol. Neoplasia 9(3), 221–236 (2004).

27 Antoniou AC, Easton DF. Models of genetic susceptibility to breast cancer. Oncogene 25(43), 5898–5905 (2006).

screening and chemoprevention are potential risk-reducing strategies. However, little is known about the impact of these risk-reducing strategies on quality of life, and more research is needed to address this issue. Moreover, potential strate-gies to lower breast cancer risk in women who carry low- or moderate-penetrance mutations have received little attention. For these patients, life-style changes will likely play an important role, and additional research is needed to better understand how alterations in diet, maintain-ing a healthy weight, exercise and reduction in alcohol intake may potentially serve to lower

breast cancer risk in women who harbor low- or moderate-penetrance mutations.

Financial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a finan-cial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or p ending, or royalties.

No writing assistance was utilized in the production of this manuscript.

2287




28 Udler MS, Meyer KB, Pooley KA et al. FGFR2 variants and breast cancer risk: fine-scale mapping using African American studies and analysis of chromatin conformation. Hum. Mol. Genet. 18(9), 1692–1703 (2009).

29 Meijers-Heijboer H, Van Den Ouweland A, Klijn J et al. Low-penetrance susceptibility to breast cancer due to CHEK2(*)1100delC in noncarriers of BRCA1 or BRCA2 mutations. Nat. Genet. 31(1), 55–59 (2002).

30 Amir E, Freedman OC, Seruga B, Evans DG. Assessing women at high risk of breast cancer: a review of risk assessment models. J. Natl Cancer Inst. 102(10), 680–691 (2010).

31 Geller G, Bernhardt BA, Doksum T, Helzlsouer KJ, Wilcox P, Holtzman NA. Decision-making about breast cancer susceptibility testing: how similar are the attitudes of physicians, nurse practitioners, and at-risk women? J. Clin. Oncol. 16(8), 2868–2876 (1998).

32 Familial Breast Cancer: Classification and Care of People at Risk of Familial Breast Cancer and Management of Breast Cancer and Related Risks in People with a Family History of Breast Cancer, NICE Clinical Guidelines, No. 164, National Collaborating Centre for Cancer, Cardiff, UK (2013).

33 Black WC, Nease RF Jr, Tosteson AN. Perceptions of breast cancer risk and screening effectiveness in women younger than 50 years of age. J. Natl Cancer Inst. 87(10), 720–731 (1995).

34 Jatoi I, Anderson WF. Cancer screening. Curr. Probl. Surg. 42(9), 620–682 (2005).

35 Thomas DB, Gao DL, Ray RM et al. Randomized trial of breast self-examination in Shanghai: final results. J. Natl Cancer Inst. 94(19), 1445–1457 (2002).

36 Semiglazov VF, Manikhas AG, Moiseenko VM et al. [Results of a prospective randomized investigation [Russia (St. Petersburg)/WHO] to evaluate the significance of self-examination for the early detection of breast cancer]. Vopr. Onkol. 49(4), 434–441 (2003).

37 Mittra I, Mishra GA, Singh S et al. A cluster randomized, controlled trial of breast and cervix cancer screening in Mumbai, India: methodology and interim results after three rounds of screening. Int. J. Cancer 126(4), 976–984 (2010).

38 Sankaranarayanan R, Ramadas K, Thara S et al. Clinical breast examination: preliminary results from a cluster randomized controlled trial in India. J. Natl Cancer Inst. 103(19), 1476–1480 (2011).

39 Moss SM, Wale C, Smith R, Evans A, Cuckle H, Duffy SW. Effect of mammographic screening from age 40 years on breast cancer mortality in the UK Age trial at 17 years’ follow-up: a randomised controlled trial. Lancet Oncol. 16(9), 1123–1132 (2015).

40 Miller AB, Wall C, Baines CJ, Sun P, To T, Narod SA. Twenty five year follow-up for breast cancer incidence and mortality of the Canadian National Breast Screening Study: randomised screening trial. BMJ 348, g366 (2014).

41 Jatoi I. The impact of advances in treatment on the efficacy of mammography screening. Prev. Med. 53(3), 103–104 (2011).

42 Shapiro S. Periodic screening for breast cancer: the HIP randomized controlled trial. Health Insurance Plan. J. Natl Cancer Inst. Monogr. (22), 27–30 (1997).

43 Pijpe A, Andrieu N, Easton DF et al. Exposure to diagnostic radiation and risk of breast cancer among carriers of BRCA1/2 mutations: retrospective cohort study (GENE-RAD-RISK). BMJ 345, e5660 (2012).

• Suggeststhatlow-dosediagnosticradiation(i.e.,mammographyscreening)mayincreasebreastcancerriskinmutationcarriers,andthisstudytherebyprovidesfurtherrationaleforuseofbreastMRIscreeninginthesepatients.

44 Jatoi I. MRI in breast cancer management: potential for benefit and harm. Int. J. Fertil. Womens Med. 50(6), 281–284 (2005).

45 Kuhl C, Weigel S, Schrading S et al. Prospective multicenter cohort study to refine management recommendations for women at elevated familial risk of breast cancer: the EVA trial. J. Clin. Oncol. 28(9), 1450–1457 (2010).

• Examinesdifferentscreeningoptionsforwomenwithanelevatedfamilialriskforbreastcancer.

46 Fisher B, Costantino JP, Wickerham DL et al. Tamoxifen for prevention of breast cancer: report of the National Surgical Adjuvant Breast and Bowel Project P-1 Study. J. Natl Cancer Inst. 90(18), 1371–1388 (1998).

47 Powles TJ, Ashley S, Tidy A, Smith IE, Dowsett M. Twenty-year follow-up of the Royal Marsden randomized, double-blinded tamoxifen breast cancer prevention trial. J. Natl Cancer Inst. 99(4), 283–290 (2007).

48 Veronesi U, Maisonneuve P, Rotmensz N et al. Tamoxifen for the prevention of breast cancer: late results of the Italian Randomized Tamoxifen Prevention Trial among women with hysterectomy. J. Natl Cancer Inst. 99(9), 727–737 (2007).

49 Cuzick J, Forbes JF, Sestak I et al. Long-term results of tamoxifen prophylaxis for breast cancer – 96-month follow-up of the randomized IBIS-I trial. J. Natl Cancer Inst. 99(4), 272–282 (2007).

50 Visvanathan K, Chlebowski RT, Hurley P et al. American society of clinical oncology clinical practice guideline update on the use of pharmacologic interventions including tamoxifen, raloxifene, and aromatase inhibition for breast cancer risk reduction. J. Clin. Oncol. 27(19), 3235–3258 (2009).

51 King MC, Wieand S, Hale K et al. Tamoxifen and breast cancer incidence among women with inherited mutations in BRCA1 and BRCA2: National Surgical Adjuvant Breast and Bowel Project (NSABP-P1) Breast Cancer Prevention Trial. JAMA 286(18), 2251–2256 (2001).

• TherewereveryfewBRCAmutationcarriersinthisstudy,butthestudysuggestsabenefitoftamoxifeninpreventingbreastcancersinBRCA2mutationcarriers.

52 Cuzick J, Sestak I, Cawthorn S et al. Tamoxifen for prevention of breast cancer: extended long-term follow-up of the IBIS-I breast cancer prevention trial. Lancet Oncol. 16(1), 67–75 (2015).

53 Vogel VG, Costantino JP, Wickerham DL et al. Update of the National Surgical Adjuvant Breast and Bowel Project Study of Tamoxifen and Raloxifene (STAR) P-2 Trial: preventing breast cancer. Cancer Prev. Res. (Phila.) 3(6), 696–706 (2010).

54 Dowsett M, Cuzick J, Ingle J et al. Meta-analysis of breast cancer outcomes in adjuvant trials of aromatase inhibitors versus tamoxifen. J. Clin. Oncol. 28(3), 509–518 (2010).

55 Mocellin S, Pilati P, Briarava M, Nitti D. Breast cancer chemoprevention: a network meta-analysis of randomized controlled trials. J. Natl Cancer Inst. 108(2), djv318 (2016).

56 Goss PE, Ingle JN, Ales-Martinez JE et al. Exemestane for breast-cancer prevention in postmenopausal women. N. Engl. J. Med. 364(25), 2381–2391 (2011).

57 Hartmann LC, Schaid DJ, Woods JE et al. Efficacy of bilateral prophylactic mastectomy in women with a family history of breast cancer. N. Engl. J. Med. 340(2), 77–84 (1999).

• Oneofthefirststudiestodemonstrateabenefitofbilateralprophylacticmastectomyinreducingbreastcancerriskinhigh-riskindividuals.

58 Kauff ND, Satagopan JM, Robson ME et al. Risk-reducing salpingo-oophorectomy in

Future Oncol. (2016) 12(19)2288



women with a BRCA1 or BRCA2 mutation. N. Engl. J. Med. 346(21), 1609–1615 (2002).

59 Rocco N, Rispoli C, Moja L et al. Different types of implants for reconstructive breast surgery. Cochrane Database Syst. Rev. (5), CD010895 (2016).

60 Chlebowski RT, Anderson GL, Prentice RL, Rossouw JE, Aragaki AK, Manson JE. Reliable evidence from placebo-controlled, randomized, clinical trials for menopausal hormone therapy’s influence on incidence and deaths from breast cancer. Climacteric 18(3), 336–338 (2015).

61 Rebbeck TR, Levin AM, Eisen A et al. Breast cancer risk after bilateral prophylactic oophorectomy in BRCA1 mutation carriers. J. Natl Cancer Inst. 91(17), 1475–1479 (1999).

62 Heemskerk-Gerritsen BA, Seynaeve C, Van Asperen CJ et al. Breast cancer risk after salpingo-oophorectomy in healthy BRCA1/2 mutation carriers: revisiting the evidence for risk reduction. J. Natl Cancer Inst. 107(5), (2015).

63 Chai X, Domchek S, Kauff N, Rebbeck T, Chen J. RE: Breast cancer risk after salpingo-oophorectomy in healthy BRCA1/2 mutation

carriers: revisiting the evidence for risk reduction. J. Natl Cancer Inst. 107(9), (2015).

64 Jatoi I. Bilateral mastectomy for unilateral breast cancer: a perplexing trend. Indian J. Surg. Oncol. 6(4), 387–389 (2015).

65 Van Den Broek AJ, Van ‘T Veer LJ, Hooning MJ et al. Impact of age at primary breast cancer on contralateral breast cancer risk in BRCA1/2 mutation carriers. J. Clin. Oncol. 34(5), 409–418 (2016).

66 Van Sprundel TC, Schmidt MK, Rookus MA et al. Risk reduction of contralateral breast cancer and survival after contralateral prophylactic mastectomy in BRCA1 or BRCA2 mutation carriers. Br. J. Cancer 93(3), 287–292 (2005).

67 Riedl CC, Luft N, Bernhart C et al. Triple-modality screening trial for familial breast cancer underlines the importance of magnetic resonance imaging and questions the role of mammography and ultrasound regardless of patient mutation status, age, and breast density. J. Clin. Oncol. 33(10), 1128–1135 (2015).

68 Veronesi U, Paganelli G, Viale G et al. A randomized comparison of sentinel-node

biopsy with routine axillary dissection in breast cancer. N. Engl. J. Med. 349(6), 546–553 (2003).

69 Freeman BS. Subcutaneous mastectomy for benign breast lesions with immediate or delayed prosthetic replacement. Plast. Reconstr. Surg. Transplant. Bull. 30 676–682 (1962).

70 Mallon P, Feron JG, Couturaud B et al. The role of nipple-sparing mastectomy in breast cancer: a comprehensive review of the literature. Plast. Reconstr. Surg. 131(5), 969–984 (2013).

71 Murthy V, Chamberlain RS. Nipple-sparing mastectomy in modern breast practice. Clin. Anat. 26(1), 56–65 (2013).

72 Agha RA, Wellstead G, Sagoo H et al. Nipple sparing versus skin sparing mastectomy: a systematic review protocol. BMJ Open 6(5), e010151 (2016).

73 Moreno L, Linossi C, Esteban I et al. Germline BRCA testing is moving from cancer risk assessment to a predictive biomarker for targeting cancer therapeutics. Clin. Transl. Oncol. doi:10.1007/s12094-015-1470-0 (2016) (Epub ahead of print).

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Editorial

Intratumor and circulating clonal heterogeneity shape the basis of precision breast cancer therapy

Demosthenes E Ziogas1,2, John Spiliotis3, Efstathios G Lykoudis4, Georgios C Zografos5 & Dimitrios H Roukos*,1,6,7

Keywords • breast cancer • clonal diversity • intratumor heterogeneity • next-generation sequencing • therapy

The recent evidence of spatiotemporal genomes and tumor evolution has led to the development of breakthrough next-generation sequencing (NGS) technolo-gies. Intratumor heterogeneity (ITH) and circulating clonal diversity represent two of the most possible explanations of primary and secondary resistance. In this edito-rial, we discuss how extensive biobanking for each individual patient with subse-quent genome sequencing can open novel horizons for precision medicine in breast cancer.

Approximately half a century following the war against cancer and the discovery of DNA double helix, millions of patients still die from the disease. The initial enthusiasm on personalized medicine after the comple-tion of the first human genome sequence draft at the beginning of this millennium, was followed by skepticism on the basis of the complexity of noncoding genome functionality and nonlinear transcription.

Although the ENCODE project [1,2] has revolutionized biomedical research high-lighting the necessity for a long-term basic research efforts, at the same time it has shaped innovative horizons in improv-ing NGS technologies and developing breakthrough methods for understanding cancer evolution and r esistance to current therapies [3,4].

Progress in personalized prevention & treatmentProgress in basic, translational and clini-cal research and implementation of new discoveries into routine clinical practice is faster in breast cancer than in any other cancer type. Advances in single gene testing have led to the development of a person-alized approach in the prevention setting and targeted therapy. Breast cancer is still a major health problem for women taking into consideration that 1.67 million new cases are diagnosed each year worldwide [5].

1Centre for Biosystems & Genomic Network Medicine, University of Ioannina, Ioannina, Greece 2Department of Surgery, ‘G Hatzikosta’ General Hospital, Ioannina, Greece 3Department of Surgery, METAXA Cancer Memorial Hospital, Botosi 51, TK 18535, Piraeus, Greece. 4Department of Plastic Surgery, Ioannina University School of Medicine, Ioannina, Greece 5First Department of Propaedeutic Surgery, Hippocration Hospital, Athens Medical School, National & Kapodistrian

University of Athens, Athens 11525, Greece 6Department of Surgery, Ioannina University Hospital, Ioannina, Greece 7Biomedical Research Foundation of the Academy of Athens (BRFAA), Athens, Greece

*Author for correspondence: Tel.: +30 265 100 7423; Fax: +30 265 100 7094; [email protected]

“...rapid developments in technological genome systems and computational network methods open new avenues in understanding

etiopathogenesis on the basis of large-scale translational research studies.”

“The recent evidence of spatiotemporal genomes and tumor evolution has led to the development of breakthrough next-generation sequencing

technologies.”

First draft submitted: 27 June 2016; Accepted for publication: 11 August 2016; Published online: 1 September 2016

Future Oncol. (Epub ahead of print) ISSN 1479-6694


editorial Ziogas, Spiliotis, Lykoudis, Zografos & Roukos


20 years after the discovery of germline muta-tions BRCA1 and BRCA2, which are associated with life-time high risk of breast and ovarian cancer, effective primary prevention, has devel-oped as a result of personalized therapies based on both family history and BRCA1/2 testing [6]. Moreover, more aggressive risk-reducing surgery including bilateral mastectomy and bilateral salpingo-oophorectomy has been suggested for BRCA1 than BRCA2 mutation carriers [6].

In the multimodal treatment setting, estab-lished clinical models on the basis of both tradi-tional and genetic criteria provide better selection of patients improving oncological and quality-of-life outcomes. For example, age, tumor size (T), node status (N), histological grade (G) and molecular features including estrogen receptor and progesterone receptor (ER/PR) status as well as HER2 status represent the modern algorithm for decision-making in adjuvant treatment of early breast cancer (M0).

Trastuzumab (Herceptin®; Genentech, Inc., CA, USA) added to chemotherapy for HER2-positive patients has improved overall survival (OS) rates in the adjuvant [7] and metastatic set-ting (M1). More recently, trastuzumab–emtasine conjugate (T-DM1, KADCYLA®, Genentech, Inc., CA, USA) prolongs mean OS [8]. Moreover, Palbociclib has recently received regulatory fast-track approval opening a new therapeu-tic horizon for postmenopausal women with ER+/HER2-negative metastatic breast cancer [9].

limitationsDespite these advances, much more work is required to reach a personalized cancer medi-cine model for the clinic. Among women with significant family history who account for 25% of all breast cancers, only 7% are caused by the known BRCA1/2 mutations, while for the remaining 18% no other high-penetrance gene has yet been identified and thus no precise risk estimation can be made. For sporadic breast cancer that accounts for 70–80% of all breast cancers, no personalized risk prediction is feasi-ble. Therefore, a generalized strategy including mammography and ultrasound has been estab-lished as an evidence-based approach for early-stage breast cancer diagnosis and OS benefit recommended by guidelines.

In the treatment setting, anti-estrogen for ER+ has long been considered as standard treat-ment. For HER2-positive women trastuzumab has been recommended in the adjuvant setting,

while trastuzumab–emtansine has been sug-gested in trastuzumab-resistant metastatic dis-ease. More recently, palbociclib plus letrozole for ER+/HER2-negative postmenopausal women has become a new standard therapy, improv-ing treatment efficacy and survival. However, despite advances and standardization of systemic chemotherapy and targeted therapy, intrinsic and acquired resistance remains an unresolved problem.

From interpatient to intrapatient heterogeneityTumor heterogeneity has long been considered as genetic variation for most solid malignan-cies. In recent years, the development of high-throughput technologies has enhanced the abil-ity of many studies to access the impact of tumor heterogeneity in the clinic. Most of these studies have suggested different genetic characteristics not only among patients with the same cancer type (interpatient heterogeneity) [10], but also between primary and metastatic tumor(s) in the same individual patient [3]. However, a few other studies have shown high similarities between primary and metastatic tumor, suggesting the pre-existence of a small cell population within the primary tumor responsible for metastasis (reviewed also in [3]).

Next-generation sequencing systemsThis relative uncertainty of individual patient’s tumor heterogeneity could be overcome in recent years by the implementation of NGS systems. NGS systems have revolutionized bio-medical research because of clinical validity and co ntinuing lowering cost of these technologies.

Numerous genomic studies using tumor-nor-mal pairs for each individual patient including whole-exome sequencing (WES) [11] and whole-genome sequencing (WGS) [12,13] have dramati-cally been increased over the last 6 years. Even in this NGS era, multiple challenges are emerg-ing, regarding their potential for improving clinical treatment and patients’ outcomes. Two NGS strategies including a conventional and a breakthrough approach have been developed providing promise for clinical implications.

Following single biopsy-based modern oncol-ogy, most NGS studies available are based on the same concept. In the largest WES study reported by Lawrence et al. [11] on 4742 tumor-normal pairs, 892 breast cancers were analyzed. In this study, the SETBD1 gene has been discovered for

“The unprecedented potential of advancing

next-generation sequencing systems and

methods to explore spatiotemporal genomes

and tumor evolution, raises for the first time rational

hope for deep understanding of

mechanisms underlying therapeutic resistance and metastasis in response to

therapy.”

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Intratumor & circulating heterogeneity in breast cancer editorial


the first time with strict statistically significant criteria (p < 0.01) [11]. Three other new genes, CBFB, RUNX1 and GATA3 involved in triple negative breast cancer were reported for the first time by Banerji et al. [14]. However, because of the relatively small number of patients enrolled in this study (n = 103), confirmation by larger studies is required to reach the statistical level of accuracy which was recommended by Lawrence et al. [11] in the discovery of new cancer driver genes.

At least four WGS breast cancer studies have been reported. In one of these studies, Ellis et al. [12] identified five genes (RUNX1, CBFB, MYH9, MLL3 and SF3B1) in 46 patients’ samples with ER+ breast cancer. These genes have previously been linked with hematopoietic disorders, but are reported for the first time in breast cancer. However, this conventional sin-gle biopsy-based WES/WGS strategy has strong limitations in understanding primary and sec-ondary resistance because of more recent evi-dence on dynamics genomic clones evolution [15] and ITH [16]. Based on this new knowledge, innovative methods and NGS applications have been developed.

intratumor & circulating clonal heterogeneityExploiting these new data from basic and trans-lational research, landmark studies open new predictive and therapeutic horizons for breast cancer. Developing new methods, dynamics of genomics clones’ evolution [16], ITH and cir-culating genomic clones diversity can now be explored. These techniques and methods pro-vide the potential not only to explain, but also to overcome therapeutic resistance and prevent metastasis.

