Molecular Classification and Prognostic Signatures of Breast Tumors

Luciane R. Cavalli and Iglenir J. Cavalli

5.1

Introduction

Breast cancer is a complex and heterogeneous disease where tumors of the same apparent prognostic type can differ widely in their responsiveness to therapy and survival rates. Traditionally, the classication of breast cancer is performed on the basis of clinicalhistopathological parameters, such as age, tumor size, histological grade, lymph node status, and analysis of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression. The evaluation of these combined factors has been widely used in clinical practice and formed the basis to classify patients into various risk categories such as the St. Gallen criteria [1] and the Nottingham Prognostic Index [2]. However the markedly extensive breast cancer heterogeneity combined with the lack of reliable predictive factors among these categories limits their ability to distinguish subtle phenotypic differences that may present relevant therapeutic implications. In the past decade, with the development of highthroughput microarray platforms, genome-wide-based methods have been widely employed and a molecular classication of breast cancer has emerged. Gene expression proling studies have showed that expression pattern analysis can rene the classication of breast tumors into different subtypes, known as intrinsic subtypes, and represented a signicant improvement over the traditional methods of tumor classication [3, 4]. In addition, several prognostic gene signatures to predict clinical outcome and

customize therapy have originated from these studies (for reviews, see [59]. In this chapter we will discuss some of these gene expression signatures and their emerging roles in providing new insights into breast cancer classication and in assessing the patients prognosis and dening therapy.

5.2

Molecular Classification: The Gene Expression Intrinsic Subtypes

L. R. Cavalli (&) Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, USA e-mail: lrc@georgetown.edu I. J. Cavalli Genetic Department, Federal University of Parana, Curitiba, Brazil e-mail: cavalli@ufpr.br

Genome-wide studies, using microarray-based methods, have allowed the analysis of the DNA copy number changes or gene expression of thousands of genes in a single experiment in a given tumor sample [1012]. These methods revealed the complexity of the notable breast cancer heterogeneity at the molecular level [1315] as clearly demonstrated by the large variation in the gene expression patterns. The pioneer study described by Perou et al. [3], based on gene expression analysis, set the basis for the current molecular classication of breast tumors known as the intrinsic subtypes. These authors performed complementary DNA microarray analysis in a set of normal and malignant human breast tissues from 42 individuals. With use of a hierarchical clustering method, the samples were clustered into four molecular subtypes according to differences in their gene expression proles (of 1,753 genes): luminal, normal breast-like, HER2, and basal-like. In a very simplistic description, luminal tumors were characterized by high expression of hormone receptors and associated genes; normal breast-like cancers were dened by poorly characterized tumors; HER2 subtypes exhibited high expression of HER2 and other genes located in the 17q amplicon and low expression of ER and associated genes; and basal tumors exhibited high expression of basal epithelial genes, basal cytokeratins, and epidermal growth factor receptor (EFGR), and low expression of ER and associated genes. The morphological and

C. Urban and M. Rietjens (eds.), Oncoplastic and Reconstructive Breast Surgery, DOI: 10.1007/978-88-470-2652-0_5, Springer-Verlag Italia 2013

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L. R. Cavalli and I. J. Cavalli

Fig. 5.1 Breast cancer classication into ve molecular subtypes. Hierarchical clustering of 115 tumor tissues and seven nonmalignant tissues using the intrinsic gene set. Experimental dendrogram

showing the clustering of the tumors into ve subgroups. Branches corresponding to tumors with low correlation to any subtype are shown in gray. (From Sorlie et al. [20])

immunohistochemical features of basal-like cancers were similar to those described for tumors arising in BRCA1 germ-line mutation carriers [1619]. In subsequent larger studies from the same group, it was demonstrated that the luminal subtype could be further divided into at least two subgroups (luminal A and luminal B) [4, 20, 21], each with different gene expression proles and different prognosis (Fig. 5.1). Luminal A tumors exhibited high levels of expression of ER-activated genes and low proliferation rates and were associated with a good prognosis, whereas luminal B tumors were more often of higher histological grade and exhibited higher proliferation rates and a worse prognosis. This initial molecular taxonomy has been validated in several other studies, which also identied a few less dened subtypes, including the interferon-rich, molecular apocrine, and claudin-low tumors [2028]. The complete molecular characterization of these less dened subtypes has not been performed and the clinical implications have not been fully identied and/or are not known. The ve major molecular subtypes identied in these studies differ not only in regard to their pattern of gene expression and clinical features but also in regard to the response to treatment and clinical outcome [5, 6, 20, 29, 30]. Patients with luminal tumors respond well to endocrine therapy; however, luminal A and luminal B tumors respond differently to the type of endocrine agent used (tamoxifen or aromatase inhibitors) and also exhibit a variable response to chemotherapy [3134]. Patients with luminal A tumors present with an overall good prognosis with a 5 year survival rate of approximately 90 %. Patients with HER2amplied tumors respond to trastuzumab antibody monoclonal therapy and to anthracycline-based chemotherapy; however, they generally have a poor prognosis and their 5 year survival rate can be as low as 20 % [35, 36]. Finally, patients with the basal-like tumor subtype have no response to endocrine therapy or trastuzumab therapy; however, they

