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ANCILLARY TOOLS IN BREAST PATHOLOGY

The role of molecular analysis in breast cancer

`* FELIPE C. GEYER*, CATERINA MARCHIO AND

JORGE S. REIS-FILHO

Molecular Pathology Laboratory, The Breakthrough Breast Cancer Research Centre, Institute of Cancer Research, London, United Kingdom; *these authors contributed equally to this review

Summary Breast cancer is a heterogeneous disease, encompassing a wide variety of histological types and clinical behaviours. Current histopathological classication systems for breast cancer are based on descriptive entities that are of prognostic signicance. Few predictive biomarkers are currently available. High throughput molecular technologies are reshaping our understanding of breast cancer and a molecular taxonomy that has stronger predictive power is slowly emerging. Novel therapeutic targets and prognostic/predictive gene signatures have been identied. This review will address the contribution of molecular methods to our understanding of breast cancer and its precursors, their use in breast cancer translational research and their impact on diagnostic breast cancer histopathology.
Key words: Microarrays, comparative genomic hybridisation, prognostic marker, predictive marker, genomic signature, histological grade, breast cancer. Received 20 July, revised 5 August, accepted 7 August 2008

Molecular pathology has already had a great impact on the diagnosis of haematological and soft tissue neoplasia. The contribution of molecular techniques to the classication of carcinomas has not yet been so profound. In the last few years, breast cancer has been the epithelial malignancy most studied by molecular techniques; therefore, it is not surprising that not only predictive markers, but also novel therapeutic targets are emerging. The focus of this review will be on the impact of molecular proling analysis and molecular genetics on our understanding of breast cancer and its precursors.

AN ARRAY OF CHANGES
The boom of high throughput technologies and the class discovery studies pioneered by the Stanford group4,5 have not only brought forward the fact that breast cancer is not a single entity but also provided a working model for a molecular taxonomy for breast cancer.48 Expression proling analyses using microarrays have demonstrated that breast cancers can be classied according to their expression patterns into at least ve groups:48 luminal A, luminal B, normal breast-like, HER2 and basallike. The most robust distinction observed by microarray analysis is between the transcriptome of ER positive and ER negative breast cancers. Luminal tumours are described as those that show expression patterns reminiscent of normal luminal epithelial cells of the breast, including consistent expression of low molecular weight cytokeratins 8/18, ER and genes associated with an active ER pathway.48 At least two subgroups of luminal tumours have been identied: luminal A, which are usually of low histological grade, have an excellent prognosis and show high levels of expression of ER-activated genes; and luminal B, which are more often of higher histological grade, have higher proliferation rates and a poorer prognosis than luminal A tumours.48 Normal breast-like cancers are rather poorly characterised tumours; one of the dening features of these lesions is that they consistently cluster together with samples of broadenomas and normal breast samples. The clinical signicance of normal breastlike tumours is yet to be determined48 and some have suggested that this subgroup may be a mere artefact of expression proling (i.e., disproportionally high content of stromal cells). HER2 tumours are usually ER negative and characterised by over-expression of HER2 and genes associated with HER2 pathway and/or HER2 amplicon on 17q12. HER2 cancers have very aggressive clinical behaviour; however, they are amenable to novel tailored

INTRODUCTION
Breast cancer is a heterogeneous disease encompassing a wide variety of pathological entities that are reported to have distinct clinical behaviours.1,2 Pathologists have acknowledged the complexity of breast cancer and endeavoured to devise classication systems to account for its diversity. However, current classication systems are descriptive, based on morphological entities that have been shown to have prognostic implications. For the success of targeted therapies and individualised medicine, a predictive, rather than purely prognostic, classication system is required. Despite the translational research eorts of the 1980s and 90s, only three biomarkers are routinely used in breast cancer patient management, namely oestrogen receptor (ER), progesterone receptor (PR) and HER2. Interestingly, all of these biomarkers have optimal negative predictive values (i.e., patients with ER negative breast cancer are highly unlikely to respond to endocrine therapy; HER2 negative breast cancers fail to respond to humanised monoclonal antibodies against HER2). However, their positive predictive value is rather limited, with a substantial proportion of patients with HER2 positive disease either harbouring de novo resistance or developing resistance to trastuzumab over time.3

Print ISSN 0031-3025/Online ISSN 1465-3931 # 2009 Royal College of Pathologists of Australasia DOI: 10.1080/00313020802563536