Identif ication of ITH, which represents genetic characteristics of different cell subpopu-lations within the primary tumor, could provide important clinical implications in overcoming resistance to current therapeutics. Multiregional NGS analysis has been suggested as the opti-mal method to reveal ITH. In the largest ITH study available, Yates et al. [16] have performed WGS and targeted sequencing of the primary tumor in 50 patients with breast cancer includ-ing 303 solid tumor samples. Sequencing data were compared in 18 patients who had under-gone neoadjuvant treatment followed by sur-gical resection. This intelligent method could reveal not only ITH but also clonal evolution

in response to neoadjuvant treatment. In 13 patients, targetable mutations were resulted from clonal evolution following neoadjuvant treatment.

Noninvasive methods with major potential clinical implications have recently been devel-oped such as the circulating tumor DNA fol-lowed by NGS analysis (ctDNA-NGS). The aim from a clinical perspective is to use this method as a biomarker to predict therapeutic resistance and relapse before it clinically occurs. Murtaza et al. [17] have performed a serial ctDNA-NGS analysis in 19 plasma samples obtained from six patients with breast, ovar-ian and lung cancer. Quantification of allele fractions in plasma identified the emergence of mutations associated with acquired therapeutic resistance. This method not only can potentially predict resistance-based metastatic relapse, but also opens new avenue for drug-development design. In breast cancer, one patient that was treated with chemotherapy, was found to have an activating mutation in PIK3CA. These data establish a proof-of-principle that ctDNA exome sequencing can be used as biomarker to predict acquired resistance but additional larger studies are required.

Future perspective & conclusionThe unprecedented potential of advancing NGS systems and methods to explore spatiotemporal genomes and tumor evolution, raises for the first time rational hope for deep understanding of mechanisms underlying therapeutic resistance and metastasis in response to therapy.

Indeed, once the vast majority of women with nonmetastatic breast cancer receive systemic chemotherapy and targeted therapy and a sub-stantial proportion among them develop relapse, it is crucial to understand the reasons why.

Exploiting recent evidence on dynamic clonal evolution [15,16] we could explain therapeutic resistance-based metastatic relapse. Preliminary data suggest that dynamic evolution depends on the kind of mutation. On one hand most data support dynamic evolution of point mutations only, while on the other large structural genome changes are pre-existed and remain stable during tumor growth and progression [18,19]. However, these finding require validation by large studies.

Clinical evidence that almost 25% of women with HER2-positive breast cancer treated with guidelines-based recommendation therapy develop relapse, clonal evolution identification

“This understanding of molecular mechanisms landscape underlying therapeutic resistance

shapes new horizons in robust biomarkers and

drug-development strategy to reach precision

cancer medicine.”

10.2217/fon-2016-0307

editorial Ziogas, Spiliotis, Lykoudis, Zografos & Roukos


following systemic therapy could reduce this resistance-based treatment failure. The potential of ITH and subclonal emergence identification in the neoadjuvant treatment and postsurgical setting [16] along with repeated ctDNA-NGS can reveal the emergence of resistant genome-wide alterations responsible for clinical relapse several months before it clinically occurs.

In summary, rapid developments in technolog-ical genome systems and computational network methods open new avenues in understanding eti-opathogenesis on the basis of large-scale trans-lational research studies. This understanding of molecular mechanisms landscape underlying

therapeutic resistance shapes new horizons in robust biomarkers and drug-development st rategy to reach precision cancer medicine.

Financial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a finan-cial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or p ending, or royalties.


references1 ENCODE Project Consortium. An integrated

encyclopedia of DNA elements in the human genome. Nature 489(7414), 57–74 (2012).

2 Gerstein MB, Kundaje A, Hariharan M et al. Architecture of the human regulatory network derived from ENCODE data. Nature 489(7414), 91–100 (2012).

3 Klein CA. Selection and adaptation during metastatic cancer progression. Nature 501(7467), 365–372 (2013).

4 Bedard PL, Hansen AR, Ratain MJ, Siu LL. Tumour heterogeneity in the clinic. Nature 501(7467), 355–364 (2013).

5 Ferlay J, Soerjomataram I, Dikshit R et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int. J. Cancer 136(5), E359–E386 (2015).

6 Bombard Y, Bach PB, Offit K. Translating genomics in cancer care. J. Natl Compr. Canc. Netw. 11(11), 1343–1153 (2013).

7 Goldhirsch A, Gelber RD, Piccart-Gebhart MJ et al. 2 years versus 1 year of adjuvant trastuzumab for HER2-positive breast cancer (HERA): an open-label, randomised

controlled trial. Lancet 382(9897), 1021–1028 (2013).

8 Verma S, Miles D, Gianni L et al. Trastuzumab emtansine for HER2-positive advanced breast cancer. N. Engl. J. Med. 367(19), 1783–1791 (2012).

9 Beaver JA, Amiri-Kordestani L, Charlab R et al. FDA approval: palbociclib for the treatment of postmenopausal patients with estrogen receptor-positive, HER2-negative metastatic breast cancer. Clin. Cancer Res. 21(21), 4760–4766 (2015).

10 Vogelstein B, Papadopoulos N, Velculescu VE, Zhou S, Diaz LA Jr, Kinzler KW. Cancer genome landscapes. Science 339(6127), 1546–1558 (2013).

11 Lawrence MS, Stojanov P, Mermel CH et al. Discovery and saturation analysis of cancer genes across 21 tumour types. Nature 505(7484), 495–501 (2014).

12 Ellis MJ, Ding L, Shen D et al. Whole-genome analysis informs breast cancer response to aromatase inhibition. Nature 486(7403), 353–360 (2012).

13 Roukos DH. Crossroad between linear and nonlinear transcription concepts in the

discovery of next-generation sequencing systems-based anticancer therapies. Drug Discov. Today 21, 663–673 (2016).

14 Banerji S, Cibulskis K, Rangel-Escareno C et al. Sequence analysis of mutations and translocations across breast cancer subtypes. Nature 486(7403), 405–409 (2012).

15 Eirew P, Steif A, Khattra J et al. Dynamics of genomic clones in breast cancer patient xenografts at single-cell resolution. Nature 518(7539), 422–426 (2015).

16 Yates LR, Gerstung M, Knappskog S et al. Subclonal diversification of primary breast cancer revealed by multiregion sequencing. Nat. Med. 21(7), 751–759 (2015).

17 Murtaza M, Dawson SJ, Tsui DW et al. Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA. Nature 497, 108–112 (2013).

18 Wang Y, Waters J, Leung ML et al. Clonal evolution in breast cancer revealed by single nucleus genome sequencing. Nature 512(7513), 155–160 (2014).

19 Fox EJ, Loeb LA. Cancer: one cell at a time. Nature 512(7513), 143–144 (2014).

10.2217/fon-2016-0307 Future Oncol. (Epub ahead of print)

1219Immunotherapy (2016) 8(10), 1219–1232 ISSN 1750-743X10.2217/imt-2016-0052 © 2016 Future Medicine Ltd

Immunotherapy

Review 2016/08/288

10

2016

HER2/neu is expressed in the majority of in situ breast cancers, but maintained in 20–30% of invasive breast cancer (IBC). During breast tumorigenesis, there is a progressive loss of anti-HER2 CD4pos Th1 (anti-HER2Th1) from benign to ductal carcinoma in situ, with almost complete loss in IBC. This anti-HER2Th1 response can predict response to neoadjuvant therapy, risk of recurrence and disease-free survival. Vaccines consisting of HER2-pulsed type I polarized dendritic cells (DC1) administered during ductal carcinoma in situ and early IBC can efficiently correct anti-HER2Th1 response and have clinical impact on the disease. In this review, we will discuss the role of anti-HER2Th1 response in the three phases of immunoediting during HER2 breast cancer development and opportunities for reversing these processes using DC1 vaccines alone or in combination with standard therapies. Correcting the anti-HER2Th1 response may represent an opportunity for improving outcomes and providing a path to eliminate escape variants.

First draft submitted: 4 May 2016; Accepted for publication: 23 June 2016; Published online: 8 September 2016

Keywords: adoptive cell therapy • checkpoint inhibitor • chemotherapy • dendritic cell • immunotherapy • multimodality • radiotherapy • targeted therapy

The rationale for targeting HER2Although HER2 (ERBB2) is transiently expressed during fetal and normal breast development, as well as during breast growth in pregnancy, overexpression of HER2/neu can contribute to breast tumorigen-esis. HER2/neu has become the most stud-ied tumor-associated antigen (TAA), as it is a molecular oncodriver overexpressed in 20–30% of invasive breast cancers (IBC) and in about 13–56% of ductal carcinoma in situ (DCIS) lesions [1].

HER2/neu is an immunogenic pro-tein that elicits both humoral and cellular immune responses in patients with HER2pos tumors, but patients with HER2/neu over-expressing tumors often demonstrate dimin-ished existing immunity directed against the protein [2]. In IBC, HER2/neu is asso-ciated with a heightened risk for i nvasion,

suggesting a crucial role for HER2/neu in stimulating a tumorigenic microenvi-ronment. Whether by ‘immunoediting’ or other immune evasion mechanisms, a diminished cellular immune response to HER2/neu in the tumor microenvironment of HER2pos breast cancer (HER2posBC) is associated with poorer prognosis [2]. On the contrary, an increased cellular and humoral response against HER2/neu has been asso-ciated with decreased tumor development and improved outcomes [3]. Clinical impli-cations of HER2/neu expression in DCIS (HER2posDCIS) are not as clear, but present data suggest that it predicts the presence of invasive foci [4], and increases risk of disease recurrence, although only approximately 20% of HER2posDCIS recurs as HER-2posBC [5]. This low rate of HER2posDCIS conversion to HER2posIBC may be due to

Restoring anti-oncodriver Th1 responses with dendritic cell vaccines in HER2/neu-positive breast cancer: progress and potential

Lucy M De La Cruz2, Nadia F Nocera2 & Brian J Czerniecki*,1

1Department of Breast Oncology, H. Lee

Moffitt Cancer Center, Tampa, FL 33617,

USA 2Department of Endocrine & Oncologic

Surgery, University of Pennsylvania

Perelman School of Medicine,

Philadelphia, PA, USA

*Author for correspondence:

Tel.: +1 813 4821596

[email protected]

part of

Review


1220 Immunotherapy (2016) 8(10) future science group

Review Cruz, Nocera & Czerniecki

pathway and phenotype instability in HER2pos can-cer cells, with natural or induced immunity possibly shaping the resulting tumor p henotype [6].

With the exception of antibody-based HER2 target-ing, HER2-directed immunotherapy in BC has not been as successful as initially expected, there has only been limited success in the setting of advanced disease where tumor cells have already escaped immunosurveillance. The future of HER2-targeted vaccination trials should be geared toward early-stage HER2posBC, potentially halting progression of HER2posDCIS to IBC (Figure 1) or situations to prevent recurrence. HER2 vaccines must be created based on immunologic principles of circum-venting tolerance, a primary mechanism of escape, by strengthening the weak pre-existent anti-HER2 CD4pos Th1 (anti-HER2Th1) immune response [6].

Loss of anti-HER2 CD4pos Th1 during breast tumorigenesisRecently, we have identified a progressive loss of anti-HER2Th1 immune response in HER2posBC patients relative to healthy controls, with an early and pro-gressive decrease in immune competence in patients with HER2posBC [7]. Specifically, there is a loss of

anti-HER2Th1 response during breast tumorigenesis, where healthy patients have a strong anti-HER2Th1 immune response that is diminished in patients with DCIS and nearly absent in patients with IBC [7]. This supports the hypothesis that the level of circulating anti-HER2Th1 response correlates to immune response in patients with HER2posBC after treatment [7]. It is likely that this immune deficit begins in DCIS and contin-ues to decline with disease progression. This depressed anti-HER2Th1 response is driven predominantly by Th1 phenotypes. In patients who had undergone neo-adjuvant therapies, preserved anti-HER2Th1 response was associated with pCR and improved disease-free sur-vival (DFS), while a depressed response was found in patients without pathologic complete response (pCR) and recurrent disease after treatment [8]. These findings may potentially lead to the use of anti-HER2Th1 moni-toring for patients receiving HER2-targeted therapies to identify those at risk of clinicopathologic failure.

Role of immune response & immunoediting during breast tumorigenesisThe breast parenchyma normally harbors immune cells such as cytolytic CD8pos T cells, Th1 cells and NK cells

Figure 1. Immunoediting process in breast cancer with the potential sites of immunotherapy and targeted therapies in HER2-positive, triple-negative and ER-positive breast cancer. AH-IR: Anti-HER2 immune response; DC: Dendritic cell; DCIS: Ductal carcinoma in situ; ER + BC: Estrogen receptor positive breast cancer; H2IBC: HER2-positive invasive breast cancer; IBC: Invasive breast cancer; IR: Immune response; TNBC: Triple-negative breast cancer.

Opportunities for DC vaccinesin breast cancer

HER2 major oncodriver insporadic DCIS –clinically undetectable disease

DC vaccines to correctequilibrium phasetargeting HER2

Combines HER2-pulsed DC vaccine therapies

Combined targeted andDC vaccine therapies tocorrect escape phase

Tumorigenesis Phase of immunoediting

No tumor

DCIS

AH-IR lossHER2+ IBC(loss of HER2response)

Antigen lossTNBCER+ IBC

Escape

Equilibrium

Elimination

AH-IR

DCIS

AH-IR

AH-IR

AH-IR

DCIS

H2-IBC

IBC

DC vaccines

HER2-pulsedDC vaccines

Target otheroncodrivers


Dendritic cell vaccines in breast cancer Review

that aid in development, lactation, involution and may participate in immunosurveillance of the breast [9]. The presence of high-density CD8pos T cells in tumors and nearby stroma is associated with improved prog-nosis in BC, suggesting that immune effector cells have identified the malignant cells and are actively mounting an appropriate immune response. Potent immune effectors act by eliminating tumors directly via exo cytosis of cytotoxic granules, or indirectly, by producing INF- γ to activate Th1 immune response. It is thought that under immunosurveillance, the host eliminates evolving tumor cells continuously during elimination, until some malignant cells manage to outnumber and stress the immune response, evad-ing immune recognition through a process described as ‘immunoediting’ [9]. Initially malignant clones are effectively eliminated by the immune response but as the tumor grows and expands a state of tumor equilib-rium is reached (Figure 1). If this process becomes inef-fective or fails, malignant cells may escape from immu-nological control to proliferate and manifest clinically as invasive and metastatic cancer [10].

The immunoediting phenomenon is evident dur-ing breast tumorigenesis mediated by HER2. HER2/neu is present in nonmalignant cells at basal levels, but overexpression leads to malignant transformation. We hypothesize that in the initial stage of malignant trans-formation, the mounted immune response is robust enough against this HER2/neu overexpression and may eliminate HER2posDCIS from becoming clini-cally apparent. If elimination is not achieved, HER2/neu overexpressing tumor cells and the immune response develop a state of equilibrium where the anti-HER2Th1 immune response is unable to completely eliminate malignant cells, but is able to prevent them from becoming invasive. This equilibrium state may be clinically manifested as HER2posDCIS presenting with microcalcifications on mammography [11]. These calcifications may be a reflection of necrosis caused by immune effectors cells (anti-HER2Th1 response) attempting to eliminate tumor cells during this period of homeostasis (Figure 1). The anti-HER2Th1 response although diminished remains somewhat active in DCIS [7]. This phase of tumor equilibrium in DCIS may be boosted using DC vaccines, which, by stimulating both CD4pos and CD8pos T cells and IL-12, can maximize INF-γ production and the functionality of Th1 cells thereby tip the equilibrium state toward elimination again (Figure 1).

If the process of equilibrium continues, the tumor may remain stagnant and suspended as DCIS with no disease progression to invasion. The alternative to equilibrium in DCIS would be tumor progression and developing invasion (IBC). If progression occurs, there

are likely two scenarios where the immune response would be ineffective at halting disease progression. First, the anti-HER2Th1 immune response further erodes over time [7] and becomes virtually absent allowing tumor escape. This hyporesponsive state may be due to a chronic exhaustion of the immune system leading to increased inhibitory receptors [11], decreased effector cytokines or impaired cytotoxic-ity, allowing for tumor escape. The result is continued expression of HER2 on the IBC. The second scenario is one where anti-HER2Th1 response is sustained, but tumor cells manage to evade immunosurveillance via phenotypic shifting. In this scenario, the immune sys-tem remains responsive, but tumor cells lose or down-regulate HER2/neu expression resulting in a new phe-notype, such as triple-negative breast cancer (TNBC) or estrogen receptor positive BC (ERposBC) (Figure 1). There are numerous examples of ERpos non-HER2 primary tumors developing systemic metastasis that are HER2pos and also examples of HER2pos primary tumors that lose expression of HER2 [12]. Using our understanding of immunoediting and immune escape, there are numerous opportunities to use DC vaccines to boost anti-HER2 immunity when needed and boost i mmunity against oncodrivers involved with escape to prevent recurrence.

Activation of type I polarized DC drive strong anticancer Th1 responsesDendritic cells (DCs) are a heterogeneous group of specialized APCs [13], found in lymphoid and nonlym-phoid tissue. They are classified according to their abil-ity to elicit a specific immune response based on polar-ization of a T-cell response. DCs are typically found in immature form, and maturation is triggered by proinflammatory cytokines such as IL-1, IL-6, IFN-γ and TNF-α [14]. Additional signals such as bacterial lipopolysaccharide and IFN-γ drive IL-12 produc-tion leading to pulsed type I polarized DC1 [15]. Once maturation and activation occurs, migration to nearby lymph nodes is facilitated [16], where activated DCs deliver costimulatory signals essential for T-cell acti-vation such as CD40, CD80, CD86, CD46 ligands and Toll-like receptors [17]. DCs signal activation and polarization of T cells is crucial for the differentia-tion of CD8pos T cells into cytotoxic T lymphocytes (CTLs) [18] and for the polarization of CD4pos T cells into their different effectors (Th1, Th2 and Th17) [19].

During BC development and progression, CD4pos T cells may be a crucial element in the tumor ‘immuno-editing’ process. Therefore, there is increasing inter-est in activating CD4pos T cells because of their role as helpers in maintaining CD8pos cells as functionally active [20] and indirect effects on other innate cells such



as NK, macrophages and DCs [21]. CD4pos are signifi-cantly increased during breast cancer and the subsets dynamically change with disease progression. In early-tumor stages, Th1 cells are the dominant population of CD4pos T cells, while in the advanced tumor stages, FoxP3pos Treg and Th17 cells become the dominant populations [22]. Th1 cells secrete high-level cytokines such as IFN-γ and TNF-α, both key components of initiating Th1-polarized response and to the antitu-mor function of DC1 vaccines [19]. DC1s are very effi-cient in the presentation of antigens and production of IL-12 [23] that polarizes T cells toward the IFN-γ Th1 phenotype [24]. IL12 has multiple roles with inherent antitumor effect, antiangiogenic capabilities, activa-tion of NK cells as well as enhancing adaptive immu-nity and improving sensitization to tumor antigens [25].

Despite their ability to prime an immune response, results have been disappointing when testing DC vac-cines in late-stage BC. One reason may be because, in advanced disease, DCs are unable to mount a strong enough immune response to overcome the overwhelm-ing immunosuppressive tumor microenvironment pres-ent in tumors that have escaped immunosurveillance. This may be one reason why, in early stages of BC, DC vaccines have enjoyed some clinical successes [3]. Another potential reason for the ineffectiveness of DC vaccines in late-stage disease is that inflammatory type DC vaccines have been used in most studies that do not necessarily drive strong Th1 response [15]. Increas-ing understanding of the dynamics of DCs activation, treatment of early-stage BC and different adjuvant set-tings will allow for improvements that warrant further investigation.

Effectiveness of DC1 vaccines administered in the equilibrium phase of DCISWe suggest that HER2posDCIS represents the equi-librium phase of immunoediting, where Th1 immune response is in homeostasis with cancer cells in the ducts. Supporting this argument, data suggest that a strong Th1 proinflammatory signature in tumor microenviroment is associated with improved out-comes [13]. Therefore, if we can stimulate a strong Th1 immune response during the equilibrium phase of dis-ease, it may be possible to prevent recurrence of the preinvasive lesions or halt disease progression to IBC.

A group of 27 patients with HER2posDCIS received HER2-pulsed DC1 vaccines in the neoadjuvant set-ting [2]. These patients all required surgical resection of their HER2posDCIS. In vaccinated subjects pCR was achieved in 18.5% of all patients (ERneg = 40 vs ERpos 5.9%) suggesting shift from equilibrium to elimina-tion. Among those patients without a pCR in about 50% of HER2 expression was eradicated in residual

DCIS (sustained HER-2/neu expression ERneg =10% vs ERpos = 47.1% [p = 0.04]). Postvaccination pheno-types were significantly different between ERpos and ERneg subjects (p =0.01). The conversion of HER2pos-

DCIS to HER2neg phenotypes after vaccination dem-onstrated the potential process of tumor escape in that a single target may not be sufficient to completely treat or eradicate disease. Interestingly, most of the DCIS lesions that changed were ERpos and subsequent trials have demonstrated this group can have similar com-plete response rate by combining antiestrogen therapy with DC1 vaccination (Lowenfeld L, Press Oncoimmu-nology 2016, Manuscript submitted). This suggests that future experimental BC vaccines may be more effective by either targeting multiple oncodrivers or combining with other therapies that block additional pathways to prevent the escape phase of immunoediting [26].