can be sensitive to platinum-based chemotherapy and poly(ADP-ribose) polymerase 1 inhibitors [3739]. These tumors are especially common in African-American women and generally also have poor prognosis [4042]. Interestingly, in the neoadjuvant setting, the intrinsic subtypes have also been found to exhibit different responses to treatment. The pathological complete response rates to standard chemotherapy based on anthracycline and taxane was approximately 7 % for the luminal A subtype, 17 % for the luminal B subtype, 36 % for the HER2-positive subtype, and 43 % for the basal-like subtype [32]. To study the utility of these subtypes in breast tumor classication, a total of 189 breast tumors across 1,906 intrinsic genes were analyzed by Parker et al. [32]. They identied a set of 50 genes that were further validated and compared for reproducibility of classication across different prediction methods and different patient cohorts. This analysis proled by quantitative real-time PCR a total of 122 breast cancers from the 189 individuals into the intrinsic subtypes luminal A, luminal B, HER2-positive, basal-like, and normal-like. Owing to its high reproducibility, a standardized method of classication was developed, the Prediction Analysis of Microarray 50 (PAM50) Breast Cancer Intrinsic Classier test, which is currently commercially available (ARUP Laboratories, Salt Lake City, UT, USA). The PAM50 assay offers the measurement of the expression level of 55 genes (50 classier genes and ve housekeepers) and is recommended for all patients diagnosed with invasive breast cancer, regardless of tumor stage or ER status. The gene expression intrinsic subtypes were discussed for consideration at the last St. Gallen International Breast Cancer Conference [42]. A simplied clinicopathological classication that denes subtypes on the basis of the immunohistochemical analysis of ER, PR, and HER2 status and Ki-67 labeling index (Ki-67 is a cell proliferation marker), similar to what was proposed by Cheang et al. [31], was

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Table 5.1 Intrinsic and immunohistochemical (IHC) subtypes and type of treatment recommended (St.Gallens conference, 2011) Intrinsic subtype Luminal A Luminal B IHC subtype Luminal A Luminal B (HER2 negative) Denition HER2 positive Ki-67 low HER2 negative ER positive PR positive Ki-67 high HER2 positive ER positive PR positive HER2 positive ER negative PR negative HER2 negative ER negative PR negative Type of treatment Endocrine therapy alone Endocrine therapy with or without cytotoxic therapy

Luminal B (HER2 positive)

Cytotoxic therapy plus anti-HER2 therapy plus hormonal therapy

HER2 overexpression

HER2 positive

Cytotoxic therapy plus anti-HER2 therapy

Basal-like

Triple negative

Cytotoxic therapy

Modied from Goldrisch et al. [42] and Perou et al. [3] ER estrogen receptor, PR progesterone receptor, HER2 human epidermal growth factor receptor 2, ki-67 antigen ki-67- protein marker for cell proliferation

endorsed (Table 5.1). The breast cancer subtypes dened by this classication are similar but not identical to the ve intrinsic subtypes and represent a convenient approximation that can be used in considerably less expensive and less complex assays. In general, the therapy recommendations for this classication follow the intrinsic subtype classication: luminal A tumor patients generally require only endocrine therapy, considering that they are mostly less responsive to chemotherapy; luminal B patients, in addition to endocrine therapy, should receive chemotherapy (both anthracycline-based and taxane-based); patients with HER2positive tumors should receive chemotherapy and 1 year of treatment with trastuzumab; and patients with triple-negative tumors should be treated with chemotherapy (also anthracycline-based and taxane-based in addition to an alkylating agent, typically cyclophosphamide). The St. Gallen panel [42] did not endorse the measurement of cytokeratin 5/6 or epidermal growth factor receptor for the determination of basal-like tumors for clinical decision making. In the future, this formal subtyping is very likely to be rened and expanded to include the measurement of novel tumor markers; presently, however, the St. Gallen consensus recommends the classication of breast cancer subtypes and the guide to therapeutic decisions to be based only on the four clinicopathological markers described above. Although gene expression proling has greatly contributed to the determination of breast cancer subtypes and their associated differential prognosis, presently this dened intrinsic molecular classication is not routinely used in clinical practice to classify the patients breast tumor subtype, and no new target therapies have yet resulted for these subtypes [4345]. Several technical challenges limit its use