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therapies using either humanised monoclonal antibodies against HER2 or HER2 tyrosine kinase inhibitors.413 Although the vast majority (480%) of HER2 cancers as dened by microarrays harbour HER2 gene amplication or HER2 3 immunohistochemical expression,6,14 not all tumours that are HER2 amplied fall into the HER2 cluster by expression arrays analysis. There is also evidence to suggest that some HER2 amplied, ER positive cancers fall within the luminal subtypes rather than the HER2 microarray subtype.6 Basal-like cancers, another group of ER negative cancers, are so named because the neoplastic cells of this tumour type consistently express genes usually expressed in normal basal/myoepithelial cells of the breast, including high molecular weight cytokeratins (5/6, 14 and 17), P-cadherin, caveolins 1 and 21522 and epidermal growth factor receptor (EGFR)17 and, in a minority of cases, harbour EGFR gene amplication.7 These tumours are usually of high histological grade and characterised by high mitotic indices, the presence of central necrotic zones, pushing borders, conspicuous lymphocytic inltrate and typical/atypical medullary features.23,24 In addition, metaplastic elements are not uncommonly found.25 The morphological and immunohistochemical features of basal-like cancers are remarkably similar to those described for tumours arising in BRCA1 germline mutation carriers21 and there is a growing body of evidence to suggest that BRCA1 pathway is dysfunctional in sporadic basal-like cancers.26 In fact, engineered mouse models targeting Brca1 and Trp53 genes in luminal or basal cells of the mouse mammary gland resulted in the development of tumours displaying morphological and immunohistochemical features that recapitulate those seen in human basal-like breast carcinomas.27,28

BASAL-LIKE CARCINOMAS: BACK TO THE BASIS

Before discussing the implications of basal-like breast carcinomas, it is worth mentioning that two misconceptions have plagued the literature on this topic. First, there is a pervasive misconception that basal-like cancers were discovered by gene expression proling analysis. In fact, the existence of a subgroup of aggressive breast carcinomas showing features of myoepithelial/basal dierentiation has been known since the 1960s16,2932 and that these tumours more often express the immunohistochemical features now known to be characteristic of basallike cancers has been known since the 1990s.33 However, it would be fair to say that basal-like breast carcinomas only gained widespread interest after their rediscovery and systematic classication by microarray-based expression proling analysis. Another important misconception is that basal-like and triple negative (ER, PR and HER2 negative) cancers are synonymous.34 Although the vast majority of triple negative cancers are of basal-like phenotype3537 and the vast majority of tumours expressing basal markers are triple negative,17,38 there is a signicant number of triple negative cancers that do not express basal markers and a small, but still signicant, subgroup of basal-like cancers that express either hormone receptors or HER2.14,37,3941 Bertucci et al.39 have addressed this issue directly and conrmed that not all triple negative tumours when

analysed by gene expression proling were classied as basal-like cancers (i.e., only 71% were of basal-like phenotype) and not all basal-like breast carcinomas classied by expression arrays displayed a triple negative phenotype (i.e., 77% were of triple negative phenotype). Taken all together, these results are in accord with the concept that the triple negative phenotype is not an ideal surrogate marker for basal-like breast cancers37,39,42 and call for caution in the interpretation of ongoing therapeutic trials whose selection of patients was made on the basis of lack of ER, PR and HER2 expression.39 Furthermore, there are several lines of evidence to suggest that the group of triple negative cancers is substantially more heterogeneous than the group encompassed by basal-like breast cancers.3739 Although the gold standard for diagnosis of basal-like carcinomas remains microarray-based expression proling, this technology is unlikely to be rolled out as a diagnostic tool in most pathology departments. In fact, several groups have endeavoured to develop surrogate markers for the intrinsic gene list molecular taxonomy. Real-time PCR based methods have already been developed and validated in archival, formalin-xed, paran-embedded tissue samples.43,44 However, for routine management of breast specimens, immunohistochemistry is likely to become the method used to identify these subtypes. Although there is no internationally accepted immunohistochemical denition for basal-like breast cancer,45 Nielsen et al. have demonstrated that, using a panel of four immunohistochemical markers, basal-like carcinomas can be detected with a sensitivity of 76% and a specicity of 100%.17 Basallike carcinomas are thus dened as being ER negative, HER2 negative and positive for expression of cytokeratin 5/6 and/or EGFR. Importantly, this immunohistochemical panel has been shown to be of prognostic signicance in dierent cohorts.38,42 Further renement of this panel by the same group38 has demonstrated that expression of basal markers (i.e., cytokeratin 5/6 and/or EGFR) in ER, PR and HER2 negative cases, identies a subgroup of triple negative cancers with a signicantly worse outcome than patients with triple negative carcinomas that are negative for basal markers. Basal-like carcinomas have been shown to display a rather aggressive clinical behaviour,15,18,41,46,47 with most recurrences happening within the rst ve years after diagnosis.41,47 Late recurrences are reported not to be as common. Basal-like cancers appear to respond to neoadjuvant chemotherapy,14,48,49 however, patients with basal-like tumours, paradoxically, still have a worse outcome when compared with those with tumours pertaining to other molecular subgroups.14,15,18,35,46,48 However, there is substantial circumstantial evidence to suggest that patients with basal-like breast cancer who evolve to pathological complete response following neoadjuvant chemotherapy have an excellent prognosis, whereas those who only display a partial response have remarkably poor outcomes.48,50 This underpins the concept that basal-like cancers are a heterogeneous group in terms of their expression proles, molecular genetic patterns51 and clinical behaviour.41,47 Interestingly, basal-like and tumours arising in BRCA1 mutation carriers have recently been shown to preferentially express markers consistent with a cancer stem cell/ cancer progenitor cell phenotype.52,53 However, it should