Postimmunization, sensitization of T cells to at least one class II peptide was observed in 22 of 25 evaluable subjects, while 11 of 13 subjects were successfully sen-sitized to class I peptides. Perhaps most importantly, anti-HER2 peptide responses were observed up to 52 months postimmunization. These data show even in the presence of early-stage BC, such DC1 are potent inducers of durable Th1-polarized immunity, suggest-ing potential clinical value for development of cancer immunotherapy [27]. There is no significant difference in immune response detected systemically after vac-cination in patients with HER2pos or ERneg and ERpos tumors, but complete tumor regression was signifi-cantly more common in patients with ERneg compared with ERposDCIS [28]. This proposed the concept of ER signaling as an escape pathway in HERposBC resistance to anti-HER2-targeted therapies. When looking at the effect of antiestrogen therapy in combination with DC1 vaccination, there is an increase rate of pCR and decreases recurrence in patients with ERpos/HERposBC. Interestingly, the addition of antiestrogen therapy increased the anti-HER2Th1 response in regional sen-tinel nodes (Lowenfeld L, Press Oncoimmunology 2016, Manuscript submitted).

The increased interest in targeted vaccination against HERposBC has shown promising results. This data provide rationale for developing vaccinations to reduce recurrence in patients with ERneg and HER2pos-

DCIS in whom there personalized therapies other than standard surgery and radiation do not exist. There is a large push in the community to develop novel, ratio-nale-targeted therapies for DCIS that also provide pro-tection against new breast events.

The other area where DC1 vaccines may have clini-cal benefit is in patients with HER2pos IBC where the anti-HER2Th1 response is severely compromised (Figure 1) [7]. These patients have substantial risk of



recurrence and include those patients with residual disease following neoadjuvant therapy, and advanced stage III and IV patients that are no evidence of dis-ease (NED). Ongoing research with DC and non-DC vaccines in early-stage trials are currently opened and results are pending. (Table 1). We have demonstrated that the anti-HER2 Th1 response can be restored using DC1 vaccines in these patients [29]. Whether or not restoration of Th1 response translates to d iminished recurrence must await larger trials.

Use of DC1 vaccines during tumor escape in combination with anti-HER2 therapiesLoss of anti-HER2Th1 immune response is associ-ated with lack of pCR to adjuvant chemotherapy and HER2-targeted therapies [7] as well as predicts increased of recurrence and diminished DFS [8]. Though DC vaccination has not yet proven to be an effective treat-ment of BC, other adjuvant treatment modalities have been shown to aid in promoting the immune response to tumor cells when given in combination with DC vaccines. These treatments often work synergisti-cally with vaccination by aiding T-cell recognition of tumor cells. Some therapies, such as c hemotherapy or

r adiation, may induce changes in the tumor microen-vironment, allowing a more complete response to DC vaccination. In combination with other therapies, such as HER2-targeted therapies, DC vaccination can aid in overcoming resistance [29,30]. Hence, pCR may be best achieved using combination therapy with DC vac-cination (Figure 2).

Adjuvant chemotherapyThe tumor microenvironment may play a role in the recognition and response of the immune system to tumor cells. It has been observed that a high level of lymphocytic infiltration in breast tumors predicts a better prognosis and a better response to chemother-apy [31]. Chemotherapy has the potential to augment the tumor microenvironment by increasing CD4pos T-cell infiltration (Figure 2), demonstrating a positive correlation to pCR [32]. Because vaccination, espe-cially with DCs, can stimulate activation of T cells and potentially improve lymphocytic infiltration of breast tumors, concomitant or sequential adminis-tration of vaccines with low-dose chemotherapy may improve treatment outcomes. Sequential administra-tion of a low dose of the chemotherapeutic drug, cyclo-

Table 1. Clinical trials of immunotherapy in early-stage breast cancer.

Phase Study Vaccine type NCI identifier Status

I Folate receptor binding peptide vaccine Peptide/protein NCT02019524 Open

I GP2-GM-CSF versus AE37 + GM-CSF versus GM-CSF alone

Peptide/protein NCT00524277 Open

I NY-EOS-1 vaccine + sirolimus Peptide/protein NCT01522820 Open

I MUC1-peptide for triple-negative breast cancer Peptide/protein NCT00986609 Open

II Trastuzumab + GM-CSF/HER-2 E75 peptide Peptide/Protein NCT01570036 Open

III NeuVax TM Peptide/protein NCT01479244 Open

I Pilot-breast cancer vaccine + Poly-ICLC Celullar vaccine NCT01532960 Open

II GSK2302024A (Wilms tumor 1-specific therapy) + adjuvant therapy

Celullar vaccine NCT01220128 Open

II Preoperative cryotherapy +/- ipilimumab (anti-CTLA-4)

Checkpoint inhibitors

NCT01502592 Open

I/II HER-2/Neu-pulsed DC1 vaccine combined with trastuzumab for patients with DCIS

Pulsed dentritic cells NCT02336984 Open

I/II Randomized trial of HER-2/Neu-pulsed DC1 vaccine for patients with DCIS

Pulsed dentritic cells NCT02061332 Open

I/II Nelipepimut-S E75 (HER2) NeuVax TM Peptide/protein Closed

I/II GP2 + GM-CSF Peptide/protein Closed

AE37 + GM-CSF Peptide/protein Closed

AE37/GP2 + GM-CSF Peptide/protein Closed

I A HER-2/Neu-pulsed DC1 vaccine for patients with DCIS

Pulsed dentritic cells NCT00107211 Closed

DCIS: Ductal carcinoma in situ; MUC-1: Mucin-1.



phosphamide, along with vaccination against HER2 has been shown to induce de novo HER2-specific T helper-dependent immunity [33]. Low-dose doxorubi-cin, paclitaxel and methotrexate have also been shown to enhance HER2-specific immunity with vaccina-tion as well as stimulate DC maturation and differen-tiation [33–35]. Therefore, chemotherapy may be better utilized in order to maximize the effectiveness of the antitumor immune response.

Combinations of vaccines with HER2-directed therapyTrastuzumab is a monoclonal antibody that targets the HER2/neu protein by binding to the extracel-lular domain, triggering HER2 internalization and degradation. The use of trastuzumab induces passive immunity to HER2, although a major limitation of this therapy after continued use is the development of drug resistance. About 70% of patients who ini-tially respond to therapy experience progression of the disease within approximately a year, suggesting the development of an acquired resistance to the anti-body [36]. Patients undergoing neoadjuvant trastu-zumab therapy plus chemotherapy may not show pCR due to depressed anti-HER2Th1 cell response [29]. Conversely, combination use of trastuzumab with vaccination against HER2 can significantly sensitize CTLs to tumor cells expressing the peptide and result in increased tumor cell lysis specific to HER2 [30,37]. In addition, it has recently been demonstrated that the anti-HER2Th1 response is positively correlated with pCR in patients given neoadjuvant chemother-apy plus trastuzumab. Those patients with a depressed anti-HER2Th1 response did not show pCR, but it was shown that this could be augmented with use of HER2-targeted DC vaccination [29]. Additionally, there is a strong association between immune gene expression and DFS following treatment with adju-vant trastuzumab, suggesting that there is a subset of HER2pos tumors with a high level of immunologic activity [38]. Thus, DC vaccination may prove to be a useful adjunct to trastuzumab therapy by restoring immune response to HER2.

Pertuzumab another anti-HER2 mAb, similar to trastuzumab, targets the extracellular dimerization domain of the HER2 receptor. Developed in attempts to combat resistance to trastuzumab, pertuzumab pre-vents HER2/HER3 heterodimerization [39,40]. Coad-ministration of the two therapies has been shown to enhance clinical effectiveness compared with either one administered singly [40,41]. Pertuzumab has not yet been studied in combination with vaccination against cancer, but, considering its similar mechanism of action and synergistic effects with trastuzumab,

DC vaccination could prove to be a clinically effective adjunct.

Similarly, lapatinib, a tyrosine kinase inhibitor (TKI) that disrupts the HER2/neu and EGFR, is used to treat HER2posBC. Like trastuzumab, patients may develop resistance fairly early. In attempts to combat this, vaccination against HER2 was given concomi-tantly with lapatinib in a clinical trial, which demon-strated safety of the combined treatment, but due to small sample size, no additional immunologic benefits were observed [42]. However, another TKI, axitinib, showed improved therapeutic efficacy when combined with DC vaccination in a preclinical model of murine melanoma [43]. Although DC vaccination combined with TKIs has not yet shown improved outcomes in breast cancer, improved anti-HER2 immunity acquired by vaccination may improve pCR and over-come resistance seen with continued use of TKIs [29,37].

RadiationRadiation is an important part of anticancer therapy, with nearly two-thirds of patients with cancer receiving it at some point during the course of their treatment [44]. Radiation is known for its ability to damage DNA of tumor cells, eventually causing enough injury to pro-hibit cellular proliferation and/or cause tumor cell death. Along with the antitumor properties that radiation has on its own, it may also have the ability to amplify the effects induction of apoptosis and necrosis; so APCs are ultimately attracted to this environment. This radiation-induced tumor microenvironment increases expression of Fas, MHC class I molecules and several other cell surface proteins [45], which provoke a DC-mediated antigen-specific immune response [46], therefore stimu-lating a CTL response. Correspondingly, the goal of vaccination is to induce an antigen-specific immune response to tumor cells, so addition of radiotherapy to vaccination should have a synergistic effect. This effect has been shown in a preclinical model of subcutaneous murine colorectal carcinoma where, after combination therapy of local radiation and vaccination, there was a massive infiltration of T cells that was not seen in either modality alone [47]. Prostate cancer patients treated with radiotherapy in combination of anti-PSA DC vaccines were able to increase immune response against PSA, but there was no clear synergistic effect of the combination of radiation with vaccination [45]. At this point, there have been no studies examining the effect of radiation therapy in combination with DC vaccines in the treat-ment of BC, however the ability of radiation to enhance MHC class I expression and consequently CTL infiltra-tion of tumor has been shown in other forms of cancer immunotherapy using monoclonal antibodies against molecules such as cytotoxic T-lymphocyte associated



antigen 4 (CTLA-4) and PD-1 [48–50]. Ultimately, radiotherapy may provide a useful adjunct to DC vac-cination in the treatment of BC to affect the tumor m icroenvironment.

Checkpoint inhibitorsTumors may utilize immune checkpoint pathways by expressing ligands that, under normal circumstances, would prevent aberrantly activated T cells from caus-ing autoimmunity [51,52]. Tumors may effectively hide from the immune system with expression of these ligands. By blocking the interaction of ligands with immune effector cells using monoclonal antibodies (mAb), the tumor cells may now be easily ‘seen’ by the immune system. Hence, two of these checkpoint path-ways that are currently areas of major investigation are the PD-1/PD-L1 and CTLA-4/B7 pathways, which have demonstrated significant success in melanoma, lung cancer and colon cancer [53–55] – all tumors with significant production of neoantigens.

PD-1 is expressed on effector immune cells – including T cells, NK cells, activated monocytes and DCs (Figure 2) [51]. Contact of PD-1 with its ligand, PDL-1, results in T-cell inactivation and apoptosis [56] and inhibits the activation of tumor-antigen-specific T cells [57]. Expression of PDL-1 on tumor cells may render them immunologically invisible. Like the PD-1 pathway, the CTLA-4 is a significant immune checkpoint receptor exploited in cancer. CTLA-4 is a CD28 homolog, which is upregulated upon T-cell activation and competes with CD28 for binding to APC ligands [58]. Tumor cells can express the CTLA-4 receptor on their surface [59] and counteract the activ-ity of the T-cell costimulatory receptor, CD28 [60] i mpairing tumor-reactive T cells.

mAb against PD-1 and CTLA4 may restore the function of disabled CD8pos T cells in cancer as well as preventing the depletion of activated memory B cells, resulting in a more robust immune response [61]. PD-1/PD-L1 blockade can neutralize the inhibi-

Figure 2. Multimodality enhancement of dentritic cells-based immunotherapy. In ex vivo manipulation, monocyte precursorsare sequentially matured and loaded with antigen to be injected. Lymphatics serve as sites of T-cell co-stimulation, DCs present antigen to T cells in the context of MHCClass I/II molecules, activating antigen-specific CD4posTh1 cells or CD8pos CTLs. These effector and helper populations migrate to the tumor bed, where they target tumor cells or elaborate cytokines (e.g., IFN-γ and TNF-α) mediating apoptosis. In conventional cytotoxic modalities radiation of tumor cells induces release of TAAs, pro-inflammatory cytokines (IL-1β, TNF-α), or endogenous TLR agonists (HMGB-1), activating DCs to prime antigen-specific CTL responses; antigens may also bepresented by stromal cells for destruction by CTLs. Chemotherapy generates an immune recovery cytokine environment and inhibits Treg and MDSC function. Endocrine and mAb-based targeted therapies halt downstream nuclear signaling and inhibit proliferation. Checkpoint inhibitors (CTLA-4 and PD-1) are aimed at recovering T-cell cytotoxicity and muting Treg and MDSCs. CTL: Cytotoxic T lymphocyte; mAb: Monoclonal antibody; MDSC: Myeloid-derived suppressor cell; TLR: Toll-like receptor.

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In ex vivo manipula�on, monocyte precursors are sequen�ally matured and loaded with an�gen to be injected. Lympha�cs serve as sites of T-cell co-s�mula�on, DCs present an�gen to T-cells in the context of MHC Class I/II molecules, ac�va�ng an�gen -specific CD4pos Th1 cells or CD8pos CTLs. These effector and helper popula�ons migrate to the tumor bed, where they target tumor cells or elaborate cytokines (e.g., IFN-γand TNF-α) media�ng apoptosis. .

Conven�onal cytotoxic modali�es: Radia�on of tumor cells induces release of TAAs, pro -inflammatory cytokines (IL-1β, TNF-α), or endogenous TLR agonists (HMGB-1), ac�va�ng DCs to prime an�gen-specific CTL responses; an�gens may also be presented by stromal cells for destruc�on by CTLs. Chemotherapy generates an immune recovery cytokine environment and inhibit Tregand MDSC func�on. Endocrine and mAb-based targeted therapies –halts downstream nuclear signaling and inhibits prolifera�on. Checkpoint inhibitors (CTLA-4 and PD-1) –aimed at recovering T-cell cytotoxicity and mu�ng Treg and MDSC.

FIGURE 2. Mul�modality enhancement of DC-based immunotherapy.

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tion of DC–T-cell interactions, restore APC function, repair T-cell function by increasing IFN-γ production, promoting T-cell proliferation [52] and T-cell targeting of tumors (Figure 2).

The restoration of T-cell function with the use of mAb against these checkpoint inhibitors presents a syn-ergistic opportunity for administration of DC vaccines. Using PD-1 mAb in conjunction with DC vaccination has shown an increase in CD4pos and CD8pos T-cell responses [56]. Along with restoring function of disabled immune cells, blocking the PD-1/PD-L1 checkpoint may further enhance the effectiveness of DC vaccina-tion by preventing suppression of DC maturation [52]. In a murine breast cancer model, administration of a monoclonal antibody against PD-L1 in combination with DC vaccination induced more potent protective immunity than just DC vaccination alone [52]. Anti-HER2 DC vaccination combined with anti-CTLA4 therapy in a murine mammary carcinoma model also exhibited a significant increase in the frequency of tumor infiltrating CD4pos and CD8pos T cells [62]. A probable mechanism for the success in this therapeutic combina-tion is the restoration and promotion of a robust Th1 response, encouraging cytotoxic CD8pos T-cell response and infiltration into the tumor [62]. Checkpoint inhibi-tors and DC vaccination seem to be an advantageous synergistic combination for activation of the immune system in cancer therapy for which there are ongoing trials [58]. Ultimately, the immune response needs help targeting oncodrivers, and the combination of check-point inhibitors with DC vaccines may prove to be the most successful combination.

DC vaccines for breast cancer escape with loss of HER2 oncodriver expressionGenetic profiling has allowed us to identify different oncodrivers associated with IBC as well as the four principal subtypes. HER2pos expressing tumor cells, as previously mentioned, may undergo phenotypic shifting or loss of immune response under anti-HER2 pressure (Figure 1) which may allow for disease pro-gression to HER2negIBC with subtypes such as basal like (TNBC) or Luminal A (ERposBC). With this in mind, modulating the immune responses using DC pulsed with antigens against other oncodrivers may aid in eliminating residual BC cells and prevent recurrence of escape variants that lose HER2/neu expression. We will discuss some of the potential oncodriver targets in the different subtypes of BC.

ER-positive breast cancer oncodriversMUC1Epithelial Mucin-1 (MUC1) is a large transmembrane protein and is the most widely expressed of the mucins,

located on the apical surface of human epithelial cells lin-ing glands or ducts. It is an overexpressed antigen found in about 90% of BC and is a cancer-specific class of vac-cine target [63]. Elevated levels of MUC1 on the tumor have been associated with invasiveness, tumor growth, metastatic properties leading to poor prognosis in colon, pancreas, breast and bladder cancer [64,65]. MUC1 has been found to stimulate ER-mediated transcription, antagonize tamoxifen and contribute to ER-induced growth and survival of BC cells [66]. These findings support for the idea that MUC1 is crucial to ER func-tion in BC. It contributes to tumorigenesis by reducing degradation of EGFR, increasing cell proliferation and inhibiting cell death by protecting against oxidative and genotoxic stress [67–69]. MUC1 has been studied as a target in cancer immunotherapy due to the ability to induce humoral immune responses in healthy subjects and in cancer patients [70]. Patients vaccinated with MUC1 core peptide-pulsed DC vaccines with MUC1-positive cancer were also found to have a reduction in tumor size or tumor marker levels as well as prolonged survival, suggesting that MUC1 is sufficiently immu-nogenic to be used as a target for DC vaccination [71]. More recently, MUC1 peptide vaccine study was able to induced INF gamma CD4pos and CD8pos T cells that recognized tumor-associated MUC1. Although only T-cell preactivation outside the tumor bed, either in cul-ture or by repetitive vaccination, showed to overcome escape phase of tumor ‘immunoediting’ [72].

HER2-positive breast cancer – other oncodrivershTERTHuman telomerase reverse transcriptase (hTERT) expression has been found to be increased in radio-resistant HER2posBC, while HER2 reduction has been shown to cause hTERT/telomerase activity (TA) downregulation. A widely expressed tumor anti-gen, hTERT, is expressed in more than 85% of all human cancers [73] and absent in most normal human cells [73], making it a favorable target for immuno-therapy. hTERT is not a cellular growth receptor or signal transducer, but rather overexpression, prolongs tumor cellular survival by maintaining chromosomal integrity and protecting telomeric DNA [74]. TA has been found in up to 75% of breast carcinoma in situ lesions. Proto-oncogenes and growth factors, such as p53 and HER2, have been associated with the regula-tion of TA. Studies have shown that HER2 mediates hTERT expression through activation of NF-κB and c-myc [75]. In the setting of immunotherapy, it has been observed that the hTERT peptide, I540, binds with high affinity to HLA-A2, and can be used to generate specific CTLs in vitro that lyse a wide range of hTERT-positive tumor cell lines [73]. Overall, hTERT-specific



immunity has been achieved with induction of CD8pos tumor infiltrating lymphocytes [76,77]. Although there was some immunity to the TAA peptide hTERT, trials have not yet proven DC vaccination against hTERT to be an effective therapeutic strategy.

HER3HER3 is another member of the EGFR family. Some studies have suggested that combination of HER2 and HER3 are crucial in cell growth, tumorigenesis and directly contribute in acquired resistance to therapies in HER2posBC and ERposBC. Although HER3 over-expression and activation is seen in invasive BC, a few studies have looked at it in the setting of DCIS, in which HER2pos status is frequently seen [78]. HER3 can channel ErbB signaling to PI3K pathway leading to its adeptness to favor tumor growth. Murine models have shown that overexpression of HER3 is seen in conjunc-tion with high levels of HER2 [79]. This is thought to occur via gene amplification, transcription or transla-tion. In cells with high HER2 and HER3, downregu-lation of HER3 affects both receptors, implying asso-ciation of the two for signal maintenance [79]. This may have impact in HER2-targeted therapies and provides resistance against pertuzumab, mTOR inhibitors and tamoxifen. In the case of tamoxifen, downregulation of ERBB3 in MCF-7 cells rendered resistant by HER2 overexpression promotes Tamoxifen-induced apopto-sis. This relationship allows for a chance at improv-ing efficacy of HER2-targeted therapies, by hindering linking of HER2 to HER3 potentially using DC vac-cines. HER-3 expression is also a poor prognostic fac-tor in TNBC [80] as it can heterodimerize with EGFR as well as transduce signals with c-MET.