in clinical practice, including not only the prohibitive costs of the equipment and reagents for the expression assays and the lack of suitable technical personnel to conduct the complex informatics data analysis, but mainly the lack of reproducibilty and uniformity among laboratories in relation to the selection of the intrinsic genes to be used. This latter limitation can be easily perceived by the existence of multiple versions of molecular classication systems developed or under development. In addition, most gene expression microarray analyses were performed by independent investigators using different methods and applied to different patient populations. Another important variability was the cellular composition of the tissue samples (stroma, tumor, and normal cells) in these studies [46, 47]. Cleator et al. [46], in evaluating the cellular composition of the classied samples, demonstrated that the percentage of invasive cancer cells within a sample inuenced the expression prole; at least 10 % of the genes (144 genes) were found to correlate with cellular composition. Other challenges include biological inherent facts, such as that the subtypes assigned by microarrays do not always correspond to the same subtype dened by the routine immunohistochemical (IHC) staining that is used in standard clinical assays [32, 48, 49]. In the retrospective analysis by de Ronde et al. [48], 195 stage II and stage III breast tumors from patients that received neoadjuvant treatment were classied by both IHC and messenger RNA expression analysis on then basis of the molecular classication. The IHC and molecular subtypes showed high concordance with the exception of the HER2 group, where 60 % of the HER2positive tumors were not classied as the HER2 molecular subtype. In addition, for the ER-positive tumors, neither the PR status nor the endocrine responsiveness index (all the

58 Table 5.2 Commonest prognostic gene expression breast cancer signatures commercially available Gene expression signatures Oncotype Dx Patient population ER positive/ negative LN negative Tamoxifen treated ER positive/ negative LN negative Tumor size \ 5 cm Age \ 61 years LN negative ER positive/ negative No systemic therapy ER positive/ negative LN positive/ negative ER positive LN negative Prediction Number of genes Material Assay

L. R. Cavalli and I. J. Cavalli

Company

Risk of recurrence

21 genes

FFPE

RT-PCR

Genomic Health (Redwood City, CA, USA)

MammaPrint

Risk of distant metastasis

70 genes

Frozen

Microarray

Agendia (Huntington Beach, CA, USA, and Amsterdam, The Netherlands)

PAM50

Risk of relapse

55 genes

Frozen/ FFPE

qRT-PCR/ microarraya/ nCounterb

ARUP Laboratories (Salt Lake City, UT, USA): (qRT-PCR format) Nanostring Technologies (Seattle, WA, USA): nCounter format Ipsogen (New Haven, CT, USA, and Marseilles, France)

MapQuant DX

Molecular grading

97 genes

Frozen/ FFPE

Microarray

Breast Cancer Index

Risk of late recurrence Response to endocrine therapy

2 genes, HOXB13:IL17R molecular grade index

FFPE

RT-PCR

BioTheranostics (San Diego, CA, USA)

ER estrogen receptor, LN lymph node, FFPE formalin-xed parafn-embedded, RT-PCR real-time PCR, qRT-PCR quantitative real-time PCR PAM50, marketed by Arup Laboratories as a breast cancer classier b In development
a

tumors showed similar degrees of response to chemotherapy) accurately distinguished the tumors into the luminal A and luminal B subtypes. In fact, several studies have suggested that these ER-positive subtypes may not be completely separate entities [5052]. Once these and others critical challenges are overcome and a universally accepted signature for identifying breast cancer subtypes is established, assays that can maintain a similar level of analytical reproducibility and clinical utility can be developed and implemented for the molecular classication of a patients breast tumor. However such assays should not be expected, at least not soon, to replace the traditional breast cancer classication systems.

5.3

Prognostic Gene Expression Signatures

In the daily management of breast cancer, the selection of the most appropriate adjuvant treatment for an individual patient remains a challenge, despite the excellent assistance of the established therapy guidelines such as those of the St. Gallen consensus [1, 42], the National Institutes of Health [53], and Adjuvant! Online (http://www.adjuvantonline.

com). The ability to identify breast cancer patients with either a very high or a very low risk of recurrence, who would need adjuvant systemic therapy, from those who could be spared such type of treatment is critical. The power of making this distinction at the time of diagnosis, from the analysis of the patients primary tumor, would substantially improve breast cancer survival. Several multigene signatures that predict outcome and response to therapy in breast cancer have been developed through the data obtained from gene expression proling (for reviews, see [59] (Table 5.2). In these studies, major prognostic factors, such as lymph node status and ER status, were addressed and have allowed subgroups of tumors with a very distinct clinical outcome that could not be predicted by conventional prognostic factors to be distinguished in the analysis of the patients primary tumors. The main objective in most of these studies was to predict which patients would benet from a more aggressive treatment and which patients would be unlikely to respond and therefore for whom there would not be a signicant survival benet. Vantveer et al. [54], some of the pioneers of these studies, proposed a prognostic gene signature to identify a group of good prognosis patients with minimal risk of