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be emphasised that not all basal-like cancers display CD44/CD24 cells.52 Interestingly, although breast cancer stem cells are reported to be resistant to chemotherapy and cancer stem cells are more prevalent in basal-like breast cancers, the highest prevalence of pathological complete response following neoadjuvant chemotherapy is found in the subgroup of basal-like cancers.48,50 Although several theories have been advanced to reconcile these contrasting results, they still remain purely theoretical.

GENE-EXPRESSION PROFILING: PROGNOSTIC SIGNATURES

Pathology-derived details have long been used to tailor therapy for cancer patients. Their diligent use has contributed to the reduction of breast cancer mortality and morbidity, with a breast pathologist having a crucial role in the treatment decision making. As an example, sentinel lymph node biopsy has been responsible for great improvement in breast cancer treatment and its recognised high negative predictive value for axillary nodal status54,55 depends on adequate histopathological examination.56,57 Adjuvant!Online (www.adjuvantonline.com, accessed July 2008), Nottingham prognostic index, St Gallen guidelines, and National Institutes of Health (NIH) criteria,58,59 tools often used by oncologists to decide which patients should receive chemotherapy, are based on tumour size, histological grade, ER status, vascular invasion and presence of lymph node metastasis. However, oncologists, pathologists and surgeons would not disagree that the parameters currently available are not sucient to capture the complexity of breast cancer and to tailor therapy for individual patients. Therefore, several research groups have carried out comprehensive microarray gene-expression proling studies with the aim of improving on traditional clinico-pathological parameters. The rst breast cancer prognostic signature described60 was developed through a class prediction analysis of a series of 78 young patients with small (55 cm), lymph node negative cancers who did not receive adjuvant chemotherapy. By comparing the expression proles of tumours from patients who developed distant metastasis within 5 years with those who did not, the authors identied a multi-gene prognostic predictive score comprising 70 genes (MammaPrint; Agendia, The Netherlands). Later the signature was validated in larger cohorts with node positive and node negative patients.61,62 A dierent approach has been undertaken by Wang et al.63 who rst identied 16 and 60 genes associated with relapse in ER positive and ER negative breast carcinomas, respectively, and assembled them into a 76-gene prognostic signature (VDX2 gene chips; Veridex LLC, USA). In the studies above, statistical analyses have arguably shown that the predictive power of these signatures was signicantly superior to the standard clinico-pathological parameters. However, subsequent statistical analyses have called into question the actual contribution of these signatures for breast cancer patient management.6466 Following the attention these two prognostic signatures attracted, several other prognostic signatures have been or are currently being developed. Fan et al.67 applied multiple signatures (i.e., intrinsic subtypes, 70-gene signature,