Triple-negative breast cancer oncodrivers

c-MET & EGFRc-MET and EGFR amplification and overexpression have been associated with TNBC and basal-like sub-groups. EGFR is found in 50% of BC and c-MET is overexpressed in 20–30% of cases. c-MET is a tyrosine kinase receptor that binds to ligand EGFR or HGF for organ development, but when an anomalous acti-vation occurs it can lead to tumor development [81]. Overexpression has been associated with increasing tumor size, increased nodal involvement [82], decreased DFS, decreased overall survival and poor response to c hemotherapy [83].

c-MET amplification is associated with treatment failure to trastuzumab and shorter time to disease progression [84] in HER2posBC, and resistance to anti-EGFR therapies in TNBC [85]. Currently, Phase II t rials of anti-c-MET therapies are underway in the set-

ting of advanced BC using targeted therapies such as tyrosine kinase inhibitors and monoclonal antibodies.

Combined c-MET/EGFR therapies have been studied in head and neck, lung and colon cancer and more recently for TNBC. EGFR/MET inhibition is synergic and therefore targeting both may provide for insight to establishing an effective therapeutic strategy in the setting on TNBC were limited therapies are available [85]. This role of c-MET and EGFR in BC merits further investigation and potentially using them as a DC v accine target to block both pathways.

P53p53 is a tumor suppressor gene, regulator of the cell cycle, cellular proliferation and apoptosis, which is found to be mutated in 25% of BC. It is most com-monly seen in TNBC in about 50–80% [86]. Over-expression is associated with poor response to chemo-therapy [87], DFS and overall survival [88]. Tumors cells are able to bypass the G1 checkpoint and complete the cell cycle, even in the presence of DNA damage in the setting of a p53 mutation [86]. Th1 response is signifi-cantly more prevalent in patients with tumors exhibit-ing high expression of p53. With this in mind, future immunotherapeutic approaches may potentially be geared toward Th1 polarization to allow a more effec-tive immunotherapy in patients with advanced disease with p53 mutation [89].

ConclusionCancer immunotherapy is an important feature in the rapidly changing landscape of cancer treatment. Because breast cancer may represent a classic example of immunoediting, DC-based cancer vaccines can har-ness the potential to reinvigorate the immune system, moving toward elimination of tumor cells and shifting away from the equilibrium and escape stages. How-ever, a one-size-fits-all theory will not likely be possible with DC vaccines. The use of vaccines in early stages of breast cancer, such as in DCIS, has claimed more suc-cess than in late-stage use, likely because, in DCIS, the tumor cells and the immune response have achieved a state of equilibrium. Vaccines may be able to more readily tip the scales in favor of the immune system, thereby eliminating equilibrium and moving toward elimination of the tumor. Although DC vaccines have enjoyed some success in early stages of breast cancer, they still have been limited success when used in later stages. This limited success has shifted focus toward using DC vaccines in the adjuvant setting to prevent recurrence. Vaccination in combination with targeted molecular and immune therapies that provide an addi-tive or synergistic response, as well as using DC with cytotoxic therapies such as chemotherapy and radiation



remain underexplored. Combinations with checkpoint inhibitors are rapidly moving forward. DC vaccination therapy may also be used to target numerous TAAs, giving it the potential to be a personalized tumor treat-ment of tumor immune escape for essentially any phe-notypic subtype of breast cancers. The versatility of DC vaccines lends itself to being a p owerful tool in the future of cancer therapy.

Future perspectiveIn the past, monotherapy with DC vaccines did not meet the expectations as a standalone therapy in

advanced breast cancer. As we move forward, the focus of DC vaccines is being shifted to early disease and use in adjuvant settings. In addition, DC vac-cines are being incorporated in combination with conventional therapies such as radiation, chemother-apy and antiestrogen therapy as well as in combina-tion with other immune adjuvants in the treatment of early-stage breast cancer. It is presumed that such uses of DC vaccines will reduce recurrence and could potentially be developed for primary prevention. The synergistic effects will yield improved outcomes as the era of p ersonalized medicine continues.

Executive summary

Targeting HER2 & loss of anti-HER2• HER2/neu is associated with a heightened risk for invasion and metastasis, suggesting a crucial role for

HER2/neu during breast tumorigenesis. We have observed a progressive loss of anti-HER2 CD4pos Th1 (anti-HER2Th1) immune response during tumorigenesis in patients with HER-2 expressing breast cancer. HER2 vaccines can restore anti-HER2Th1 immune responses.

Role of immune response & immunoediting during breast tumorigenesis• Since healthy women have robust anti-HER-2 CD4 Th1 responses, we hypothesize that in the initial stage

of malignant transformation, the mounted immune response is robust enough against this HER2/neu overexpression and may eliminate HER2posDCIS or invasive breast cancer (IBC) from becoming clinically apparent. If elimination is not achieved, HER2/neu overexpressing tumor cells and the immune response develop into a stalemate of equilibrium where the anti-HER2Th1 immune response is unable to completely eliminate malignant cells, but is able to prevent them from becoming invasive. This equilibrium state may be clinically manifested as HER2posDCIS and may remain in this state of equilibrium and the anti-HER-2 Th1 response further erodes allowing tumor escape as HER-2posIBC or anti-HER2Th1 response is sustained but ineffectual and tumor cells manage to evade immunosurveillance via phenotypic shifting.

Activation of type I polarized DC drive strong anticancer Th1 responses• CD4pos T cells may be a crucial element in the antitumor immune response because of their role as helpers in

keeping CD8pos cells functionally active. Th1 cells secrete high-level cytokines such as IFN-γ and TNF-α, both key components in antitumor function. These cytokines can induce apoptosis of HER-2 expressing breast cancer cells and upregulate MHC class I expression making the cells more susceptible to CD8-mediated cytolysis. Dendritic cell (DC) polarized toward high levels of IL-12 production specifically drive anti-HER-2 CD4 Th1.

Effectiveness of DC1 vaccines administered in the equilibrium phase of DCIS• We suggest that HER2posDCIS represents the equilibrium phase of immunoediting where Th1 immune response

is in homeostasis with cancer cells in the ducts. Stimulating a strong Th1 immune response may lead to eradication or return to the elimination phase. Results from ductal carcinoma in situ patients treated with HER-2-pulsed DC1 demonstrated that some patients who received HER2-pulsed DC1 vaccines alone achieved pCR. In addition, others have remaining tumor with loss of HER-2 suggesting tumors can escape. Combining vaccines targeting multiple oncodrivers or combination therapies may prevent immunoediting.

Use of DC1 vaccines during tumor escape in combination with anti-HER2 therapies• Persistent diminished anti-HER2Th1 immune response is associated with lack of pCR to neoadjuvant

chemotherapy and HER2-targeted therapies. Ongoing clinical trials as being performed to assess whether restoring anti-HER-2 CD4 Th1 can prevent recurrence or combined with initial therapies to cause more complete responses to standard therapy. Combining the anti-HER-2 therapies and other immune modulating agents such as checkpoint inhibitors are subjects of future trials to augment effective anti-HER-2 CD4 Th1 responses.

DC vaccines for breast cancer escape with loss of HER2 oncodriver expression• Ineffectual anti-HER-2 Th1 responses may lead HER2pos expressing tumor cells to undergo phenotypic shifting

or loss of immune response under anti-HER2 pressure which may allow for disease progression to HER2negIBC with subtypes such as basal like (triple-negative breast cancer) or Luminal A (ERposBC). Modulating immune responses using DC pulsed with antigens against other oncodrivers such as Mucin-1, c-MET, human telomerase reverse transcriptase, HER3 may aid in eliminating residual BC cells and prevent recurrence of escape variants that lose HER2/neu expression.



Financial & competing interests disclosureWork from our laboratory cited in this review was funded

by NIH R01 CA096997, Henle Fund Research Fellowship (*)

and Pennies in Action® Foundation. No external funding was

secured for this study. The authors have no financial relation-

ships or conflicts of interest relevant to this article to disclose.

Work from our laboratory cited in this review was funded by

National Institutes of Health R01 CA096997, Henle Fund Re-

search Fellowship (*) and Pennies in Action® Foundation. The

authors have no other relevant affiliations or financial involve-

ment with any organization or entity with a financial interest

in or financial conflict with the subject matter or materials dis-

cussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this

manuscript.

ReferencesPapers of special note have been highlighted as: •• of considerable interest

1 Witton CJ, Reeves JR, Going JJ, Cooke TG, Bartlett JM. Expression of the HER1–4 family of receptor tyrosine kinases in breast cancer. J. Pathol. 200(3), 290–297 (2003).

2 Czerniecki BJ, Koski GK, Koldovsky U et al. Targeting HER-2/neu in early breast cancer development using dendritic cells with staged interleukin-12 burst secretion. Cancer Res. 67(4), 1842–1852 (2007).

3 Disis ML, Calenoff E, Mclaughlin G et al. Existent T-cell and antibody immunity to HER-2/neu protein in patients with breast cancer. Cancer Res. 54(1), 16–20 (1994).

4 Roses RE, Paulson EC, Sharma A et al. HER-2/neu overexpression as a predictor for the transition from in situ to invasive breast cancer. Cancer Epidemiol. Biomarkers Prev. 18(5), 1386–1389 (2009).

5 Hassett MJ, Jiang W, Habel LA et al. Characteristics of second breast events among women treated with breast-conserving surgery for DCIS in the community. Breast Cancer Res. Treat. 155(3), 541–549 (2016).

6 Harada S, Mick R, Roses RE et al. The significance of HER-2/neu receptor positivity and immunophenotype in ductal carcinoma in situ with early invasive disease. J. Surg. Oncol. 104(5), 458–465 (2011).

7 Datta J, Rosemblit C, Berk E et al. Progressive loss of anti-HER2 CD4+ T-helper type 1 response in breast tumorigenesis and the potential for immune restoration. OncoImmunology 4(10), e1022301 (2015).

•• FirstdescriptionoftheprogressiveandspecificlossofCD4+Th1immunitytoamolecularoncodriverduringbreasttumorigenesis.Pointsintothenegativeclinicalandpathologicoutcomesassociatedwithdepressedanti-HER2Th1immunityandimpliesthatimmunerestorationwithvaccinationorotherimmunemodulatingstrategiesmaybeanoptiontodiminishtumorprogressionorpreventrecurrence.

8 Datta J, Fracol M, Mcmillan MT et al. Association of depressed anti-HER2 T-helper type 1 response with recurrence in patients with completely treated HER2-positive breast cancer: role for immune monitoring. JAMA Oncol.

2(2), 242–246 (2015).

9 Hennighausen L, Robinson GW. Signaling pathways in mammary gland development. Dev. Cell 1, 467–475 (2001).

10 Schreiber RD, Old LJ, Smyth MJ. Cancer immunoediting: integrating immunity’s roles in cancer suppression and promotion. Science 331(6024), 1565–1570 (2011).

11 Wherry E. T cell exhaustion. Nat. Immunol. 12(6), 492–499 (2011).

12 Zhu YY SW, Ji TF, Guo XQ, Hu Y, Yang Jl. The variation and clinical significance of hormone receptors and HER-2 status from primary to metastatic lesions in breast cancer patients. Tumour Biol. 37(6), 675–684 (2015).

13 Merad M, Sathe P, Helft J, Miller J, Mortha A. The dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting. Annu. Rev. Immunol. 31, 563–604 (2013).

14 Bancherau J, Briere F, Caux C et al. Immunobiology of dendritic cells. Annu. Rev. Immunol. 18, 767–811 (2000).

15 Kono M, Nakamura Y, Suda T et al. Enhancement of protective immunity against intracellular bacteria using type-1 polarized dendritic cell (DC) vaccine. Vaccine 30(16), 2633–2639 (2012).

16 Sallusto F, Schaerli P, Loetscher P et al. Rapid and coordinated switch in chemokine receptor expression during dendritic cell maturation. Eur. J. Immunol. 28(9), 2760–2769 (1998).

17 Austyn J. Death, destruction, danger and dendritic cells. Nat. Med. 5(11), 1232–1233 (1999).

18 Curtsinger JM, Johnson CM, Mescher MF. CD8 T cell clonal expansion and development of effector function require prolonged exposure to antigen, costimulation, and signal 3 cytokine. J. Immunol. 171(10), 5165–5171 (2003).

19 Kaiko Ge HJ, Beagley KW, Hansbro PM. Immunological decision-making: how does the immune system decide to mount a helper T-cell response? Immunology 123(3), 326–338 (2008).

20 Antony PA, Piccirillo CA, Akpinarli A et al. CD8+ T cell immunity against a tumor/self-antigen is augmented by CD4+ T helper cells and hindered by naturally occurring T regulatory cells. J. Immunol. 174(5), 2591–2601 (2005).

21 Gao FG, Khammanivong V, Liu WJ, Leggatt GR, Frazer IH, Fernando GJ. Antigen-specific CD4+ T-cell help is required to activate a memory CD8+ T cell to a fully functional tumor killer cell. Cancer Res. 62(22), 6438–6441 (2002).

22 Huang Y, Ma C, Zhang Q et al. CD4+ and CD8+ T cells have opposing roles in breast cancer progression and outcome. Oncotarget 6(19), 17462–17478 (2015).

23 Scott P. IL-12: initiation cytokine for cell-mediated immunity. Science 260(5107), 496–497 (1993).

24 Pulendran B. Pulendran modulating TH1/TH2 responses with microbes, dendritic cells, and pathogen recognition receptors. Immunol. Res. 29(1), 187–196 (2004).

25 Cintolo JA, Datta J, Mathew SJ, Czerniecki BJ. Dendritic cell-based vaccines: barriers and opportunities. Future Oncol. 8(10), 1273–1299 (2012).



26 Sharma A, Koldovsky U, Xu S et al. HER-2 pulsed dendritic cell vaccine can eliminate HER-2 expression and impact ductal carcinoma in situ. Cancer 118(17), 4354–4362 (2012).

•• ResultsfromthisstudysuggestthattargetingtheHER-2/neupathwayinearlybreastcancerandductalcarcinomain situusingDC1pulsedwithHER-2/neupeptidesboostedanti-HER-2CD4Th1responsesandcausedregressioninsomeoftheselesions,especiallythosewithestrogen-independentHER-2/neuposductalcarcinomain situ.

27 Koski GK, Koldovsky U, Xu S et al. A novel dendritic cell-based immunization approach for the induction of durable Th1-polarized anti-HER-2/neu responses in women with early breast cancer. J. Immunother. 35(1), 54–65 (2012).

28 Fracol M, Xu S, Mick R et al. Response to HER-2 pulsed DC1 vaccines is predicted by both HER-2 and estrogen receptor expression in DCIS. Ann. Surg. Oncol. 20(10), 3233–3239 (2013).

29 Datta J, Berk E, Xu S et al. Anti-HER2 CD4 (+) T-helper type 1 response is a novel immune correlate to pathologic response following neoadjuvant therapy in HER2-positive breast cancer. Breast Cancer Res. 17, 71 (2015).

30 Mittendorf EA, Storrer CE, Shriver CD, Ponniah S, Peoples GE. Investigating the combination of trastuzumab and HER2/neu peptide vaccines for the treatment of breast cancer. Ann. Surg. Oncol. 13(8), 1085–1098 (2006).

31 Denkert C, Loibl S, Noske A et al. Tumor-associated lymphocytes as an independent predictor of response to neoadjuvant chemotherapy in breast cancer. J. Clin. Oncol. 28(1), 105–113 (2010).

32 García-Martínez E, Gil GL, Benito AC et al. Tumor-infiltrating immune cell profiles and their change after neoadjuvant chemotherapy predict response and prognosis of breast cancer. Breast Cancer Res. 16, 1–17 (2014).

33 Emens LA, Asquith JM, Leatherman JM et al. Timed sequential treatment with cyclophosphamide, doxorubicin, and an allogeneic granulocyte-macrophage colony-stimulating factor-secreting breast tumor vaccine: a chemotherapy dose-ranging factorial study of safety and immune activation. J. Clin. Oncol. 27(35), 5911–5918 (2009).

34 Kaneno R, Shurin GV, Tourkova IL, Shurin MR. Chemomodulation of human dendritic cell function by antineoplastic agents in low noncytotoxic concentrations. J. Transl. Med. 7, 58 (2009).

35 Pfannenstiel LW, Lam SS, Emens LA, Jaffee EM, Armstrong TD. Paclitaxel enhances early dendritic cell maturation and function through TLR4 signaling in mice. Cell. Immunol. 263(1), 79–87 (2010).

36 Gajria D, Chandarlapaty S. HER2-amplified breast cancer: mechanisms of trastuzumab resistance and novel targeted therapies. Expert Rev. Anticancer Ther. 11(2), 263–275 (2011).

37 Disis ML, Wallace DR, Gooley TA et al. Concurrent trastuzumab and HER2/neu-specific vaccination in patients with metastatic breast cancer. J. Clin. Oncol. 27(28), 4685–4692 (2009).

•• Reportedsafetyandtolerabilityofcombinationtherapy

oftrastuzumabandaHER2/neu-specificvaccine,aswellasdemonstratedthepotentialuseofvaccinestoboostandmaintainpre-existingimmunitytoHER2/neuwithimmunization.Moreover,theyshowedepitopespread,whichcouldbeinvolvedinmodulatingsystemicmediatorsoftumor-inducedimmunesuppression.

38 Perez EA, Thompson EA, Ballman KV et al. Genomic analysis reveals that immune function genes are strongly linked to clinical outcome in the North Central Cancer Treatment Group n9831 Adjuvant Trastuzumab Trial. J. Clin. Oncol. 33(7), 701–708 (2015).

•• Discussedtheimportanceofidentifyingpatientswhoarelikelytobenefitfromtrastuzumabonthebasisofevaluationofanimmuneresponsegenesignature.Patientswithlowimmuneresponsesignaturedidnotobtainassignificantbenefitfromtrastuzumabasthosewithhighimmuneresponsesignature.ThisopensthepossibilityforfutureclinicaltrialsdesignedtoevaluatetherapeuticapproachesthatmightenhancetheimmunesignaturewithinHER2-positivetumorsandtherebysensitizethetumorstobiologictherapies.HER-2-pulsedDC1vaccinesmaybeonewaytoboostthetumorimmunesignature.

39 Zanardi E, Bregni G, De Braud F, Di Cosimo S. Better together: targeted combination therapies in breast cancer. Semin. Oncol. 42(6), 887–895 (2015).

40 Gollamudi J, Parvani JG, Schiemann WP, Vinayak S. Neoadjuvant therapy for early-stage breast cancer: the clinical utility of pertuzumab. Cancer Manag. Res. 8, 21–31 (2016).

41 Gianni L, Pienkowski T, Im YH, et al. Efficacy and safety of neoadjuvant pertuzumab and trastuzumab in women with locally advanced, inflammatory, or early HER2-positive breast cancer (NeoSphere): a randomised multicentre, open-label, Phase II trial. Lancet Oncol. 13(1), 25–32 (2012).

42 Hamilton E, Blackwell K, Hobeika AC et al. Phase 1 clinical trial of HER2-specific immunotherapy with concomitant HER2 kinase inhibition [corrected]. J. Transl. Med. 10, 28 (2012).

43 Bose A, Lowe DB, Rao A, Storkus WJ. Combined vaccine+axitinib therapy yields superior antitumor efficacy in a murine melanoma model. Melanoma Res. 22(3), 236–243 (2012).

44 Verbrugge I, Hagekyriakou J, Sharp LL et al. Radiotherapy increases the permissiveness of established mammary tumors to rejection by immunomodulatory antibodies. Cancer Res. 72(13), 3163–3174 (2012).

45 Gulley JL, Arlen PM, Bastian A, et al. Combining a recombinant cancer vaccine with standard definitive radiotherapy in patients with localized prostate cancer. Clin. Cancer. Res. 11(9), 3353–3362 (2005).

46 Friedman E. Immune modulation by ionizing radiation and its implications for cancer immunotherapy. Curr. Pharm. Des. 8(19), 1765–1780 (2002).

47 Chakraborty MA, Scott I, Coleman N et al. External beam radiation of tumors alters phenotype of tumor cells to render them susceptible to vaccine-mediated T-cell killing. Cancer Res. 64, 4328–4337 (2004).



48 Demaria S, Kawashima N, Yang AM et al. Immune-mediated inhibition of metastases after treatment with local radiation and CTLA-4 blockade in a mouse model of breast cancer. Clin. Cancer. Res. 11, 728–734 (2005).

49 Vanpouille-Box C, Diamond JM, Pilones KA et al. TGFbeta Is a master regulator of radiation therapy-induced antitumor immunity. Cancer Res. 75(11), 2232–2242 (2015).

50 Reits EA, Hodge JW, Herberts CA et al. Radiation modulates the peptide repertoire, enhances MHC class I expression, and induces successful antitumor immunotherapy. J. Exp. Med. 203(5), 1259–1271 (2006).