Molecular Classification and Prognostic Signatures of Breast Tumors

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development of distant metastasis within 5 years after diagnosis. The expression of 25,000 genes was analyzed in primary breast tumors, and a set of 70 genes with differential expression proles separated the patients into two categories, poor and good signature groups, on the basis of their risk of developing distant metastasis. Among the genes that were upregulated in the poor signature group were genes involved in the cell cycle, angiogenesis, invasion and metastasis, and signal transduction, such as CCNE2, MCM6, MMP9, MP1, RAB6B, PK428, ESM1, and the vascular endothelial growth factor receptor FLT1. Subsequent studies conrmed the reproducibility of the initial 70-gene signature as a predictor of outcome independently of traditional clinicalhistopathological prognostic markers [5557]. This validation analysis led to the development of the commercial test MammaPrint developed by Agendia (Amsterdam, The Netherlands). This test is approved by the Food and Drug Administration for use in patients less than 61 years old, who are lymph-node-negative, and who present with a tumor smaller than 5 cm in size. This signature is currently being evaluated in a large clinical trial, MINDACT (Microarray In Node-Negative and 13 Positive Lymph-Node Disease May Avoid Chemotherapy Trial), which is performed in breast cancer patients with ER-positive, lymph-node-negative disease with long-term follow-up and known clinical outcome. The primary end point is to test its robustness and clinical applicability in identifying patients who could be spared the use of chemotherapy without affecting the survival outcome. On the basis of an independent validation study [58], this trial now also includes patients with one to three positive axillary lymph nodes. The other prognostic signature also commercially available is the Oncotype DX breast cancer assay (Genomic Health, Redwood City, CA, USA). This assay was developed on the basis of the identication of 250 selected genes with different expression proles [5961], initially tested in patients from the National Surgical Adjuvant Breast and Bowel Project (NSABP) B-20 clinical trial [62]. After statistical analysis and clinical validation, 21 genes (16 cancerrelated genes and ve reference genes) were selected and their expression analysis was translated into a recurrence score (RS), which was then used to assign the patients to one of three groups, on the basis of the risk of developing distant metastasis: low risk (RS \ 18), intermediate risk (RS C 18 and RS \ 31), and high risk (RS C 31) [63]. This validation study was performed in lymph-node-negative, ER-positive breast cancer patients who were treated with tamoxifen in the large, multicenter NSABP B-14 trial [64]. Subsequent studies have demonstrated its clinical utility as an independent prognostic parameter in ER-positive and lymph-node-positive patients who received adjuvant chemotherapy [65] and also in postmenopausal patients with

ER-positive tumors who were treated with aromatase inhibitors [66]. An ongoing large prospective clinical trial, TAILORX [Trial Assigning Individualized Options for Treatment (Rx)], is further testing the clinical utility of Oncotype DX with the primary end point of accessing whether adjuvant chemotherapy plus hormonal therapy produces a better outcome when compared with hormonal therapy alone in patients who have a low or intermediate score (RS between 11 and 25). In contrast to MammaPrint, which is performed by a microarray assay in frozen tumor tissue samples, Oncotype DX can be performed by real-time PCR in formalin-xed parafn-embedded samples, not requiring therefore the highest-quality RNA material. The Oncotype DX prognostic test has been endorsed by the American Society of Clinical Oncology [67] for clinical use and was included in the last National Comprehensive Cancer Network guidelines (Breast Cancer version 1.201) and the St. Gallen International Expert Consensus [42]. The recommendation for its use is limited to newly diagnosed patients with lymph-node-negative, ER-positive breast cancer who were treated with tamoxifen. The clinical utility and appropriate application recommendations for other multigene assays, such as MammaPrint, are under investigation. The PAM50 multigene gene-expression-based assay, described above as an intrinsic subtype classication assay, is also used to predict prognosis. This assay can predict relapse-free survival, based on a risk of relapse (ROR) score, for patients with lymph-node-negative tumors who were not treated with adjuvant systemic therapy [32]. The prediction value of the ROR was evaluated in an independent set of 786 patients with ER-positive tumors who were treated only with tamoxifen [33]. For both lymphnode-negative and lymph-node-positive patients, the ROR together with tumor size outperformed standard clinicopathological variables, such as Ki-67, PR, and histological grade. For the lymph-node-negative patients the PAM50 ROR identied a group with more than 95 % 10 year survival who had not been submitted to chemotherapy. Several other prognostic signatures were developed, including ones that take into consideration the patients tumor grade, such as MapQuant Dx (Ipsogen, Marseille, France), a microarray-based assay originally based on 97 differentially expressed genes, which was validated as strongly associated with risk of recurrence among patients with grade 2 tumors [68, 69], and the Theros Breast Cancer Index (BCI BioTheranostics, San Diego, CA, USA), which is based on a quantitative real-time PCR assay and provides an assessment of the likelihood of distant recurrence in patients diagnosed with ER-positive and lymph-node-negative breast cancer. It uses a combination of indices (HOXB13:IL17BR two-gene ratio) and a proliferation-related ve-gene molecular grade index, which discriminates grade