wound response signature and 21-gene recurrence score) to a cohort of breast cancer patients and conrmed their prognostic value.67 On the other hand, others have failed to nd a superior predictive value for the 70-gene signature when compared with that of regular clinico-pathological features.14,64,65 Although all of these signatures are based on the expression of genes related to similar pathways and to some extent correlate with tumour proliferation,67,68 many were surprised by the fact that the overlap in terms of genes belonging to these signatures is negligible. As a consequence, both pathologists and clinicians face the challenge of which signature should be used. Disappointingly, combining dierent signatures does not seem to result in a signicant improvement of accuracy.67 To complicate matters even further, many doubts regarding the reliability and reproducibility of the technique have arisen.7,69,70 One of the reasons for this apparent failure of microarrays in realising their potential stems from the disparate paces of microarray technology development and the development of bioinformatics and statistics applied to microarray analysis. For instance, in the beginning of this century, data over-tting was a poorly understood concept and methods for power calculation for microarray analysis were yet to be described.71 Fortunately, this eld is maturing very rapidly and currently there are clear guidelines as to how a therapeutically signicant gene signature should be developed and validated.72,73 Unfortunately, none of the microarray-based signatures described to date fulls all criteria required. Although we believe that microarray-based technology, or most likely one of its derivatives, is likely to be incorporated in breast pathology practice, we argue that there are practical issues that need to be resolved before gene expression proling can be translated to routine clinical use. First of all, there are data to suggest that the prognostic power of the gene signatures reported to date is rather limited in ER negative, high grade disease.68 In addition, although microarrays are quite reproducible and can certainly be applied to class discovery studies and preclinical analysis, their accuracy and reproducibility are not sucient for clinical use. For instance, up to 15% of error in qualitative assessment of gene expression has been demonstrated when the optimal samples were analysed with the same platform and protocols in dierent laboratories.74 Furthermore, the applicability of microarrays to readily available, formalin-xed, paran-embedded material is limited, as this technology has been shown to perform suboptimally when RNA extracted from this type of sample is used. Although Illumina (USA; http://www.illumina. com/, accessed July 2008) has recently provided a method for microarray-based gene expression analysis of formalinxed, paran-embedded samples (DASL gene expression), which has been shown to produce reasonable results when applied to matched frozen and formalin-xed, paranembedded specimens,75 neither the 70-gene nor the 76-gene signature has been converted into this platform. As stated above, we anticipate that one of the derivatives of microarray analysis, rather than microarrays themselves, is likely to be incorporated into clinical practice. In fact, quantitative reverse transcriptase PCR (qRT-PCR)-based signatures and immunohistochemical panels have already been developed as alternative methods for expression proling analysis. The prototype of this approach is

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Oncotype DX (Genomic Health, USA),76 which is based on the mRNA expression levels of 21 genes (16 cancer related genes and ve reference genes). These 16 genes comprise components of ER pathway (ER, PGR, BCL2 and SCUBE2), proliferation (Ki67, STK15, Survivin, CCNB1 and MYBL2), HER2 amplicon (HER2 and GRB7), invasion (MMP11 and CTSL2) and GSTM1, CD68 and BAG1. The analysis of the gene expression by qRT-PCR gives rise to the recurrence score, which has been shown to be an independent prognostic factor in early stage, endocrine responsive, tamoxifen treated patients. Patients with a high recurrence score are thought to benet from chemotherapy. Intermediate and low scores are assembled under the good prognosis group and chemotherapy would be of limited value. This assay has proven suitable for the analysis of formalin-xed paran-embedded samples, however the recurrence score was not validated in two studies.77,78 Furthermore, some have argued that it only recapitulates the information provided by grade, ER, HER2 and MIB179 and it remains to be determined if this signature will be of any prognostic value for patients receiving aromatase inhibitors or anthracyline- and taxanebased therapeutic regimens.80

GRADING OF BREAST CANCER: THE END OF NOTTINGHAM GRADE?

Molecular studies have conrmed the importance of histological grade in breast carcinomas and this should not be surprising, given that morphological features visible in H&E sections such as nuclear atypia, tubule formation and mitosis represent the expression of thousands of genes and the interactions of tens of thousands of proteins.81 It has been demonstrated that grade, more than any other clinico-pathological parameter or tumour intrinsic characteristic, is associated with the type, pattern and complexity of molecular changes seen in breast cancer and its precursors.1,2 Ma et al. analysed matched normal, preinvasive and invasive breast lesions of dierent grades by means of expression proling analysis and observed that samples preferentially clustered according to histological grade rather than stage.82 Currently, histological grading of breast carcinomas is one of the cornerstones of treatment decision making. It is performed by histopathological analysis using the modied Bloom and Richardson method,83 also known as Nottingham grade. Despite its importance, concordance between pathologists has been reported to be suboptimal.84 In fact intra- and inter-observer agreement ranges from 50% to 86%. The clinical signicance of grade I and grade III are well dened, but only about half of breast carcinomas are classied as such. Often oncologists have to care for a patient with a grade II invasive ductal carcinoma of no special type, which is not helpful in clinical decision making. This has prompted several groups to investigate methods that could rene the current grading system, making it more reproducible and assigning high and low risk subgroups. Sotiriou et al. undertook a hypothesis-driven approach focusing on histological grade.85 They analysed grade I and grade III ER positive breast carcinomas using expression arrays and proposed the gene expression