51 Keir ME, Butte MJ, Freeman GJ, Sharpe AH. PD-1 and its ligands in tolerance and immunity. Annu. Rev. Immunol. 26, 677–704 (2008).

52 Ge Y, Xi H, Ju S, Zhang X. Blockade of PD-1/PD-L1 immune checkpoint during DC vaccination induces potent protective immunity against breast cancer in hu-SCID mice. Cancer Lett. 336(2), 253–259 (2013).

53 Ribasa C, Comin-Anduix B, Chmielowski B et al. Dendritic cell vaccination combined with CTLA4 blockade in patients with metastatic melanoma. Clin. Cancer. Res. 15(19), 6267–6276 (2009).

54 Topalian SL, Hodi FS, Brahmer JR et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N. Engl. J. Med. 366(26), 2443–2454 (2012).

55 Hamid O, Robert C, Daud A et al. Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma. N. Engl. J. Med. 369(2), 134–144 (2013).

56 Rosenblatt J, Glotzbecker B, Mills H et al. PD-1 blockade by CT-011, anti-PD-1 antibody, enhances ex vivo T-cell responses to autologous dendritic cell/myeloma fusion vaccine. J. Immunother. 34(5), 409–418 (2011).

57 Ashley C-M, Jeremy BF, Leisha AE. Immune targeting in breast cancer. Oncology 29(5), 375 (2015).

58 Datta J, Berk E, Cintolo JA, Xu S, Roses RE, & Czerniecki BJ. Rationale for a multimodality strategy to enhance the efficacy of dendritic cell-based cancer immunotherapy. Front. Immunol. 6, 271 (2015).

59 Katz T, Avivi I, Benyamini N, Rosenblatt J, Avigan D. Dendritic cell cancer vaccines: from the bench to the bedside. Rambam Maimonides Med. J. 5(4), e0024 (2014).

60 Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat. Rev. Cancer 12(4), 252–264 (2012).

61 Zitvogel L, Kroemer G. Targeting PD-1/PD-L1 interactions for cancer immunotherapy. Oncoimmunology 1(8), 1223–1225 (2012).

62 Linch S, Kasiewicz MJ, Mcnamara MJ, Hilgart-Martiszus IF, Farhad M, & Redmond WL. Combination OX40 agonism/CTLA-4 blockade with HER2 vaccination reverses T-cell anergy and promotes survival in tumor-bearing mice. Proc. Natl. Acad. Sci. USA 113(3), E319–E327 (2016).

63 Bafna S, Kaur S, Batra SK. Membrane-bound mucins: the mechanistic basis for alterations in the growth and survival of cancer cells. Oncogene 29(20), 2893–2904 (2010).

64 Suzuki H, Shoda J, Kawamoto T et al. Expression of MUC1 recognized by monoclonal antibody MY.1E12 is a useful biomarker for tumor aggressiveness of advanced colon

carcinoma. Clin. Exp. Metastasis 21(4), 321–329 (2004).

65 Ghosh SK, Pantazopoulos P, Medarova Z, & Moore A. Expression of underglycosylated MUC1 antigen in cancerous and adjacent normal breast tissues. Clin. Breast Cancer 13(2), 109–118 (2013).

66 Kufe DW. MUC1-C oncoprotein as a target in breast cancer: activation of signaling pathways and therapeutic approaches. Oncogene 32(9), 1073–1081 (2013).

67 Rivalland G, Loveland B, Mitchell P. Update on Mucin-1 immunotherapy in cancer: a clincal perspective. Expert Opin. Biol. Ther. 15(12), 1773–1787 (2015).

68 Burchell JM, Mungul A, Taylor-Papadimitriou J. O-linked glycosylation in the mammary gland: changes that occur during malignancy. J. Mammary Gland Biol. Neoplasia 6(3), 355–364 (2001).

69 Blixt O, Bueti D, Burford B et al. Autoantibodies to aberrantly glycosylated MUC1 in early stage breast cancer are associated with a better prognosis. Breast Cancer Res. 13(2), R25 (2011).

70 Hanisch FG. Design of a MUC1-based cancer vaccine. Biochem. Soc. Trans. 33(4), 705–708 (2005).

71 Keiichi K, Taguchi O, Ozaki Y et al. Dendritic cell vaccine immunotherapy of cancer targeting MUC1 mucin. Int. J. Mol. Med. 12, 493–502 (2003).

72 Lakshmiarayanan V, Supekar NT, Wei J et al. MUC1 vaccines, comprised of glycosylated or non-glycosylated peptides or tumor- derived MUC1, can circumvent immunoediting to control tumor growth in MUC1 transgenic mice. PLoS One 11(1), e0145920 (2016).

73 Domchek SM, Recio A, Mick R et al. Telomerase-specific T-cell immunity in breast cancer: effect of vaccination on tumor immunosurveillance. Cancer Res. 67(21), 10546–10555 (2007).

74 William CH, Matthew M. Telomerase activation, cellular imortalization and cancer. Ann. Med. 33, 123–129 (2001).

75 Papanikolaou V, Athanassiou E, Dubos S et al. hTERT regulation by NF-jB and c-myc in irradiated HER2-positive breast cancer cells. Int. J. Radiat. Biol. 87(6), 609–621 (2011).

76 Märten A, Sievers E, Albers P et al. Telomerase-pulsed dendritic cells: preclinical results and outcome of a clinical Phase I/II trial in patients with metastatic renal cell carcinoma. GMS German Med. Sci. 4, Doc02 (2006).

77 Vonderheide RH, Domchek SM, Schultze JL et al. Vaccination of cancer patients against telomerase induces functional antitumor CD8+ T lymphocytes. Clin. Cancer. Res. 10, 828–839 (2004).

78 Holbro T, Beerli RR, Maurer F, Koziczak M, Barbas CF 3rd, Hynes NE. The ErbB2/ErbB3 heterodimer functions as an oncogenic unit: ErbB2 requires ErbB3 to drive breast tumor cell proliferation. Proc. Natl. Acad. Sci. USA 100(15), 8933–8938 (2003).

79 Siegel P, Ryan ED, Cardiff RD, Muller WJ. Elevated expression of activated forms of Neu/ErbB-2 and ErbB-3 are involved in the induction of mammary tumors in transgenic mice: implications for human breast cancer. . EMBO J. 18, 2149–2164 (1999).



80 Green AR, Barros FF, Abdel-Fatah TM et al. HER2/HER3 heterodimers and p21 expression are capable of predicting adjuvant trastuzumab response in HER2+ breast cancer. Breast Cancer Res. Treat. 145(1), 33–44 (2014).

81 Siziopikou KP, Cobleigh M. The basal subtype of breast carcinomas may represent the group of breast tumors that could benefit from EGFR-targeted therapies. Breast (Edinburgh, Scotland) 16(1), 104–107 (2007).

82 Kim YJ, Choi JS, Seo J et al. MET is a potential target for use in combination therapy with EGFR inhibition in TNBC. Int. J. Cancer 134(10), 2424–2436 (2014).

83 Lengyel E, Prechtel D, Resau JH et al. c-met expression in node positive breast cancer identifies patients with poor clinical outcomes independent of HER2/neu. Int. J. Cancer 113(4), 678–682 (2005).

84 Minuti G, Cappuzzo F, Duchnowska R et al. Increased MET and HGF gene copy numbers are associated with trastuzumab failure in HER2 positive metastatic cancer. Br. J. Cancer 107(5), 793–799 (2012).

85 Sohn J, Liu S, Parinyanitikul N et al. cMET activation and EGFR-directed therapy resistance in triple-negative breast cancer. J. Cancer 5(9), 745–753 (2014).

86 Turner N, Moretti E, Siclari O et al. Targeting triple negative breast cancer: is p53 the answer? Cancer Treat. Rev. 39(5), 541–550 (2013).

87 Kandioler-Eckersberger D, Ludwig C, Rudas M et al. TP53 mutation and p53 overexpression for prediction of response to neoadjuvant treatment in breast cancer patients. Clin. Cancer. Res. 6(1), 50–56 (2000).

88 Miller LD, Smeds J, George J et al. An expression signature for p53 status in human breast cancer predicts mutation status, transcriptional effects, and patient survival. Proc. Natl. Acad. Sci. USA 102(38), 13550–13555 (2005).

89 Met O, Balslev E, Flyger H, Svane IM. High immunogenic potential of p53 mRNA-transfected dendritic cells in patients with primary breast cancer. Breast Cancer Res. Treat. 125(2), 395–406 (2011).

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Breast CancerManagement

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PersPective

Use of complementary therapies for side effect management in breast cancer: evidence and rationaleKelly Roe1, Moira Kirvan Visovatti1, Trevor Brooks1, Mohamad Baydoun1, Patricia Clark1 & Debra L Barton*,1

1School of Nursing, University of Michigan, Ann Arbor, MI 48109, USA

*Author for correspondence: [email protected]

Women diagnosed with breast cancer can experience chronic side effects after curative treatment concludes, negatively impacting survivorship. The most prevalent side effects addressed in the medical and nursing literature include symptoms such as hot flashes, fatigue, myalgias/arthralgias and cognitive impairment. Complementary therapies, particularly natural products including herbs, dietary supplements, vitamins, minerals, and probiotics, and mind–body techniques that include such modalities as yoga, meditation, massage, acupuncture, relaxation, tai chi and hypnosis show promise for treatment of some of these symptoms associated with cancer care. However, the research in this area is nascent and much more work is needed to understand symptom physiology and mechanisms of action of complementary therapies. The purpose of this paper was to summarize key evidence from Phase II and III randomized clinical trials in order to provide guidance to distinguish promising versus nonpromising interventions for symptom management.

First draft submitted: 2 March 2016; Accepted for publication: 13 July 2016; Published online: 31 August 2016

Keywords • AIMSS • arthralgias/myalgias • cognitive impairment • complementary therapies • fatigue • ginseng • hot flashes • mind and body techniques • yoga • Ginkgo biloba

For approximately 3.1 million breast cancer survivors in the USA [1], side effects from treatment may lead to symptoms that persist long after active treatment [2]. These symptoms, such as cancer-related fatigue (CRF), hot flashes, myalgias/arthralgias and cognitive impairment, affect many women even a decade into their recovery thus diminishing overall health-related quality of life [3]. For difficult to manage symptoms, the use of complementary therapy modalities has increased within the past three decades, particularly within the cancer population [4]. Complementary

Practice points

● Complementary therapy use is common among women with a history of breast cancer and too often, this use is not reported to the medical care team. Therefore, it is important to ask patients if they are using any over-the-counter products or mind–body therapies and to ask with in an open, accepting manner.

● Women with breast cancer who use complementary therapy are typically younger, well-educated, of higher socioeconomic status and not currently receiving chemotherapy or hormone therapy.

● Some of the most prevalent physical symptoms experienced by women having been treated for breast cancer include hot flashes, fatigue, myalgias/arthralgias and cognitive impairment.

● Few natural products, also called dietary supplements, have been shown to be effective for symptoms in well-designed, placebo-controlled trials, and mechanisms of action are often not known.

● There is evidence that hypnosis can reduce hot flashes and may reduce cancer-related fatigue.

● Yoga has evidence for improving fatigue and cognitive impairment.

● Arthralgias/myalgias from aromatase inhibitors may be improved with acupuncture.

Breast Cancer Manag. (Epub ahead of print) ISSN 1758-1923


PersPective Roe, Visovatti, Brooks, Baydoun, Clark & Barton


therapies are defined by the National Center for Complementary and Integrative Health (NCCIH) as natural products, including herbs, dietary supplements, vitamins, minerals and probiotics, and mind–body techniques that include such modalities as yoga, meditation, massage, acupuncture, relaxation, tai chi and hypnosis [5]. According to the NCCIH, more research is needed surrounding long-term effects of cancer treatment and these challenges facing patients well into their survivorship [5].

Prevalence of use of complementary therapies in women with breast cancerWomen with breast cancer express interest in complementary therapies, but historically underreport usage due to fear of dismissive-ness from their providers [6]. Overall, patients report improved satisfaction when a provider can educate regarding options, integrate care and encourage patients to actively participate in their treatment course [7]. This facilitates a healthy patient-provider relationship while pro-moting hopefulness and empowerment toward self-care [6]. Integrating complementary thera-pies with conventional care has long existed in other countries around the world. Currently in European countries, complementary therapy use ranges from 14.9 to 73.1% of women with breast cancer [6]. Within the USA, the NCCIH [5] esti-mates that up to 90% of women with breast can-cer have utilized some form of complementary therapy during their treatment course.

Women with breast cancer who use comple-mentary therapy are typically younger, well-educated, of higher socioeconomic status (SES) and not currently receiving chemotherapy or hormone therapy [8,9]. Women with breast can-cer tend to favor natural products (21%) and mind–body therapies (13%) for relief of long-term symptoms, improved overall health or for increased health-related quality of life [8,10–11].

common side effects/toxicities women with breast cancer experiencePatients undergo a variety of treatments dur-ing their breast cancer experience that elicit a wide range of side effects and symptoms spe-cific to that particular stage of care. During the surgical phase (mastectomy, lumpectomy or reconstruction), patients are at increased risk of postoperative symptoms such as infection, pain, swelling or impaired sleep. During chemother-apy, patients suffer from nausea and vomiting,

neuropathy, fatigue and ‘chemo brain’ or cog-nitive impairment [1]. For patients undergoing radiation therapy, common side effects include skin irritation, soreness or localized swelling [1,8]. Patients continuing with long-term adjuvant endocrine therapy continue to experience fatigue (15–90%), vaginal changes (21–48%), hot flashes (50–70%) and musculoskeletal changes, arthralgias and myalgias [2,12]. Hence, survi-vors of breast cancer can face many side effects indefinitely into their survivorship [2].

summaryGiven the growing population of breast can-cer survivors, and the prevalence of ongoing cancer-related symptoms [4], further knowledge regarding effective treatment of these symptoms is warranted. While complementary therapies are commonly used, the majority have not been rigorously tested for overall safety or efficacy [13]. There is preliminary research that attempts to elucidate the benefit, or lack of effect, of sev-eral complementary therapies. This paper will review the evidence for complementary thera-pies, specifically focusing on natural products and mind–body therapies, for some of the most common symptoms women with breast cancer experience, namely, hot flashes, fatigue, arthral-gias/myalgias and cognitive impairment [4]. The literature search for this paper was performed from December 2015 to February 2016 and included PubMed, CINAHL, EMBASE and MEDLINE databases. First, the literature was searched for manuscripts that described the prevalence of symptoms in breast cancer survi-vorship. The most prevalent symptoms formed the basis for the next level of the literature search. Search terms included combinations of the terms ‘breast cancer’, ‘complementary therapy’, ‘alter-native therapy’ and ‘natural products’ with the following symptom terms: hot flashes, fatigue, menopausal symptoms, joint pain, arthral-gias, myalgias, AIMSS and cognitive function. Studies included in this paper were published in English and included randomized, controlled clinical trials or pilot studies with pre–post intervention testing conducted in ambulatory settings in which the primary outcome of the study was the symptom of interest. Populations included breast cancer only and mixed popula-tions in which the majority of participants had a breast cancer diagnosis. Manuscripts in which the symptom of interest was not the primary outcome or included pharmacologic therapy that

bmt-2016-0013 Breast Cancer Manag. (Epub ahead of print)

Use of complementary therapies for side effect management in breast cancer PersPective


was not a dietary supplement were excluded. The reference lists for manuscripts selected for inclusion were scanned for relevant studies.

Hot flashesHot flashes are a prevalent symptom after men-opause (natural and surgical), and also after treatment for breast cancer. They manifest as a sudden feeling of warmth, with or without sweating, that involves much of the body and may be associated with feelings of panic and anxiety [14]. It is estimated that 96% of women with breast cancer report hot flashes soon after beginning anticancer therapy [15] and up to 73% reporting hot flashes 6 years after diag-nosis, even after estrogen deprivation therapy has been completed [16]. Hot flashes in women with breast cancer are particularly distressing since they can occur much earlier than their peer group. Estrogen deprivation therapies such as tamoxifen or aromatase inhibitors (AIs) can cause hot flashes and these therapies are taken for at least 5 or more years. Hot flash physiology is not definitively known, however, the current hypothesis suggests that dysregu-lation of the autonomic nervous system may contribute to hot flash symptomatology, both in terms of objective rises in temperature, and

in subjective discomfort [17,18]. Management of hot flashes is critical for the maintenance of a high quality of life as they can interrupt sleep and thereby cause mood and cognitive alterations. There are some medications that are effective for reducing hot flashes, but these are mostly antidepressants that are associated with side effects and for some women, negative stigma. Therefore, women often seek comple-mentary and alternative therapies for the relief of hot flashes, hoping for effectiveness without unwanted side effects.

●● Natural productsStudies of dietary supplements have been informed by the broad evidence base that sero-tonergic antidepressants have been effective in reducing hot flashes. Physiologic hypotheses for the etiology of hot flashes include low levels of serotonin as well as over activity of the sympa-thetic nervous system [17,19]. Natural products that have been evaluated for hot flashes include soy [20], black cohosh [21], vitamin E [22], mag-nesium oxide [23] and f laxseed (table 1) [24]. Despite encouraging preliminary data, mostly in single arm trials, numerous randomized con-trolled trials (RCTs) evaluating various dietary supplements for hot flash reduction have been

table 1. randomized placebo controlled trials with natural products for hot flashes.

study (year), study size

Natural product Dose and duration Primary outcome measure

results ref.

Barton et al. (1998), n = 120

Vitamin E Vitamin E 800 IU Placebo 4-week crossover design

HF score; severity × frequency per prospective HF diary, self-report

Statistically significant favoring vitamin E on crossover analysis but not clinically meaningful

[22]

Quella et al. (2000), n = 177

Phytoestrogens Phytoestrogens 600 mg with 50 mg soy isoflavones: genistein, diadzein, glycitein Placebo 4-week crossover design


No statistically significant difference, placebo looked a little better than the soy

[20]

Pruthi et al. (2012), n = 188

Flaxseed Flaxseed bars 40 g flaxseed; 410 mg lignans Placebo bars 1 bar daily for 6 weeks


HF reduction flaxseed: 29% HF reduction placebo: 28% Not statistically significant

[24]

Pockaj et al. (2006), n = 132

Black cohosh Black cohosh 20 mg Placebo 1 pill twice per day 4-week crossover design


HF reduction black cohosh: 20% HF reduction placebo: 27% Not statistically significant

[21]

Park et al. (2015), n = 289

Mg oxide Mg oxide 800 mg Mg oxide 1200 mg Placebo Daily for 8 weeks


HF reduction Mg oxide: 25% (800)–41% (1200) HF reduction placebo: 34% Not statistically significantly different

[23]

HF: Hot flash; Mg: Magnesium.

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negative. More recently, a dietary supplement believed to increase concentrations of a serotonin metabolite and thus exhibiting an antidepres-sant effect, S-adenosylmethionine or SAMe, was evaluated for hot flash relief in a single arm pre–post-design study to determine if there was any activity supporting a larger Phase III trial. The study population included women with and without a history of breast cancer. The results were not supportive of any ability to reduce hot flashes, providing an effect equivalent to what is expected from a placebo [25].

●● Mind–body techniquesMind–body approaches have also been studied for hot flashes, namely, paced breathing, cogni-tive–behavioral therapy (CBT) and hypnosis. These modalities target the relaxation response that can potentially decrease sympathetic acti-vation, negative cognitions and stress that can be triggers for hot flashes [17,19,26–27]. One CBT strategy has been studied in women with and without a history of breast cancer and evaluated in both a group delivered therapy and individu-ally self-help delivered therapy [28,29]. The inter-vention included several components, namely, relaxation, paced breathing, cognitive restruc-turing, behavioral strategies (such as planning activities when energy levels are the highest) and stress/anxiety management. Sessions lasted 90 min for 6 weeks [28] or 120 min for 4 weeks [29] when delivered in a group. The self-help delivery utilized a workbook over 4 weeks and included two contacts with a clinical psy-chologist. Results of these studies demonstrated reductions in rating hot flashes and night sweats as a problem in the groups that received the intervention by group or through the self-help delivery, but actual frequency of hot flashes was not reduced. A four-arm RCT includ-ing over 400 women with a history of breast cancer compared physical exercise to the same CBT intervention described above (relaxation, paced breathing, cognitive restructuring), compared with both interventions (exercise and CBT) together versus a waitlist (WL) control group [30]. Similar results were shown with those receiving CBT reporting a decreased problem rating with hot flashes but actual fre-quency not decreasing. Hypnosis, on the other hand, has demonstrated improvements in the frequency and severity of hot flashes as well as a decrease in interference with activities and improvements in emotional parameters of

anxiety and depressed mood [31,32]. Hypnosis for hot flashes is a mind–body therapy that uses relaxation and imagery to induce a deep relaxa-tion where suggestions for cooling and stress reduction can be delivered to the subconscious. Studies have been completed in women with a history of breast cancer [31] as well as in women with natural or surgical menopause without breast cancer [32]. The study in breast cancer survivors used a WL control group, but in the study with a nonbreast cancer population, an attention control group was used as the control. Both studies demonstrated approximately 70% reductions in the frequency and severity of hot flashes, with additional benefits in reducing anxiety, depressed mood and improving sleep.