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L. R. Cavalli and I. J. Cavalli

1 from grade 3 breast tumors [70, 71]. Several other prognostic signatures that predict clinical outcome were developed on the basis of cancer cell characteristics and processes, including wound healing, hypoxia, stem cells, and stromabroblast interactions [7277]. Although the importance of the gene expression signature of breast tumors has been well established and may be a more accurate prognostic marker than other well-established clinicalhistopathological criteria, one cannot assume that all the genes present in these gene expression signature panels are equally important or have an independent role in breast cancer pathogenesis and recurrence. Successful enrichment, reliable identication, and molecular proling of pure epithelial tumor cells are still key issues to be addressed [78, 79]. Interestingly, although there is very little overlap among these signatures in relation to the gene composition, most of them are related to proliferation and ER-signaling cellular processes [52, 80, 81]. It is no coincidence that the prediction power of most of these signatures is more robust and indicated for ER-positive tumors/luminal subtype and less for the ER-negative subtypes [81], hence the specic clinical indication of most of these prognostic gene-expressionbased assays for patients with ER-positive, lymph-nodenegative disease for whom it is safer to recommend a treatment based only on hormonal therapy. Recent studies have provided evidence that other genes related to cellular processes, including the expression of immune-response genes, have the potential to predict survival, especially within the HER2-positive and basal-like subtypes of tumors [8286].

identication of new therapeutically targetable markers, leading to the development of novel diagnostic tests to guide the most appropriate and individualized cancer therapy. Finally, considering the current dissemination of the genomically based testing and treatment strategies for cancer, it is imperative to address the use of these tests on the impact of the therapeutic decision making and the patients health outcome, taking into consideration the social and economic variants of specic breast cancer patient populations.

References
1. Goldhirsch A, Ingle JN, Gelber RD et al (2009) Thresholds for therapies: highlights of the St. Gallen international expert consensus on the primary therapy of early breast cancer. Ann Oncol 8:13191329 2. Galea MH, Blamey RW, Elston CE et al (1992) The Nottingham Prognostic Index in primary breast cancer. Breast Cancer Res Treat 3:207219 3. Perou CM, Sorlie T, Eisen MB et al (2000) Molecular portraits of human breast tumors. Nature 6797:747752 4. Sorlie T, Perou CM, Tibshirani R et al (2001) Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci USA 19:1086910874 5. Sotiriou C, Pusztai L (2009) Gene-expression signatures in breast cancer. N Engl J Med 360:790800 6. Weigelt B, Baehner FL, Reis-Filho JS (2010) The contribution of gene expression proling to breast cancer classication, prognostication and prediction: a retrospective of the last decade. J Pathol 220:263280 7. Colombo PE, Milanezi F, Weigelt B et al (2011) Microarrays in the 2010 s: the contribution of microarray-based gene expression proling to breast cancer classication, prognostication and prediction. Breast Cancer Res 13:212227 8. Fumagalli D, Desmedt C, Ignatiadis M et al (2011) Gene proling assay and application: the predictive role in primary therapy. J Natl Cancer Inst Monogr 124127 9. Reis-Filho JS, Pusztai L (2011) Gene expression proling in breast cancer: classication, prognostication, and prediction. Lancet 378:18121823 10. Pinkel D, Segraves R, Sudar D et al (1998) High resolution analysis of DNA copy number variation using comparative genomic hybridization to microarrays. Nat Genet 20:207211 11. Duggan DJ, Bittner M, Chen Y et al (1999) Expression proling using cDNA microarrays. Nat Genet 21:1014 12. Pollack JR, Srlie T, Perou CM et al (2002) Microarray analysis reveals a major direct role of DNA copy number alteration in the transcriptional program of human breast tumors. Proc Natl Acad Sci USA 99:1296312968 13. Shipitsin M, Campbell LL, Argani P et al (2007) Molecular denition of breast tumor heterogeneity. Cancer Cell 11:259273 14. Stingl J, Caldas C (2007) Molecular heterogeneity of breast carcinomas and the cancer stem cell hypothesis. Nat Rev Cancer 7:791799 15. Weigelt B, Reis-Filho JS (2009) Histological and molecular types of breast cancer: is there a unifying taxonomy? Nat Rev Clin Oncol 6:718730 16. Laakso M, Loman N, Borg A et al (2005) Cytokeratin 5/14positive breast cancer: true basal phenotype conned to BRCA1 tumors. Mod Pathol 18:13211328