grade index (GGI). GGI is based on 97 genes, most of them are associated to cell cycle progression and proliferation. Indeed, proliferation has been reported to be the most important predictor of outcome in breast cancer, especially in ER positive patients.86 GGI could identify grade I and grade III tumours in the validation set with an accuracy of *90% and was more strongly associated with relapse-free survival than histological grade. Importantly, it was shown that GGI could stratify grade II tumours into genomic low grade and genomic high grade and that these groups were of prognostic signicance: histological grade II GGI low tumours had outcomes similar to GGI low cancers, whereas histological grade II GGI high tumours had outcomes similar to GGI high cancers. GGI has been subsequently validated by the same group.87 Another genetic grade signature was proposed by Ivshina et al.88 who proled 347 breast invasive tumours with expression arrays. Using two class prediction algorithms, six grade-associated genes were identied and accurately classied grade I and grade III tumours in low and high genomic grade, respectively. Grade II tumours were not a molecularly distinct group but could be separated into grade IIa (low grade-like) or grade IIb (high grade-like). Grade IIa patients had a signicantly better outcome than grade IIb patients, whereas no dierence was observed between the grade IIa and grade I survival curves, or the grade IIb and grade III curves. These results have been further validated by the authors in datasets generated with dierent microarray platforms.88 However, further analyses have shown that there were signicant molecular and clinico-pathological dierences between the grade I and grade IIa tumours and the grade IIb and grade III tumours. More recently, using a similar approach, Ma et al. reported a ve-gene molecular grade index.89 In a way akin to the signatures described above, this molecular grade index could classify grade I and grade III tumours with 89% accuracy and grade II tumours were stratied into two clinically signicant groups. The results were validated in a qRT-PCR assay suitable for formalin-xed, paranembedded clinical samples. Therefore, the authors argued that this signature could be incorporated in the daily practice, as it removes the subjectivity and inter-/intraobserver variability associated with conventional histological grading. It has also been proposed that grading signatures could replace histological grade in currently used prognostic models such as Adjuvant!Online and Nottingham prognostic index.68,86,88 Ivshina et al. have described that incorporating their genetic grade signature into the Nottingham prognostic index, some patients classied as having worse prognosis shifted to the good prognosis group. Hence, the use of genetic grade may help improve the identication of patients who could be spared toxic adjuvant systemic therapy.88 Although there is a great enthusiasm about genomic grading, additional independent validation of the above genomic grade signatures is still required and their discriminatory power in ER negative tumours remains to be determined. Therefore, despite the issues related to reproducibility of Nottingham histological grade, histological grade still remains a useful and less expensive tool to help tailor the therapy of breast cancer patients.

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PREDICTIVE SIGNATURES AND BIOMARKERS

Hormone receptors and HER2 are the only mandatory biomarkers to be tested for breast cancer patient decision making, and tamoxifen and trastuzumab represent two of the few successful examples of targeted therapy in breast cancer. In recent years, a paradigm shift has occurred in the way mechanisms of response or resistance to therapy and the predictive value of a biomarker are perceived. There are data to suggest that the signicance of predictive markers may depend on the molecular subtype of the tumour.90,91 Biological pathways might be important in one particular molecular subtype, such as TP53 mutation in ER positive chemotherapy resistant tumours;92 whereas the impact of TP53 mutation in ER negative disease may dier. Therefore, studies designed to discover new predictive markers of response to systemic therapy should take into account the distinctive molecular features of the dierent subtypes, otherwise they might under- or over-estimate their performance (Fig. 1).86,90,91 Several groups have tried to identify reliable predictive factors in the context of tamoxifen treated patients.93,94 Tamoxifen is the most frequently prescribed anti-oestrogen agent in women with ER positive breast carcinomas in both