●● summaryIn summary, there are no natural products, dietary supplements or herbs that have shown a benefit in hot flash reduction to date in rigor-ously designed trials. Compelling evidence exists for behavioral interventions that incorporate cognitive restructuring, relaxation and cooling suggestions as strategies to reduce hot flashes; hypnosis, in particular, has evidence leading to its recent inclusion as a treatment in a recent position statement by the North American Menopause Society [33].

FatigueFatigue is a common side effect of cancer treat-ment. The National Comprehensive Cancer Network (2003) [34] defines CRF as “an unusual, persistent, subjective sense of tiredness related to cancer or cancer treatment that interferes with usual functioning.” CRF is multifactorial with cancer pathology and treatment toxicity to be the two key factors. Other contributing factors may include anemia, depression, infection and sleep disorders [35]. It is proposed that in response to cancer and its treatments, inf lammatory cytokines signal the CNS and generate fatigue by altering neural processes [36]. CRF generally improves following treatment completion, but some level of fatigue may persist for years after that [37]. Studies in women with breast cancer indicate that fatigue is reported in up to 94% of patients undergoing cancer treatment [38–40]. Additionally, fatigue may be a persistent problem long into breast cancer survivorship. Numerous studies have reported fatigue prevalence of 20–66%, occurring up to 5–10 years after com-pletion of adjuvant chemotherapy [41–44].




●● Natural productsThere are no US FDA-approved pharmacologic agents for treating or preventing CRF and little evidence of benefit for commonly used drugs such as psychostimulants. Thus, studying natu-ral products for fatigue is common. Preliminary evidence supports the use of Wisconsin ginseng for fatigue treatment as reported by two stud-ies completed by the same investigators and evaluated American ginseng for the reduction of fatigue in a heterogeneous group of cancer survivors, both in those getting chemotherapy and/or radiation and in those who have com-pleted all treatments [45,46]. Among these stud-ies, a Phase III randomized, double-blind trial comparing 2000 mg of American ginseng to placebo for 8 weeks in 364 cancer patients, pre-dominantly breast cancer, found a trend toward mean change in fatigue scores at 4 weeks and a statistically significant between-group mean change at 8 weeks, favoring ginseng over pla-cebo [46]. Another plant product, this one from Brazil, is believed to have stimulant and aph-rodisiac properties. It is called guarana and it is being studied for fatigue, although thus far the data are mixed. In one randomized, double-blind, placebo-controlled crossover trial includ-ing 36 breast cancer patients, 75 mg of guarana twice a day was not able to significantly improve CRF during radiation therapy [47]. However, in a more recent randomized, double-blind, placebo-controlled crossover study evaluating 50 mg of guarana twice daily in 75 women with breast cancer and receiving chemotherapy, guarana was shown to have a positive effect on CRF [48]. So, while data are not yet definitive on these two plant products, more research, particularly relating to their mechanisms of action, is needed to better understand their role in treating CRF.

●● Mind–body approachesMind–body techniques are among the most prevalent interventions studied for fatigue and some have shown promising results. Among these therapies, yoga has been the most studied in the breast cancer population. Two system-atic reviews summarized findings from studies evaluating various types of yoga and provide support that yoga, from Iyengar to restora-tive, may be beneficial for reducing fatigue in women with breast cancer [49,50], with one of the reviews reporting effect sizes from individ-ual trials ranging from 0.3 to 1.5 [49]. Another type of mind–body therapy that has been tested

to improve CRF is mindfulness meditation, a program-based intervention that involves bring-ing attention to an individual’s present moment experiences with openness and acceptance. In breast cancer, three clinical trials testing mind-fulness, all including fatigue as a secondary out-come, found significant improvements in fatigue scores [51–53], suggesting promise for mindful-ness as an effective intervention for reducing fatigue in this population. In a study that tested yet another therapy, acupuncture, in 302 breast cancer patients with at least moderate fatigue, the study showed significant improvement in fatigue scores [54], warranting further research to establish the effectiveness of acupuncture for reducing CRF. Acupuncture has had mixed effects due to the lack of a standardized sham treatment, which has made this area of research challenging.

Furthermore, mind and body techniques such as CBT showed some evidence in reduc-ing fatigue among women with breast cancer. In a meta-analysis on the impact of behavioral techniques, including CBT and psychosocial support, in breast cancer patients, the authors showed a very small, yet statistically significant, effect on fatigue (effect size: -0.158) [55]. More recently, a clinical trial of 200 breast cancer patients comparing attention control to a group receiving a combined intervention (CBT and hypnosis) suggested that this combination has promise in treating CRF [56].

●● summaryResearch in breast cancer indicates that yoga appears to show the most efficacy for reduc-ing fatigue as well as the dietary supplement, American ginseng, but more research is needed. Other mind and body techniques such as mind-fulness and hypnosis with CBT, need more research before definitive conclusions about their effectiveness can be made.

Arthralgias/myalgiasTreatment for hormone receptor-positive breast cancer has improved with the use of third-gen-eration AIs, as estrogen-deprivation therapies. Compared with tamoxifen (a selective estrogen receptor modulator), AIs are associated with disease-free survival [57] and forgoes serious side effects of tamoxifen such as increased risk for thromboembolic events and endometrial can-cer [58]. However, AIs present with their own disruptive side effects, and because a patient may

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be prescribed an AI for over 5 years, they present a major concern for patients and providers alike in terms of adherence and quality of life. One of the most common and debilitating side effects is the occurrence of AI-induced musculoskel-etal symptoms (AIMSS). AIMSS encompasses a variety of symptoms but are generally char-acterized as aches and pains in the joints and muscles after beginning AI use; these symptoms are commonly noted in the hands, knees and back, among others. [59]. There also appears to be changes in tenosynovial structures of affected areas [60] and possible development of carpal tun-nel syndrome; moreover, patients may describe stiffness or a feeling of sudden aging that is dif-ficult to manage [61]. Notably, musculoskeletal symptoms are defined and reported differently from study to study depending on the primary outcome and the measurements used.

Phase III clinical trials indicate that mus-culoskeletal symptoms occur between 11 and 36% [62] during AI use; using patient-reported outcomes, though, this percentage may increase to over half [63]. There is some evidence to indi-cate that the presence of AIMSS is actually asso-ciated with greater rates of disease-free survival than those who do not experience these symp-toms [57,64]. However, the discontinuation rate due to AIMSS is clinically significant at approxi-mately 30% [65,66] and symptoms will not neces-sary disappear over time while remaining on an AI [67]. Therefore, there is a compelling reason to treat and manage AIMSS to improve overall rates of adherence and, ultimately, survival.

One factor limiting treatment for AIMSS is that its etiology is unknown. Many hypotheses point to the severe estrogen depletion that occurs during AI use, although there are likely other significant etiologic factors [66,68]. Furthermore, patients that have pre-existing joint pain [67–70], are younger or more recently experienced meno-pause [63,66], were treated with systemic taxa-nes [70–72] and have insufficient levels of vita-min D [73–75], are potentially more at risk for AIMSS. There does also appear to be a genetic component to AIMSS; Mao et al. [63] observed that polymorphisms in CYP19A1, a gene known for its role in encoding aromatase, is correlated with the incidence of AIMSS. These risk fac-tors may lead to a shorter mean time to discon-tinuation particularly if a patient is younger, has received chemotherapy with taxanes, or pain is severe [66]. Primary conventional treat-ment for AIMSS includes the use of NSAIDS

or switching to another AI or tamoxifen [59]. In spite of relatively similar levels of estrogen depletion between AIs, the act of switching does appear to improve adherence rates and reduced severity of AIMSS [66]. However, more effective treatments are needed.

●● Natural productsOne notable dietary supplement linked with AIMSS outcomes is vitamin D. Descriptive data suggest that those with inadequate or deficient vita-min D levels below 30 ng/ml are potentially more likely to be symptomatic [74,75]. Khan et al. [73] pro-pose that vitamin D3 supplementation at 50,000 IU per week is safe and may reduce functional dis-ability resulting from AIMSS. It is unclear, how-ever, how strong the relationship between vitamin D and AIMSS; in spite of many postmenopausal women having inadequate serum levels of vitamin D, Singh et al. [76] did not find a significant cor-relation between AIMSS and vitamin D levels. From these studies, it is clear that vitamin D is only one potential component contributing to the cause of AIMSS and that there are other underly-ing factors.

Popular supplements such as glucosamine with chondroitin and omega-3-fatty acids (O3-FAs) have also been investigated based on their role in arthritis pain. In a single-arm, Phase II study by Greenlee et al. [77], participants (n = 39) took 1500 mg/day of glucosamine-sulfate and 1200 mg/day of chondroitin-sulfate over the course of 24 weeks; at follow-up, there were sig-nificant, moderate improvements in joint pain severity, interference and functioning. However, these results cannot necessarily be generalized without a larger, placebo-controlled trial.

Hershman et al. [78] conducted a randomized, placebo-controlled trial (n = 249) of 3.3 g/day of O3-FA compared with a soybean and corn oil placebo taken over 24 weeks. At 12 and 24 weeks, there were statistically and clinically significant changes in worst pain/stiffness scores (over 50%) for both groups, but no significant difference between the two groups was found. While O3-FAs may be beneficial for lower-ing abnormally high triglyceride levels in this group; with the current evidence, it is unlikely that O3-FAs provide a viable option for AIMSS treatment above placebo.

●● Mind–body techniquesEvidence for mind and body therapies for AIMSS is limited even though patients often




express interest in complementary therapies that can provide relief from pain and improve qual-ity of life [79,80]. Acupuncture is one option that has been associated with little to no known side effects. Crew et al. [81] provided promising results in a randomized, sham-controlled acupuncture intervention of 38 women for AIMSS that con-sisted of twice weekly treatments over 6 weeks; there was approximately a 50% improvement in worst pain score for the manual acupuncture group as well as significant differences in pain severity and interference compared with the con-trol group. Conversely, Mao et al. [82] randomized 67 women to an electro-acupuncture intervention versus a sham acupuncture versus WL control for ten sessions over 8 weeks. Both the electro-acu-puncture and sham groups reported significant reductions in pain severity and interference at 8 and 12 weeks compared with the WL control group, yet there were no significant differences between the two groups themselves. Though the results suggest that acupuncture is no better than placebo, there is an ongoing debate as to whether or not sham acupuncture provides unintended physiologic benefits beyond placebo effects [82]. Nevertheless, a recent review and meta-analysis of acupuncture randomized, controlled trials by Chien et al. [83] found that there is no standard-ized intervention for AIMSS and that results trend toward reduced symptoms but conclusive evidence does not exist. Beyond acupuncture, there exist few other mind–body interventions that have been evaluated for AIMSS; feasibility studies conducted by Galantino et al. [79,84] pro-vide promising evidence for Yoga and Tai-Chi in reducing pain severity and improving qual-ity of life, respectively. Taken together, it is clear that much of the research on mind and body techniques remains in its preliminary stages.

●● summaryBased on the studies reviewed above, effec-tive complementary therapies for AIMSS are unclear beyond feasibility and preliminary data. Therefore, it is recommended that randomized controlled interventions be conducted with par-ticular attention to larger sample sizes, blinding (if possible), consistent measures of AIMSS and long-term follow-up data. It is also important that research into the etiology and character-istics that predict the risk for more severe pain with AI myalgias/arthralgias continue, as this will lead to more options for effective prevention and treatment.

cognitive impairmentIndividuals with breast cancer can experience changes in cognitive function across the disease trajectory beginning after diagnosis and before any treatment to 20-year post-treatment [85,86]. Estimates of the prevalence of cognitive problems have been reported to be as high as 33% before treatment (range: 10–33%) and up to 61% after surgery, radiation therapy and/or chemotherapy (range: 10–61%) [87–97]. Variation among these estimates may be related to a number of study-related factors including differences in sample sizes, patient characteristics, treatment regimens, timing of assessments and definitions of impair-ment. Patient characteristics that may affect the expression of cognitive symptoms include age, cognitive reserve, presence of co-morbid condi-tions and genetic variations (APOE and COMT genotypes) [95–99]. Cognitive processes affected include attention, memory, processing speed and executive function [100]. These processes are necessary for everyday functioning including planning and achieving personal goals, learn-ing and interacting with others [101,102]. Possible mechanisms underlying cognitive changes in breast cancer include central neurotoxicity from chemotherapy, immune and other physiologic responses to the cancer and cancer treatments (surgery, radiation therapy, chemotherapy, hor-mone therapy), mental or attentional fatigue and psychological and symptom distress [103–105]. Currently, there are no empirically validated interventions to optimize cognitive function in individuals with breast cancer. Complementary therapies are a promising group of interven-tions that may reduce cognitive symptoms and improve quality of life through a variety of mechanisms including neurologic modification, immune regulation, restoring attentional func-tion, cognitive training and stress reduction. At least 13 research reports examined the benefits of complementary therapies in preventing cog-nitive decline or improving cognitive function (primary outcomes) in community dwelling women with breast cancer. These studies will be discussed below; table 2 provides a detailed description of the therapies evaluated.

●● Natural productsOnly one study examined the effects of a natural product on cognitive function in women with breast cancer. The study by Barton et al. [106] used a randomized, placebo-controlled, dou-ble-blind study design to assess the efficacy of

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table 2. complementary therapy approaches to optimize cognitive function in breast cancer.

study (year) complementary therapy

Dose Program ref.

Barton et al. (2013)

Ginkgo biloba 60-mg capsules p.o. b.i.d Ginkgo biloba administered orally before second cycle of adjuvant chemotherapy and continued until 1-month postadjuvant chemotherapy

[106]

Milbury et al. (2014)

Tibetan sound mediation

Two 60-min classes a week for 6 weeks. Home practice encouraged

Each session included focus on concentration and mindfulness. Practices included breathing exercises, visualizations and vocalization of meditation sounds

[107]

Lesiuk (2015)

Mindfulness-based music therapy

One 60-min class a week for 4 weeks plus 15–20 min daily home practice

Each session included focusing on a sound, focusing on music and a discussion of focus. Home practice included music exercises and written reflections

[108]

Cimprich (1993)

Restorative therapy

Three 20–30-min sessions per week for 3 months

Sessions included participating in restorative activities including spending time with nature (watch sunset), exploring things of interest (reading) and engaging in enjoying activities (making things)

[109]

Cimprich and Ronis (2003)

Restorative therapy – nature

Exposure to the natural environment for 120-min per week

Sessions included participating in restorative activities that focus on nature including visiting a scenic spot, bird watching, listening to sounds of nature, watching sunset and tending or plants/gardens

[110]

Alvarez et al. (2013)

Neurofeedback Two 33-min sessions a week for 10 weeks

NeurOptimal System (Zengar Institute). Session includes placing sensors on head to analyze an EEG and identify phase state changes. Participants listen to music and experience brief interruptions to signal feedback

[111]

Komatsu et al. (2015)

Yoga One 30-min orientation and 90-min yoga class. Home practice 15-min session using DVD encouraged

Hatha Yoga. Participants selected DVD(s) with differing themes (warm-up, low-intensity, high-intensity) for home practice after orientation and first yoga class. Yoga logs kept to record dose (no recommended dose)

[112]

Ferguson et al. (2007 and 2012)

Memory and Attention Adaptation Training

One 30–50-min session per month for 4 months, three phone contacts between monthly visits and daily home practice

Monthly sessions included education on chemotherapy-associated problems, self-awareness and identifying situations where cognitive problems occur and providing tailored self-regulation and compensatory strategies

[113,114]

Kesler et al. (2013)

Executive Function Training Program

Four 20–30-min online sessions a week for 12 weeks

Program included 13 different exercises from Lumos Labs. Exercises included switching games, mental rotation games, n-back memory games, spatial sequencing games, route planning and rule-based problem solving. Exercises tailored to individual’s ability and increased in difficulty over time

[115]

Von Ah et al. (2012)

Memory and speed processing program

Ten 60-min classes over 6–8 weeks

Memory training program included education on strategies for remembering word lists, sequences and text and practice (adapted from ACTIVE trial [Jobe et al., 2001]) Information Processing training program used the Insight program by Posit Science that was derived from the ACTIVE trial

[116]

Ercoli et al. (2015)

Cognitive training

One 120-min class a week for 5 weeks plus 20 min a week of daily home practice

Program focused on attention, memory and executive function. Program included education (in-class), practice with three levels of difficulty (in class and home practice using a training manual, CD and/or stopwatch), and goal setting

[117]

Damholdt et al. (2016)

Cognitive training

Five 30-min sessions a week for 6 weeks plus two phone contacts after randomization

Program included 12 tasks from HAPPYneuron Pro© in Danish. Tasks included training in focusing attention, vigilance, verbal and visual memory and problem solving, learning strategies and maintaining and processing perceptions simultaneously. Difficulty levels increase after two error-free trials. Feedback about speed and accuracy given

[118]

b.i.d.: Twice daily; CD: Compact disc; DVD: Digital video disc; p.o.: Per orem (orally).




Ginkgo biloba in preventing cognitive changes in 166 women with breast cancer receiv-ing adjuvant chemotherapy [106]. Researchers found no differences between individuals tak-ing Ginkgo versus placebo in cognitive perfor-mance or self-report from baseline to 24-month postchemotherapy [106].

●● Mind–body techniquesTwo studies examined the benefits of medita-tion on cognitive function in women with breast cancer [107,108]. Lesiuk [108] used a pre–post-study design to assess the efficacy of a mindfulness-based music therapy intervention in preventing cognitive changes in 15 women with stage I–III breast cancer currently receiving chemotherapy and found significant improvement in atten-tional performance and perceived function. The second study by Milbury et al. [107] used a RCT with a WL control group to assess the feasibility and preliminary efficacy of Tibetan Sound Meditation in optimizing cognitive func-tion in 47 women with early-stage breast cancer (stage I–III) previously treated with chemo-therapy (6–60 months prior). Researchers found Tibetan Sound Meditation to be feasible and enjoyable for participants and observed trends in cognitive improvement.

Two studies assessed the use of activities that restore attention in mentally fatigued individuals newly diagnosed with stage 0–III breast can-cer [109,110]. Specifically, Cimprich [109] used a RCT to assess a restorative therapy intervention in 32 women after primary breast cancer sur-gery and found that the intervention group had a significantly greater mean gain in attentional function over four time points from 3- to 90-day postsurgery. A subsequent RCT by Cimprich and Ronis [110] used a pre–post design to assess a restorative therapy intervention focused on the natural environment in 157 women before and after breast cancer surgery. Researchers found that the intervention group had a signifi-cant greater gain in attentional function after controlling for key covariates.

Alvarez et al. [111] used a repeated measure design with WL control period before the active intervention period to assess the efficacy of a neu-rofeedback intervention on optimizing cognitive function in 23 women with breast cancer previ-ously treated with chemotherapy (6–60 months). Researchers found a significant improvement in cognitive function by self-report from before to 1-month after the neurofeedback intervention [111].

Komatsu et al. [112] used a pre–post-study design to assess the feasibility and preliminary efficacy of a home yoga program on perceived cognitive function in 18 women with breast cancer receiving adjuvant and neoadjuvant chemotherapies. Researchers found the yoga program to be safe and feasible but did not observe any improvement self-report cognitive functioning [112].

Six studies examined the effects of CBT on improving cognitive function in women with breast cancer. Three studies used investigator developed cognitive behavioral programs. In particular, two studies by Ferguson et al. [113,114] evaluated the efficacy of a brief CBT inter-vention, Memory and Attention Adaptation Training or MAAT, in women reporting cogni-tive problems postchemotherapy for stage I–II breast cancer. In a pilot study, these investiga-tors used a repeated measures design to assess MAAT in 29 women and found it to be satisfac-tory for participants and found MAAT signifi-cantly improved objectively measured cognitive performance and self-reported cognitive symp-toms [113]. A follow-up RCT with a WL control group assessed MAAT in 40 women and found that the intervention group had a significant mean gain in verbal memory performance [114]. The third study by Ercoli et al. [117] used a repeated measures design to assess a cognitive training program in 27 women reporting cogni-tive post-treatment for stage 0–III breast cancer. Researchers found a significant improvement in attention, memory and processing performance and self-report of cognitive functioning.