5.4

Conclusions

The rapid advances in the DNA microarray technology and the ability of performing large-scale validations have allowed the development of gene expression signatures that can be used to identify breast cancer molecular subtypes and predict response to therapy and clinical outcome. It is without question that the continued improvement of molecular tumor proling in gene-expression-based assays and the development of next-generation technologies, such as large-scale sequencing, will lead to successful application of these and newly developed gene signatures in the clinical setting. These efforts will certainly be reected in the stratication of breast cancer disease into a newly rened taxonomy, allowing for the understanding of the genetic diversity in the different breast cancer subtypes. In addition, considering that the success of a treatment largely depends on the ability to match a particular tumor phenotype to a specic tumor genomic target, these new technologies will provide the

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17. Lakhani SR, Reis-Filho JS, Fulford L et al (2005) Prediction of BRCA1 status in patients with breast cancer using estrogen receptor and basal phenotype. Clin Cancer Res 11:51755180 18. Turner NC, Reis-Filho JS (2006) Basal-like breast cancer and the BRCA1 phenotype. Oncogene 25:58465853 19. Eerola H, Heinonen M, Heikkila P et al (2008) Basal cytokeratins in breast tumours among BRCA1, BRCA2 and mutation-negative breast cancer families. Breast Cancer Res 10:R17 20. Sorlie T, Tibshirani R, Parker J et al (2003) Repeated observation of breast tumor subtypes in independent gene expression data sets. Proc Natl Acad Sci USA 100:84188423 21. Sorlie T, Wang Y, Xiao C et al (2006) Distinct molecular mechanisms underlying clinically relevant subtypes of breast cancer: gene expression analyses across three different platforms. BMC Genomics 7:127 22. Farmer P, Bonnefoi H, Becette V et al (2005) Identication of molecular apocrine breast tumours by microarray analysis. Oncogene 24:46604671 23. Doane AS, Danso M, Lal P et al (2006) An estrogen receptornegative breast cancer subset characterized by a hormonally regulated transcriptional program and response to androgen. Oncogene 25:39944008 24. Hu Z, Fan C, Oh DS et al (2006) The molecular portraits of breast tumors are conserved across microarray platforms. BMC Genomics 7:96 25. Teschendorff AE, Caldas C (2008) A robust classier of high predictive value to identify good prognosis patients in ER-negative breast cancer. Breast Cancer Res 10:R73 26. Hennessy BT, Gonzalez-Angulo AM, Stemke-Hale K et al (2009) Characterization of a naturally occurring breast cancer subset enriched in epithelial-to-mesenchymal transition and stem cell characteristics. Cancer Res 69:41164124 27. Banneau G, Guedj M, MacGrogan G et al (2010) Molecular apocrine differentiation is a common feature of breast cancer in patients with germline PTEN mutations. Breast Cancer Res 12:R63 28. Prat A, Parker JS, Karginova O et al (2010) Phenotypic and molecular characterization of the claudin-low intrinsic subtype of breast cancer. Breast Cancer Res 12:R68 29. Rouzier R, Perou CM, Symmans WF et al (2005) Breast cancer molecular subtypes respond differently to preoperative chemotherapy. Clin Cancer Res 11:56785685 30. Martin M, Romero A, Cheang MC et al (2011) Genomic predictors of response to doxorubicin versus docetaxel in primary breast cancer. Breast Cancer Res Treat 128:127136 31. Cheang MCU, Chia SK, Voduc D et al (2009) Ki67 index, HER2 status, and prognosis of patients with luminal B breast cancer. J Natl Cancer Inst 101:736750 32. Parker JS, Mullins M, Cheang MC et al (2009) Supervised risk predictor of breast cancer based on intrinsic subtypes. J Clin Oncol 27:11601167 33. Nielsen TO, Parker JS, Leung S et al (2010) A comparison of PAM50 intrinsic subtyping with immunohistochemistry and clinical prognostic factors in tamoxifen-treated estrogen receptor-positive breast cancer. Clin Cancer Res 16:52225232 34. Iwamoto T, Bianchini G, Booser D et al (2011) Gene pathways associated with prognosis and chemotherapy sensitivity in molecular subtypes of breast cancer. J Natl Cancer Inst 103:264272 35. Arteaga CL, Sliwkowski MX, Osborne CK et al (2011) Treatment of HER2-positive breast cancer: current status and future perspectives. Nat Rev Clin Oncol 9:1632 36. Mukohara T. Role of HER2-targeted agents in adjuvant treatment for breast cancer. Chemother Res Pract 730360, 2011 37. Banerjee S, Reis-Filho JS, Ashley S et al (2006) Basal-like breast carcinomas: clinical outcome and response to chemotherapy. J Clin Pathol 59:729735

38. Rakha EA, Reis-Filho JS, Ellis IO (2008) Basal-like breast cancer: a critical review. J Clin Oncol 26:25682581 39. Fong PC, Boss DS, Yap TA et al (2009) Inhibition of poly(ADPribose) polymerase in tumors from BRCA mutation carriers. N Engl J Med 361:123134 40. Carey LA, Dees EC, Sawyer L et al (2007) The triple negative paradox: primary tumor chemosensitivity of breast cancer subtypes. Clin Cancer Res 13:23292334 41. Millikan RC, Newman B, Tse CK et al (2008) Epidemiology of basal-like breast cancer. Breast Cancer Res Treat 109:123139 42. Goldhirsch A, Wood WC, Coates AS et al (2011) Strategies for subtypes-dealing with the diversity of breast cancer: highlights of the St. Gallen international expert consensus on the primary therapy of early breast cancer. Ann Oncol 22:17361747 43. Desmedt C, Ruz-Garca E, Andr F (2008) Gene expression predictors in breast cancer: current status, limitations and perspectives. Eur J Cancer 44:27142720 44. van der Vegt B, de Bock GH, Hollema H et al (2009) Microarray methods to identify factors determining breast cancer progression: potentials, limitations, and challenges. Crit Rev Oncol Hematol 70:111 45. Weigelt B, Pusztai L, Ashworth A et al (2011) Challenges translating breast cancer gene signatures into the clinic. Nat Rev Clin Oncol 9:5864 46. Cleator SJ, Powles TJ, Dexter T et al (2006) The effect of the stromal component of breast tumours on prediction of clinical outcome using gene expression microarray analysis. Breast Cancer Res 8:R32 47. Mackay A, Weigelt B, Grigoriadis A et al (2011) Microarraybased class discovery for molecular classication of breast cancer: analysis of interobserver agreement. J Natl Cancer Inst 103:662 673 48. de Ronde JJ, Hannemann J, Halfwerk H et al (2010) Concordance of clinical and molecular breast cancer subtyping in the context of preoperative chemotherapy response. Breast Cancer Res Treat 119:119126 49. Weigelt B, Mackay A, AHern R et al (2010) Breast cancer molecular proling with single sample predictors: a retrospective analysis. Lancet Oncol 4:339349 50. Shak S, Baehner FL, Palmer G et al (2006) Subtypes of breast cancer dened by standardized quantitative RTPCR analysis of 10 618 tumors. Breast Cancer Res Treat 100:S295S295 51. Desmedt C, Haibe-Kains B, Wirapati P et al (2008) Biological processes associated with breast cancer clinical outcome depend on the molecular subtypes. Clin Cancer Res 14:51585165 52. Wirapati P, Sotiriou C, Kunkel S et al (2008) Meta-analysis of gene expression proles in breast cancer: toward a unied understanding of breast cancer subtyping and prognosis signatures. Breast Cancer Res 10:R65 53. Eifel P, Axelson JA, Costa J et al (2001) National institutes of health consensus development conference statement: adjuvant therapy for breast cancer. November 13, 2000. J Natl Cancer Inst 93:979989 54. vant Veer LJ, Dai H, van de Vijver MJ et al (2002) Gene expression proling predicts clinical outcome of breast cancer. Nature 6871:530536 55. van de Vijver MJ, He YD, vant Veer LJ, et al (2002) A gene expression signature as a predictor of survival in breast cancer. N Engl J Med 347:19992009 56. Buyse M, Loi S, vant Veer L, et al (2006) Validation and clinical utility of a 70-gene prognostic signature for women with nodenegative breast cancer. J Natl Cancer Inst 98:11831192 57. Bueno-de-Mesquita JM, van Harten WH, Retel VP et al (2007) Use of 70-gene signature to predict prognosis of patients with node-negative breast cancer: a prospective community-based feasibility study (RASTER). Lancet Oncol 8:10791087