The remaining three studies examined the benefit of commercially available cognitive train-ing programs [115–116,118]. Damholdt et al. [118] used a RCT with WL controls to assess a cus-tomized online cognitive training program from HAPPYneuron Pro in 41 women with a history of breast cancer reporting cognitive problems. Researchers found improvement in objectively measured verbal and working mem-ory performance but not cognitive self-report. Kesler et al. [115] used a RCT study design with WL controls in 41 women to assess a custom-ized online cognitive training program from Lumos Labs in 41 women postchemotherapy for stage I–III breast cancer and found a significant improvement in attention and information pro-cessing function and a trend to improved cogni-tive self-report. Von Ah et al. [116] used a three-group RCT to assess the efficacy of a memory

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or commercially available information process-ing speed training program (Insight) versus WL controls in 82 women reporting cognitive problems postchemotherapy for stage I–II breast cancer. Researchers found a significant improve-ment in cognitive performance and self-report postintervention in the memory training group versus controls and the information processing speed group versus controls.

●● summaryStudies in the area of cognition and complemen-tary therapy are many, but most can be consid-ered preliminary due to the lack of active con-trol groups, no blinding of the study hypothesis, small sample sizes and a failure to control for important confounding variables. No natural products appeared to benefit individuals with breast cancer; however, a variety of mind and body techniques appeared to have some promise.

conclusionDespite the prevalence of complementary ther-apy use, there continues to be limited under-standing on overall safety, efficacy and long-term effects [9]. While vitamins, dietary supplements and herbs are described as ‘natural products’, if effective, these agents have biologic properties that can pose a risk of toxicity, drug interactions and often, unknown dose effects [9,11]. Further research is needed that matches proposed mechanisms of action with symptom biology in well-designed trials to move this area of science forward.

Preliminary data from this review suggest positive evidence especially surrounding mind and body interventions for hot f lashes and fatigue, specifically hypnosis yoga and mind-fulness [31,49–50]. Regarding other common side effects, such as AI-induced myalgias/arthral-gias and cognitive dysfunction related to cancer treatment, potential benefit has been found in early studies surrounding acupuncture, restora-tive therapy and cognitive training, but these studies lack conclusive evidence due to various limitations. Studies with mind and body tech-niques need to control for the nonspecific effects of group and provider interaction at the very least, and to match strategies with mechanisms specific to the symptom under study.

Use of complementary therapies continues to be under-reported with only approximately 50% of women openly discussing complemen-tary therapy with their clinician [11]. Particularly

concerning are patients self-treating with bio-logic-based supplements, agents not screened by the FDA, with little clinical oversight. This presents a safety concern for clinicians, who may not be aware of potential risks due to lack of disclosure from the patient. Providers who are knowledgeable about the current evidence, as well as limitations of existing studies, have more options in their arsenal to improve thera-peutic outcomes for their patients suffering from cancer-related symptoms [9,11].

While some mind–body therapies have the potential for efficacy, they are not without the investment of time, resources and commitment. The most well-studied behavioral interventions, such as hypnosis, CBT, yoga or mindfulness, require trained professionals to instruct, lead and conduct a series of sessions. Further well-designed studies are still needed to optimize active components, to elucidate adequate dosing and to inform the integration into clinical prac-tice. The ability to match mechanisms of inter-ventions to the etiology of symptoms will help to develop mind–body techniques that can more efficiently improve outcomes with a reasonable amount of time and resources.

Future perspectiveComplementary therapies, such as mind–body techniques and natural products, have specific physiologic effects that need to be elucidated through rigorous clinical trials with transla-tional objectives. Understanding mechanisms will facilitate the evaluation of interventions that match symptom physiology. Advancing the sci-ence in this way will have tremendous impli-cations for the integration of complementary therapies into clinical practice.

AcknowledgementsThe authors acknowledge C Kent for her critical assistance in the preparation of this manuscript.

Financial & competing interests disclosurePartial support for this manuscript was provided by the NIH’s National Institute of Nursing Research, Grant P20-NR015331 and The Breast Cancer Research Foundation (BCRF). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.





referencesPapers of special note have been highlighted as: • of interest

1 American Cancer Society. Cancer facts & figures 2016 (2016). www.cancer.org/acs/groups

2 Stan D, Loprinzi CL, Ruddy KJ. Breast cancer survivorship issues. Hematol. Oncol. Clin. North Am. 27(4), 805–827, ix (2013).

• Overviewofsurvivorshipissuesandevidence-basedtreatment.

3 Appling SE, Scarvalone S, MacDonald R, McBeth M, Helzlsouer KJ. Fatigue in breast cancer survivors: the impact of a mind-body medicine intervention. Oncol. Nurs. Forum 39(3), 278–286 (2012).

4 Yildirim Y. Patterns of the use of complementary and alternative medicine in women with metastatic cancer. Cancer Nurs. 33(3), 194–200 (2010).

5 National Center for Complementary and Integrative Health. Complementary, alternative, or integrative health: what’s in a name? (2015). https://nccih.nih.gov

6 Lo-Fo-Wong DN, Ranchor AV, De Haes HC, Sprangers MA, Henselmans I. Complementary and alternative medicine use of women with breast cancer: self-help CAM attracts other women than guided CAM therapies. Patient Educ. Couns. 89(3), 529–536 (2012).

7 Citrin DL, Bloom DL, Grutsch JF, Mortensen SJ, Lis CG. Beliefs and perceptions of women with newly diagnosed breast cancer who refused conventional treatment in favor of alternative therapies. Oncologist 17(5), 607–612 (2012).

8 Moran MS, Ma S, Jagsi R et al. A prospective, multicenter study of complementary/alternative medicine (CAM) utilization during definitive radiation for breast cancer. Int. J. Radiat. Oncol. Biol. Phys. 85(1), 40–46 (2013).

9 Saghatchian M, Bihan C, Chenailler C, Mazouni C, Dauchy S, Delaloge S. Exploring frontiers: use of complementary and alternative medicine among patients with early-stage breast cancer. Breast 23(3), 279–285 (2014).

10 Molassiotis A, Fernadez-Ortega P, Pud D et al. Use of complementary and alternative medicine in cancer patients: a European survey. Ann. Oncol. 16(4), 655–663 (2005).

11 Navo MA, Phan J, Vaughan C et al. An assessment of the utilization of complementary and alternative medication in women with gynecologic or breast

malignancies. J. Clin. Oncol. 22(4), 671–677 (2004).

12 Athena Health Service. Epocrates essentials: epocrates (version 15.12.1), mobile application (2015). www.epocrates.com

13 Hann D, Baker F, Denniston M, Entrekin N. Long-term breast cancer survivors’ use of complementary therapies: perceived impact on recovery and prevention of recurrence. Integr. Cancer Ther. 4(1), 14–20 (2005).

14 Finck G, Barton DL, Loprinzi CL, Quella SK, Sloan JA. Definitions of hot flashes in breast cancer survivors. J. Pain Symptom Manage. 16(5), 327–333 (1998).

15 Gupta P, Sturdee DW, Palin SL et al. Menopausal symptoms in women treated for breast cancer: the prevalence and severity of symptoms and their perceived effects on quality of life. Climacteric 9(1), 49–58 (2006).

16 Davis SR, Panjari M, Robinson PJ, Fradkin P, Bell RJ. Menopausal symptoms in breast cancer survivors nearly 6 years after diagnosis. Menopause 21(10), 1075–1081 (2014).

17 Thurston RC, Christie IC, Matthews KA. Hot flashes and cardiac vagal control during women’s daily lives. Menopause 19(4), 406–412 (2012).

18 Morrow PK, Mattair DN, Hortobagyi GN. Hot flashes: a review of pathophysiology and treatment modalities. Oncologist 16(11), 1658–1664 (2011).

19 Freedman RR, Kruger ML, Wasson SL. Heart rate variability in menopausal hot flashes during sleep. Menopause 18(8), 897–900 (2011).

20 Quella SK, Loprinzi CL, Barton DL et al. Evaluation of soy phytoestrogens for the treatment of hot flashes in breast cancer survivors: a North Central Cancer Treatment Group Trial. J. Clin. Oncol. 18(5), 1068–1074 (2000).

21 Pockaj BA, Gallagher JG, Loprinzi CL et al. Phase III double-blind, randomized, placebo-controlled crossover trial of black cohosh in the management of hot flashes: NCCTG Trial N01CC1. J. Clin. Oncol. 24(18), 2836–2841 (2006).

22 Barton DL, Loprinzi CL, Quella SK et al. Prospective evaluation of vitamin E for hot flashes in breast cancer survivors. J. Clin. Oncol. 16(2), 495–500 (1998).

23 Park H, Qin R, Smith TJ et al. North Central Cancer Treatment Group N10C2 (Alliance): a double-blind placebo-controlled study of magnesium supplements to reduce menopausal hot flashes. Menopause 22(6), 627–632 (2015).

24 Pruthi S, Qin R, Terstreip SA et al. A Phase III, randomized, placebo-controlled, double-blind trial of flaxseed for the treatment of hot flashes: North Central Cancer Treatment Group N08C7. Menopause 19(1), 48–53 (2012).

25 Kadakia KC, Loprinzi CL, Atherton PJ, Fee-Schroeder KC, Sood A, Barton DL. Phase II evaluation of S-adenosyl-L-methionine (SAMe) for the treatment of hot flashes. Support. Care Cancer 24(3), 1061–1069 (2016).

26 Bernardi L, Porta C, Spicuzza L et al. Slow breathing increases arterial baroreflex sensitivity in patients with chronic heart failure. Circulation 105(2), 143–145 (2002).

27 Hunter MS, Mann E. A cognitive model of menopausal hot flushes and night sweats. J. Psychosom. Res. 69(5), 491–501 (2010).

28 Mann E, Smith MJ, Hellier J et al. Cognitive behavioural treatment for women who have menopausal symptoms after breast cancer treatment (MENOS 1): a randomised controlled trial. Lancet Oncol. 13(3), 309–318 (2012).

29 Ayers B, Smith M, Hellier J, Mann E, Hunter MS. Effectiveness of group and self-help cognitive behavior therapy in reducing problematic menopausal hot flushes and night sweats (MENOS 2): a randomized controlled trial. Menopause 19(7), 749–759 (2012).

30 Duijts SF, Van Beurden M, Oldenburg HS et al. Efficacy of cognitive behavioral therapy and physical exercise in alleviating treatment-induced menopausal symptoms in patients with breast cancer: results of a randomized, controlled, multicenter trial. J. Clin. Oncol. 30(33), 4124–4133 (2012).

31 Elkins G, Marcus J, Stearns V et al. Randomized trial of a hypnosis intervention for treatment of hot flashes among breast cancer survivors. J. Clin. Oncol. 26(31), 5022–5026 (2008).

32 Elkins GR, Fisher WI, Johnson AK, Carpenter JS, Keith TZ. Clinical hypnosis in the treatment of postmenopausal hot flashes: a randomized controlled trial. Menopause 20(3), 291–298 (2013).

33 Nonhormonal management of menopause-associated vasomotor symptoms: 2015 position statement of The North American Menopause Society. Menopause 22(11), 1155–1172; quiz 1173–1174 (2015).

34 National Comprehensive Cancer Network. Cancer-related fatigue. Clinical practice guidelines in oncology J. Natl Compr. Canc. Netw. 1(3), 308–331 (2003).

bmt-2016-0013

www.cancer.org/acs/groups/content/@research/documents/document/acspc-047079.pdf

https://nccih.nih.gov/health/integrative-health

www.epocrates.com

http://www.futuremedicine.com/action/showLinks?pmid=11790690&crossref=10.1161%2Fhc0202.103311

http://www.futuremedicine.com/action/showLinks?pmid=21522045&crossref=10.1097%2Fgme.0b013e31820ac941

http://www.futuremedicine.com/action/showLinks?pmid=23045575&crossref=10.1200%2FJCO.2012.41.8525

http://www.futuremedicine.com/action/showLinks?pmid=25423327&crossref=10.1097%2FGME.0000000000000374


http://www.futuremedicine.com/action/showLinks?pmid=24529905&crossref=10.1016%2Fj.breast.2014.01.009

http://www.futuremedicine.com/action/showLinks?pmid=19761067

http://www.futuremedicine.com/action/showLinks?pmid=20955869&crossref=10.1016%2Fj.jpsychores.2010.04.005

http://www.futuremedicine.com/action/showLinks?pmid=10694559&coi=1%3ACAS%3A528%3ADC%252BD3cXitVCgsb8%253D

http://www.futuremedicine.com/action/showLinks?pmid=15695473&crossref=10.1177%2F1534735404273723

http://www.futuremedicine.com/action/showLinks?pmid=22464017&crossref=10.1016%2Fj.pec.2012.02.019


http://www.futuremedicine.com/action/showLinks?pmid=21900849&crossref=10.1097%2Fgme.0b013e318223b021

http://www.futuremedicine.com/action/showLinks?pmid=22095062&crossref=10.1097%2Fgme.0b013e3182337166

http://www.futuremedicine.com/action/showLinks?pmid=15699021&crossref=10.1093%2Fannonc%2Fmdi110&coi=1%3ASTN%3A280%3ADC%252BD2M7kvVWnuw%253D%253D

http://www.futuremedicine.com/action/showLinks?pmid=22543386&crossref=10.1188%2F12.ONF.278-286

http://www.futuremedicine.com/action/showLinks?pmid=22340966&crossref=10.1016%2FS1470-2045%2811%2970364-3


http://www.futuremedicine.com/action/showLinks?pmid=9846028&crossref=10.1016%2FS0885-3924%2898%2900090-6&coi=1%3ASTN%3A280%3ADyaK1M%252Fmt1Cmug%253D%253D

http://www.futuremedicine.com/action/showLinks?pmid=22531358&crossref=10.1634%2Ftheoncologist.2011-0468


http://www.futuremedicine.com/action/showLinks?pmid=26248653&crossref=10.1007%2Fs00520-015-2878-3

http://www.futuremedicine.com/action/showLinks?pmid=22042786&crossref=10.1634%2Ftheoncologist.2011-0174&coi=1%3ACAS%3A528%3ADC%252BC38XmtF2ruw%253D%253D


http://www.futuremedicine.com/action/showLinks?pmid=20357652&crossref=10.1097%2FNCC.0b013e3181c295ac

http://www.futuremedicine.com/action/showLinks?pmid=22336748&crossref=10.1097%2Fgme.0b013e31823fe835

http://www.futuremedicine.com/action/showLinks?pmid=9469333&coi=1%3ACAS%3A528%3ADyaK1cXhtFShtL8%253D

http://www.futuremedicine.com/action/showLinks?pmid=16428125&crossref=10.1080%2F13697130500487224&coi=1%3ASTN%3A280%3ADC%252BD28%252FjvFWkuw%253D%253D

http://www.futuremedicine.com/action/showLinks?pmid=22658441&crossref=10.1016%2Fj.ijrobp.2012.03.025


http://www.futuremedicine.com/action/showLinks?pmid=23915746&crossref=10.1016%2Fj.hoc.2013.05.005



35 National Cancer Institute. Fatigue overview (2015). www.cancer.gov/about-cancer

36 Bower JE. Cancer-related fatigue – mechanisms, risk factors, and treatments. Nat. Rev. Clin. Oncol. 11(10), 597–609 (2014).

37 National Cancer Institute. Treatment side effects: fatigue (2016). www.cancer.gov/about-cancer

38 Sitzia J, Huggins L. Side effects of cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) chemotherapy for breast cancer. Cancer Pract. 6(1), 13–21 (1998).

39 Manir KS, Bhadra K, Kumar G, Manna A, Patra NB, Sarkar SK. Fatigue in breast cancer patients on adjuvant treatment: course and prevalence. Indian J. Palliat. Care 18(2), 109–116 (2012).

40 Jacobsen PB, Hann DM, Azzarello LM, Horton J, Balducci L, Lyman GH. Fatigue in women receiving adjuvant chemotherapy for breast cancer: characteristics, course, and correlates. J. Pain Symptom Manage. 18(4), 233–242 (1999).

41 Nieboer P, Buijs C, Rodenhuis S et al. Fatigue and relating factors in high-risk breast cancer patients treated with adjuvant standard or high-dose chemotherapy: a longitudinal study. J. Clin. Oncol. 23(33), 8296–8304 (2005).

42 Meeske K, Smith AW, Alfano CM et al. Fatigue in breast cancer survivors two to five years post diagnosis: a HEAL study report. Qual. Life Res. 16(6), 947–960 (2007).

43 Bower JE, Ganz PA, Desmond KA et al. Fatigue in long-term breast carcinoma survivors: a longitudinal investigation. Cancer 106(4), 751–758 (2006).

44 Kim SH, Son BH, Hwang SY et al. Fatigue and depression in disease-free breast cancer survivors: prevalence, correlates, and association with quality of life. J. Pain Symptom Manage. 35(6), 644–655 (2008).

45 Barton DL, Soori GS, Bauer BA et al. Pilot study of Panax quinquefolius (American ginseng) to improve cancer-related fatigue: a randomized, double-blind, dose-finding evaluation: NCCTG trial N03CA. Support. Care Cancer 18(2), 179–187 (2010).

46 Barton DL, Liu H, Dakhil SR et al. Wisconsin Ginseng (Panax quinquefolius) to improve cancer-related fatigue: a randomized, double-blind trial, N07C2. J. Natl Cancer Inst. 105(16), 1230–1238 (2013).

47 Da Costa Miranda V, Trufelli DC, Santos J et al. Effectiveness of guarana (Paullinia

cupana) for postradiation fatigue and depression: results of a pilot double-blind randomized study. J. Altern. Complement. Med. 15(4), 431–433 (2009).

48 De Oliveira Campos MP, Riechelmann R, Martins LC, Hassan BJ, Casa FB, Del Giglio A. Guarana (Paullinia cupana) improves fatigue in breast cancer patients undergoing systemic chemotherapy. J. Altern. Complement. Med. 17(6), 505–512 (2011).

49 Harder H, Parlour L, Jenkins V. Randomised controlled trials of yoga interventions for women with breast cancer: a systematic literature review. Support. Care Cancer 20(12), 3055–3064 (2012).

50 Sadja J, Mills PJ. Effects of yoga interventions on fatigue in cancer patients and survivors: a systematic review of randomized controlled trials. Explore 9(4), 232–243 (2013).

51 Bower JE, Crosswell AD, Stanton AL et al. Mindfulness meditation for younger breast cancer survivors: a randomized controlled trial. Cancer 121(8), 1231–1240 (2015).

52 Hoffman CJ, Ersser SJ, Hopkinson JB, Nicholls PG, Harrington JE, Thomas PW. Effectiveness of mindfulness-based stress reduction in mood, breast- and endocrine-related quality of life, and well-being in stage 0 to III breast cancer: a randomized, controlled trial. J. Clin. Oncol. 30(12), 1335–1342 (2012).

53 Lengacher CA, Reich RR, Post-White J et al. Mindfulness based stress reduction in post-treatment breast cancer patients: an examination of symptoms and symptom clusters. J. Behav. Med. 35(1), 86–94 (2012).

54 Molassiotis A, Bardy J, Finnegan-John J et al. Acupuncture for cancer-related fatigue in patients with breast cancer: a pragmatic randomized controlled trial. J. Clin. Oncol. 30(36), 4470–4476 (2012).

55 Duijts SF, Faber MM, Oldenburg HS, Van Beurden M, Aaronson NK. Effectiveness of behavioral techniques and physical exercise on psychosocial functioning and health-related quality of life in breast cancer patients and survivors – a meta-analysis. Psychooncology 20(2), 115–126 (2011).

56 Montgomery GH, David D, Kangas M et al. Randomized controlled trial of a cognitive–behavioral therapy plus hypnosis intervention to control fatigue in patients undergoing radiotherapy for breast cancer. J. Clin. Oncol. 32(6), 557–563 (2014).

57 Huober J, Cole BF, Rabaglio M et al. Symptoms of endocrine treatment and outcome in the BIG 1– 98 study. Breast Cancer Res. Treat. 143(1), 159–169 (2014).

58 Braithwaite RS, Chlebowski RT, Lau J, George S, Hess R, Col NF. Meta-analysis of vascular and neoplastic events associated with tamoxifen. J. Gen. Intern. Med. 18(11), 937–947 (2003).

59 Younus J, Kligman L. Management of aromatase inhibitor-induced arthralgia. Curr. Oncol. 17(1), 87–90 (2010).

60 Morales L, Pans S, Verschueren K et al. Prospective study to assess short-term intra-articular and tenosynovial changes in the aromatase inhibitor-associated arthralgia syndrome. J. Clin. Oncol. 26(19), 3147–3152 (2008).

61 Shanmugam VK, McCloskey J, Elston B, Allison SJ, Eng-Wong J. The CIRAS study: a case–control study to define the clinical, immunologic, and radiographic features of aromatase inhibitor-induced musculoskeletal symptoms. Breast Cancer Res. Treat. 131(2), 699–708 (2012).

62 Khan QJ, O’Dea AP, Sharma P. Musculoskeletal adverse events associated with adjuvant aromatase inhibitors. J. Oncol. 2010, pii: 654348 (2010).

63 Mao JJ, Su HI, Feng R et al. Association of functional polymorphisms in CYP19A1 with aromatase inhibitor associated arthralgia in breast cancer survivors. Breast Cancer Res. 13(1), R8 (2011).