62 58. Mook S, Schmidt MK, Viale G et al (2009) The 70-gene prognosis-signature predicts disease outcome in breast cancer patients with 13 positive lymph nodes in an independent validation study. Breast Cancer Res Treat 116:295302 59. Cobleigh MA, Bitterman P, Baker J et al (2003) Tumor gene expression predicts distant disease-free survival (DDFS) in breast cancer patients with 10 or more positive nodes: high throughout RT-PCR assay of parafn-embedded tumor tissues. Prog Proc Am Soc Clin Oncol 22:850850 60. Esteban J, Baker J, Cronin M et al (2003) Tumor gene expression and prognosis in breast cancer: multi-gene RT-PCR assay of parafn-embedded tissue. Prog Proc Am Soc Clin Oncol 22:850 61. Paik S, Shak S, Tang G et al (2003) Multi-gene RT-PCR assay for predicting recurrence in node negative breast cancer patients: NSABP studies B-20 and B-14. Breast Cancer Res Treat 82:A16 62. Fisher B, Dignam J, Wolmark N et al (1997) Tamoxifen and chemotherapy for lymph node-negative, estrogen receptor-positive breast cancer. J Natl Cancer Inst 89:16731682 63. Paik S, Shak S, Tang G et al (2004) A multigene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer. N Engl J Med 351:28172826 64. Fisher B, Costantino J, Redmond C et al (1989) A randomized clinical trial evaluating tamoxifen in the treatment of patients with node-negative breast cancer who have estrogen-receptor-positive tumors. N Engl J Med 320:479484 65. Habel LA, Shak S, Jacobs MK et al (2006) A population based study of tumor gene expression and risk of breast cancer death among lymph node-negative patients. Breast Cancer Res 8:R25 66. Dowsett M, Cuzick J, Wale C et al (2010) Prediction of risk of distant recurrence using the 21-gene recurrence score in nodenegative and node-positive postmenopausal patients with breast cancer treated with anastrozole or tamoxifen: a TransATAC study. J Clin Oncol 11:18291834 67. Harris L, Fritsche H, Mennel R et al (2007) American Society of Clinical Oncology 2007 update of recommendations for the use of tumor markers in breast cancer. J Clin Oncol 33:52875312 68. Loi S, Haibe-Kains B, Desmedt C et al (2007) Denition of clinically distinct molecular subtypes in estrogen receptor-positive breast carcinomas through genomic grade. J Clin Oncol 25:1239 1246 69. Ma XJ, Salunga R, Dahiya S et al (2008) A ve-gene molecular grade index and HOXB13:IL17BR are complementary prognostic factors in early stage breast cancer. Clin Cancer Res 14:26012608 70. Jerevall PL, Ma XJ, Li H et al (2011) Prognostic utility of HOXB13:IL17BR and molecular grade index in early-stage breast cancer patients from the Stockholm trial. Br J Cancer 104: 17621769 71. Chang HY, Sneddon JB, Alizadeh AA et al (2004) Gene expression signature of broblast serum response predicts human

L. R. Cavalli and I. J. Cavalli cancer progression: similarities between tumors and wounds. PLoS Biol 2:E7 West RB, Nuyten DS, Subramanian S et al (2005) Determination of stromal signatures in breast carcinoma. PLoS Biol 3:e187 Chi JT, Wang Z, Nuyten DS et al (2006) Gene expression programs in response to hypoxia: cell type specicity and prognostic signicance in human cancers. PLoS Med 3:e47 Liu R, Wang X, Chen GY et al (2007) The prognostic role of a gene signature from tumorigenic breast-cancer cells. N Engl J Med 356:217226 Finak G, Bertos N, Pepin F et al (2008) Stromal gene expression predicts clinical outcome in breast cancer. Nat Med 14:518527 Reyal F, van Vliet MH, Armstrong NJ et al (2008) A comprehensive analysis of prognostic signatures reveals the high predictive capacity of the proliferation, immune response and RNA splicing modules in breast cancer. Breast Cancer Res 10:R93 Desmedt C, Michiels S, Haibe-Kains B et al (2011) Time to move forward from rst-generation prognostic gene signatures in early breast cancer. Breast Cancer Res Treat 128:643645 Espinosa E, Vara J, Navarro IS et al (2011) Gene proling in breast cancer: time to move forward. Cancer Treat Rev 37:416 421 Fan C, Oh DS, Wessels L et al (2006) Concordance among geneexpression-based predictors for breast cancer. N Engl J Med 355:560569 Fan C, Prat A, Parker JS, et al (2011) Building prognostic models for breast cancer patients using clinical variables and hundreds of gene expression signatures. BMC Med Genomics 9(4):3 Miglietta A, Toselli M, Ravarino N et al (2010) COX-2 expression in human breast carcinomas: correlation with clinicopathological features and prognostic molecular markers. Expert Opin Ther Targets 14:655664 Schmidt M, Gehrmann M, Hengstler JG et al (2010) New prognostic and predictive factors in breast cancer. Minerva Ginecol 62:599611 Barsk NO, Keser SH, Gul AE et al (2011) The value of COX-2 expression in the prognostic parameters of invasive ductal carcinoma of the breast. Med Oncol 28:703708 Rody A, Karn T, Liedtke C, et al (2011) A clinically relevant gene signature in triple negative and basal-like breast cancer. Breast Cancer Res 13:R97 Hallett RM, Dvorkin-Gheva A, Bane A et al (2012) A gene signature for predicting outcome in patients with basal-like breast cancer. Sci Rep 2:227 Schmidt M, Hellwig B, Hammad S, et al (2012) A comprehensive analysis of human gene expression proles identies stromal immunoglobulin kappa C as a compatible prognostic marker in human solid tumors. Clin Cancer Res

72. 73.

74.

75. 76.

77.

78.

79.

80.

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82.

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