64 Hadji P, Kieback DG, Tams J, Hasenburg A, Ziller M. Correlation of treatment-emergent adverse events and clinical response to endocrine therapy in early breast cancer: a retrospective analysis of the German cohort of TEAM. Ann. Oncol. 23(10), 2566–2572 (2012).

65 Briot K, Tubiana-Hulin M, Bastit L, Kloos I, Roux C. Effect of a switch of aromatase inhibitors on musculoskeletal symptoms in postmenopausal women with hormone-receptor-positive breast cancer: the ATOLL (articular tolerance of letrozole) study. Breast Cancer Res. Treat 120(1), 127–134 (2010).

66 Henry NL, Azzouz F, Desta Z et al. Predictors of aromatase inhibitor discontinuation as a result of treatment-emergent symptoms in early-stage breast cancer. J. Clin. Oncol. 30(9), 936–942 (2012).

67 Castel LD, Hartmann KE, Mayer IA et al. Time course of arthralgia among women initiating aromatase inhibitor therapy and a postmenopausal comparison group in a prospective cohort. Cancer 119(13), 2375–2382 (2013).

68 Shi Q, Giordano SH, Lu H, Saleeba AK, Malveaux D, Cleeland CS. Anastrozole-associated joint pain and other symptoms in


www.cancer.gov/about-cancer/treatment/side-effects/fatigue/fatigue-hp-pdq

www.cancer.gov/about-cancer/treatment/side-effects/fatigue

http://www.futuremedicine.com/action/showLinks?pmid=25537522&crossref=10.1002%2Fcncr.29194

http://www.futuremedicine.com/action/showLinks?pmid=18358687&crossref=10.1016%2Fj.jpainsymman.2007.08.012

http://www.futuremedicine.com/action/showLinks?pmid=20336645&crossref=10.1002%2Fpon.1728

http://www.futuremedicine.com/action/showLinks?pmid=21612429&crossref=10.1089%2Facm.2010.0571

http://www.futuremedicine.com/action/showLinks?pmid=16219926&crossref=10.1200%2FJCO.2005.10.167&coi=1%3ACAS%3A528%3ADC%252BD2MXhtlWhtrjI

http://www.futuremedicine.com/action/showLinks?pmid=22331951&crossref=10.1200%2FJCO.2011.38.0261&coi=1%3ACAS%3A528%3ADC%252BC38Xmt1Sgs7g%253D

http://www.futuremedicine.com/action/showLinks?pmid=20179809&crossref=10.3747%2Fco.v17i1.474&coi=1%3ASTN%3A280%3ADC%252BC3c7jt1elsA%253D%253D



http://www.futuremedicine.com/action/showLinks?pmid=9460322&crossref=10.1046%2Fj.1523-5394.1998.1998006013.x&coi=1%3ASTN%3A280%3ADyaK1c7itFKhtg%253D%253D

http://www.futuremedicine.com/action/showLinks?pmid=21251330&crossref=10.1186%2Fbcr2813&coi=1%3ACAS%3A528%3ADC%252BC3MXhvVKltLc%253D


http://www.futuremedicine.com/action/showLinks?pmid=23052917&crossref=10.1007%2Fs00520-012-1611-8&coi=1%3ASTN%3A280%3ADC%252BC3s%252FktVSisA%253D%253D

http://www.futuremedicine.com/action/showLinks?pmid=23575918&crossref=10.1002%2Fcncr.28016&coi=1%3ACAS%3A528%3ADC%252BC3sXpvVeisrs%253D




http://www.futuremedicine.com/action/showLinks?pmid=23853057&crossref=10.1093%2Fjnci%2Fdjt181

http://www.futuremedicine.com/action/showLinks?pmid=23093826&crossref=10.4103%2F0973-1075.100826

http://www.futuremedicine.com/action/showLinks?pmid=22467902&crossref=10.1093%2Fannonc%2Fmds055&coi=1%3ASTN%3A280%3ADC%252BC38rhsVynsg%253D%253D

http://www.futuremedicine.com/action/showLinks?pmid=24305979&crossref=10.1007%2Fs10549-013-2792-7&coi=1%3ACAS%3A528%3ADC%252BC3sXhvFKgsrbP

http://www.futuremedicine.com/action/showLinks?crossref=10.1016%2Fj.explore.2013.04.005

http://www.futuremedicine.com/action/showLinks?pmid=23452648&crossref=10.1016%2Fj.jpain.2012.11.010&coi=1%3ACAS%3A528%3ADC%252BC3sXmslehs7k%253D



http://www.futuremedicine.com/action/showLinks?pmid=25113839&crossref=10.1038%2Fnrclinonc.2014.127&coi=1%3ACAS%3A528%3ADC%252BC2cXhtlKktr7E



http://www.futuremedicine.com/action/showLinks?pmid=10534963&crossref=10.1016%2FS0885-3924%2899%2900082-2&coi=1%3ASTN%3A280%3ADC%252BD3c%252FgsVylsQ%253D%253D

http://www.futuremedicine.com/action/showLinks?pmid=20035381&crossref=10.1007%2Fs10549-009-0692-7&coi=1%3ACAS%3A528%3ADC%252BC3cXntlalsQ%253D%253D

http://www.futuremedicine.com/action/showLinks?pmid=14687281&crossref=10.1046%2Fj.1525-1497.2003.20724.x



patients with breast cancer. J. Pain 14(3), 290–296 (2013).

69 Chim K, Xie SX, Stricker CT et al. Joint pain severity predicts premature discontinuation of aromatase inhibitors in breast cancer survivors. BMC Cancer 13, 401 (2013).

70 Lombard JM, Zdenkowski N, Wells K et al. Aromatase inhibitor induced musculoskeletal syndrome: a significant problem with limited treatment options. Support. Care Cancer 24(5), 2139–2146 (2015).

71 Fenlon D, Powers C, Simmonds P, Clough J, Addington-Hall J. The JACS prospective cohort study of newly diagnosed women with breast cancer investigating joint and muscle pain, aches, and stiffness: pain and quality of life after primary surgery and before adjuvant treatment. BMC Cancer 14, 467 (2014).

72 Saibil S, Fitzgerald B, Freedman OC et al. Incidence of taxane-induced pain and distress in patients receiving chemotherapy for early-stage breast cancer: a retrospective, outcomes-based survey. Curr. Oncol. 17(4), 42–47 (2010).

73 Khan QJ, Reddy PS, Kimler BF et al. Effect of vitamin D supplementation on serum 25-hydroxy vitamin D levels, joint pain, and fatigue in women starting adjuvant letrozole treatment for breast cancer. Breast Cancer Res. Treat. 119(1), 111–118 (2010).

74 Singer O, Cigler T, Moore AB, Levine AB, Do HT, Mandl LA. Hypovitaminosis D is a predictor of aromatase inhibitor musculoskeletal symptoms. Breast J. 20(2), 174–179 (2014).

75 Waltman NL, Ott CD, Twiss JJ, Gross GJ, Lindsey AM. Vitamin D insufficiency and musculoskeletal symptoms in breast cancer survivors on aromatase inhibitor therapy. Cancer Nurs. 32(2), 143–150 (2009).

76 Singh S, Cuzick J, Mesher D, Richmond B, Howell A. Effect of baseline serum vitamin D levels on aromatase inhibitors induced musculoskeletal symptoms: results from the IBIS-II, chemoprevention study using anastrozole. Breast Cancer Res. Treat. 132(2), 625–629 (2012).

77 Greenlee H, Crew KD, Shao T et al. Phase II study of glucosamine with chondroitin on aromatase inhibitor-associated joint symptoms in women with breast cancer. Support. Care Cancer 21(4), 1077–1087 (2013).

78 Hershman DL, Loprinzi C, Schneider BP. Symptoms: aromatase inhibitor induced arthralgias. Adv. Exp. Med. Biol. 862, 89–100 (2015).

79 Galantino ML, Greene L, Archetto B et al. A qualitative exploration of the impact of yoga

on breast cancer survivors with aromatase inhibitor-associated arthralgias. Explore 8(1), 40–47 (2012).

80 Hervik J, Mjaland O. Quality of life of breast cancer patients medicated with anti-estrogens, 2 years after acupuncture treatment: a qualitative study. Int. J. Womens Health 2, 319–325 (2010).

81 Crew KD, Capodice JL, Greenlee H et al. Randomized, blinded, sham-controlled trial of acupuncture for the management of aromatase inhibitor-associated joint symptoms in women with early-stage breast cancer. J. Clin. Oncol. 28(7), 1154–1160 (2010).

82 Mao JJ, Xie SX, Farrar JT et al. A randomised trial of electro-acupuncture for arthralgia related to aromatase inhibitor use. Eur. J. Cancer 50(2), 267–276 (2014).

83 Chien TJ, Liu CY, Chang YF, Fang CJ, Hsu CH. Acupuncture for treating aromatase inhibitor-related arthralgia in breast cancer: a systematic review and meta-analysis. J. Altern. Complement. Med. 21(5), 251–260 (2015).

84 Galantino ML, Callens ML, Cardena GJ, Piela NL, Mao JJ. Tai chi for well-being of breast cancer survivors with aromatase inhibitor-associated arthralgias: a feasibility study. Altern. Ther. Health Med. 19(6), 38–44 (2013).

85 Ahles TA, Root JC, Ryan EL. Cancer- and cancer treatment-associated cognitive change: an update on the state of the science. J. Clin. Oncol. 30(30), 3675–3686 (2012).

86 Koppelmans V, Breteler MM, Boogerd W, Seynaeve C, Gundy C, Schagen SB. Neuropsychological performance in survivors of breast cancer more than 20 years after adjuvant chemotherapy. J. Clin. Oncol. 30(10), 1080–1086 (2012).

87 Buchanan ND, Dasari S, Rodriguez JL et al. Post-treatment neurocognition and psychosocial care among breast cancer survivors. Am. J. Prev. Med. 49(6 Suppl. 5), S498–S508 (2015).

88 Ahles TA, Saykin AJ, Mcdonald BC et al. Cognitive function in breast cancer patients prior to adjuvant treatment. Breast Cancer Res. Treat. 110(1), 143–152 (2008).

89 Collins B, Mackenzie J, Stewart A, Bielajew C, Verma S. Cognitive effects of chemotherapy in post-menopausal breast cancer patients 1 year after treatment. Psychooncology 18(2), 134–143 (2009).

90 Hermelink K, Untch M, Lux MP et al. Cognitive function during neoadjuvant chemotherapy for breast cancer: results of a prospective, multicenter, longitudinal study. Cancer 109(9), 1905–1913 (2007).

91 Hurria A, Rosen C, Hudis C et al. Cognitive function of older patients receiving adjuvant chemotherapy for breast cancer: a pilot prospective longitudinal study. J. Am. Geriatr. Soc. 54(6), 925–931 (2006).

92 Jansen CE, Cooper BA, Dodd MJ, Miaskowski CA. A prospective longitudinal study of chemotherapy-induced cognitive changes in breast cancer patients. Support. Care Cancer 19(10), 1647–1656 (2011).

93 Stewart A, Collins B, Mackenzie J, Tomiak E, Verma S, Bielajew C. The cognitive effects of adjuvant chemotherapy in early stage breast cancer: a prospective study. Psychooncology 17(2), 122–130 (2008).

94 Wefel JS, Saleeba AK, Buzdar AU, Meyers CA. Acute and late onset cognitive dysfunction associated with chemotherapy in women with breast cancer. Cancer 116(14), 3348–3356 (2010).

95 Ahles TA, Saykin AJ, Noll WW et al. The relationship of APOE genotype to neuropsychological performance in long-term cancer survivors treated with standard dose chemotherapy. Psychooncology 12(6), 612–619 (2003).

96 Ahles TA, Saykin AJ, McDonald BC et al. Longitudinal assessment of cognitive changes associated with adjuvant treatment for breast cancer: impact of age and cognitive reserve. J. Clin. Oncol. 28(29), 4434–4440 (2010).

97 Cimprich B, So H, Ronis DL, Trask C. Pre-treatment factors related to cognitive functioning in women newly diagnosed with breast cancer. Psychooncology 14(1), 70–78 (2005).

98 Merriman JD, Jansen C, Koetters T et al. Predictors of the trajectories of self-reported attentional fatigue in women with breast cancer undergoing radiation therapy. Oncol. Nurs. Forum 37(4), 423–432 (2010).

99 Small BJ, Rawson KS, Walsh E et al. Catechol-O-methyltransferase genotype modulates cancer treatment-related cognitive deficits in breast cancer survivors. Cancer 117(7), 1369–1376 (2011).

100 Wefel JS, Vardy J, Ahles T, Schagen SB. International Cognition and Cancer Task Force recommendations to harmonise studies of cognitive function in patients with cancer. Lancet Oncol. 12(7), 703–708 (2011).

101 Boykoff N, Moieni M, Subramanian SK. Confronting chemobrain: an in-depth look at survivors’ reports of impact on work, social networks, and health care response. J. Cancer Surviv. 3(4), 223–232 (2009).

102 Selamat MH, Loh SY, Mackenzie L, Vardy J. Chemobrain experienced by breast cancer

bmt-2016-0013

http://www.futuremedicine.com/action/showLinks?pmid=22198469&crossref=10.1007%2Fs10549-011-1911-6&coi=1%3ACAS%3A528%3ADC%252BC38XktFOisLs%253D

http://www.futuremedicine.com/action/showLinks?pmid=24004677&crossref=10.1186%2F1471-2407-13-401


http://www.futuremedicine.com/action/showLinks?crossref=10.1016%2Fj.amepre.2015.08.013

http://www.futuremedicine.com/action/showLinks?pmid=25259847&crossref=10.1371%2Fjournal.pone.0108002

http://www.futuremedicine.com/action/showLinks?pmid=21151679&crossref=10.2147%2FIJWH.S12809

http://www.futuremedicine.com/action/showLinks?pmid=19655244&crossref=10.1007%2Fs10549-009-0495-x&coi=1%3ACAS%3A528%3ADC%252BD1MXhsFaqtr3P

http://www.futuremedicine.com/action/showLinks?pmid=16776787&crossref=10.1111%2Fj.1532-5415.2006.00732.x

http://www.futuremedicine.com/action/showLinks?pmid=21425136&crossref=10.1002%2Fcncr.25685&coi=1%3ACAS%3A528%3ADC%252BC3MXkslWiu7w%253D


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http://www.futuremedicine.com/action/showLinks?pmid=17674194&crossref=10.1007%2Fs10549-007-9686-5&coi=1%3ACAS%3A528%3ADC%252BD1cXmslyjsrY%253D

http://www.futuremedicine.com/action/showLinks?pmid=20100963&crossref=10.1200%2FJCO.2009.23.4708&coi=1%3ACAS%3A528%3ADC%252BC3cXktF2ltLY%253D

http://www.futuremedicine.com/action/showLinks?pmid=24467395&crossref=10.1111%2Ftbj.12227&coi=1%3ACAS%3A528%3ADC%252BC2cXktlCitb8%253D


http://www.futuremedicine.com/action/showLinks?pmid=21354373&crossref=10.1016%2FS1470-2045%2810%2970294-1

http://www.futuremedicine.com/action/showLinks?pmid=23008308&crossref=10.1200%2FJCO.2012.43.0116&coi=1%3ACAS%3A528%3ADC%252BC38XhvVKgsbnO

http://www.futuremedicine.com/action/showLinks?pmid=26059931&crossref=10.1007%2F978-3-319-16366-6_7

http://www.futuremedicine.com/action/showLinks?pmid=24964929&crossref=10.1186%2F1471-2407-14-467




http://www.futuremedicine.com/action/showLinks?pmid=24210070&crossref=10.1016%2Fj.ejca.2013.09.022&coi=1%3ACAS%3A528%3ADC%252BC3sXhs1OqtLrI

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http://www.futuremedicine.com/action/showLinks?crossref=10.1016%2Fj.explore.2011.10.002

http://www.futuremedicine.com/action/showLinks?pmid=20697513&crossref=10.3747%2Fco.v17i4.562&coi=1%3ASTN%3A280%3ADC%252BC3cjjs1Whsw%253D%253D

http://www.futuremedicine.com/action/showLinks?pmid=17351951&crossref=10.1002%2Fcncr.22610&coi=1%3ACAS%3A528%3ADC%252BD2sXlsF2msr4%253D





survivors: a meta-ethnography study investigating research and care implications. PLoS ONE 9(9), e108002 (2014).

103 Jansen C, Miaskowski C, Dodd M, Dowling G, Kramer J. Potential mechanisms for chemotherapy-induced impairments in cognitive function. Oncol. Nurs. Forum 32(6), 1151–1163 (2005).

104 Jung MS, Zhang M, Askren MK et al. Cognitive dysfunction and symptom burden in women treated for breast cancer: a prospective behavioral and fMRI analysis. Brain Imaging Behav. doi:10.1007/s11682-016-9507-9508 (2016) (Epub ahead of print).

105 Merriman JD, Von Ah D, Miaskowski C, Aouizerat BE. Proposed mechanisms for cancer- and treatment-related cognitive changes. Semin. Oncol. Nurs. 29(4), 260–269 (2013).

• Animportantoverviewaboutthephysiologicchangesknowntoberelatedtocognition.

106 Barton DL, Burger K, Novotny PJ et al. The use of Ginkgo biloba for the prevention of chemotherapy-related cognitive dysfunction in women receiving adjuvant treatment for breast cancer, N00C9. Support. Care Cancer 21(4), 1185–1192 (2013).

107 Milbury K, Chaoul A, Biegler K et al. Tibetan sound meditation for cognitive dysfunction: results of a randomized controlled pilot trial. Psychooncology 22(10), 2354–2363 (2013).

108 Lesiuk T. The effect of mindfulness-based music therapy on attention and mood in women receiving adjuvant chemotherapy for breast cancer: a pilot study. Oncol. Nurs. Forum 42(3), 276–282 (2015).

109 Cimprich B. Development of an intervention to restore attention in cancer patients. Cancer Nurs. 16(2), 83–92 (1993).

110 Cimprich B, Ronis DL. An environmental intervention to restore attention in women with newly diagnosed breast cancer. Cancer Nurs. 26(4), 284–292; quiz 293–294 (2003).

111 Alvarez J, Meyer FL, Granoff DL, Lundy A. The effect of EEG biofeedback on reducing postcancer cognitive impairment. Integr. Cancer Ther. 12(6), 475–487 (2013).

112 Komatsu H, Yagasaki K, Yamauchi H, Yamauchi T, Takebayashi T. A self-directed home yoga programme for women with breast cancer during chemotherapy: a feasibility study. Int. J. Nurs. Pract. 22(3), 258–266 (2016).

113 Ferguson RJ, Ahles TA, Saykin AJ et al. Cognitive–behavioral management of

chemotherapy-related cognitive change. Psychooncology 16(8), 772–777 (2007).

114 Ferguson RJ, McDonald BC, Rocque MA et al. Development of CBT for chemotherapy-related cognitive change: results of a waitlist control trial. Psychooncology 21(2), 176–186 (2012).

115 Kesler S, Hadi Hosseini SM, Heckler C et al. Cognitive training for improving executive function in chemotherapy-treated breast cancer survivors. Clin. Breast Cancer 13(4), 299–306 (2013).

116 Von Ah D, Carpenter JS, Saykin A et al. Advanced cognitive training for breast cancer survivors: a randomized controlled trial. Breast Cancer Res. Treat. 135(3), 799–809 (2012).

117 Ercoli LM, Petersen L, Hunter AM et al. Cognitive rehabilitation group intervention for breast cancer survivors: results of a randomized clinical trial. Psychooncology 24(11), 1360–1367 (2015).

118 Damholdt MF, Mehlsen M, O’Toole MS, Andreasen RK, Pedersen AD, Zachariae R. Web-based cognitive training for breast cancer survivors with cognitive complaints – a randomized controlled trial. Psychooncology doi:10.1002/pon.4058 (2016) (Epub ahead of print).


http://www.futuremedicine.com/action/showLinks?pmid=23647804&crossref=10.1016%2Fj.clbc.2013.02.004


http://www.futuremedicine.com/action/showLinks?pmid=26643264&crossref=10.1111%2Fijn.12419


http://www.futuremedicine.com/action/showLinks?pmid=8477404&crossref=10.1097%2F00002820-199304000-00001&coi=1%3ASTN%3A280%3ADyaK3s3jvVaisA%253D%253D




http://www.futuremedicine.com/action/showLinks?pmid=25759235&crossref=10.1002%2Fpon.3769&coi=1%3ASTN%3A280%3ADC%252BC2MnivFehsA%253D%253D

http://www.futuremedicine.com/action/showLinks?pmid=12886119&crossref=10.1097%2F00002820-200308000-00005


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http://www.futuremedicine.com/action/showLinks?pmid=23584550&crossref=10.1177%2F1534735413477192

http://www.futuremedicine.com/action/showLinks?pmid=24183157&crossref=10.1016%2Fj.soncn.2013.08.006

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