early stage and advanced disease; however, a signicant proportion of patients relapse due to intrinsic or acquired resistance. Ma et al. analysed the gene expression prole of 60 early-stage tamoxifen-treated patients and identied three genes associated with outcome: HOXB13, IL17BR and CHDH.94 High levels of HOXB13 mRNA and low levels of IL17BR mRNA are associated with recurrence. A simple two-gene ratio HOXB13:IL17BR was then proposed as a novel biomarker to predict recurrence in tamoxifen treated patients. This group developed a qRT-PCR assay and achieved comparable results. Although initial validation studies failed to demonstrate the predictive power of this two-gene signature,95 subsequent studies validated the results in two dierent cohorts and showed the ratio has prognostic power and is predictive of tamoxifen response.96,97 In both cohorts the ratio was not signicant in node-positive patients,96,97 explaining its lack of predictive value in the study by Reid et al.95 Moreover, it has also been shown that the ratio has complementary prognostic value when combined with the ve-gene molecular grade index described above.89 HER2 over-expression and HER2 gene amplication are both predictors of response to humanised monoclonal antibodies anti-HER2 or to HER2 tyrosine kinase

FIG. 1 Schematic illustration of data integration for prognostic/predictive markers and therapeutic targets identication based on high-throughput molecular methods. The term oncogene addiction has been used to describe the phenomenon in which tumour cells become reliant upon the activity of specic oncogenes for growth and progression.163 Consequently, identifying and inhibiting the activity or expression of the proteins encoded by these addictive oncogenes is a highly attractive therapeutic strategy in human cancer.164 (A) Representative genome plot of DNA copy number and chromosome position of a HER2 amplied breast carcinoma. Log2 ratios are plotted on the Y axis against each clone according to genomic location on the X axis. The centromere is represented by a vertical dotted line. BACs categorised as displaying genomic gains or amplication are plotted in green and those categorised as genomic losses in red. (B) Representative image of a scanned microarray chip. Red, relative high expression; green, relative low expression. (C) Representative micrograph of an invasive ductal carcinoma harbouring HER2 amplication by CISH. (D) Representative micrograph of an invasive ductal carcinoma displaying HER2 over-expression by IHC. ArrayCGH, microarray comparative genomic hybridisation; FISH, uorescent in situ hybridisation; CISH, chromogenic in situ hybridisation; qRT-PCR, quantitative reverse transcriptase PCR; IHC, immunohistochemistry; siRNA, small interfering RNA; shRNA, short hairpin RNA.

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inhibitors.413 However, there is strong evidence to suggest that current methods to determine which patients should receive trastuzumab or HER2 tyrosine kinase inhibitors have good negative predictive value, but suboptimal positive predictive values. In the last years, several mechanisms of de novo and acquired resistance to trastuzumab have been identied. It is currently accepted that PIK3CA activating mutations, PTEN inactivation, IGF1R overexpression or expression of p95 HER2 isoform may all play roles in both de novo or acquired resistance to therapies that target HER2.3,98 Furthermore, it has recently been suggested that HER2 amplied breast cancers that harbour a basal-like transcriptome are less sensitive to anti-HER2 therapies.99 Therefore, one can anticipate that additional molecular testing of HER2 positive cancers is likely to be incorporated as predictive markers for patients with tumours pertaining to this molecular subgroup. In addition, HER2 has been reported to predict response to anthracycline-based chemotherapy.100,101 Although HER2 is considered to be the target gene for amplication at chromosome 17q12-q21, the HER2 amplicon encompasses several other genes, such as TOPO2A. TOPO2A gene encodes for topoisomerase-IIa, the direct molecular target of anthracycline,102 it is often co-amplied with HER2 and its amplication seems to be restricted to HER2 amplied tumours.103106 In this context TOPO2A has emerged as a new putative predictor biomarker of response, as it has been shown that tumours with HER2/TOPO2A co-amplication show a signicantly better response to anthracycline-based chemotherapy.104,107,108

TUMOUR MICROENVIRONMENT AND STROMAL SIGNATURES

The major focus of cancer research has been on the malignant cell itself. Most of the analyses described above were performed using whole tissue samples and those judged to possess insucient tumour epithelial cell content were generally excluded. Thus, the specic contribution of epithelial cells, stromal cells and extra-cellular matrix to these tumour classiers and prognostic/predictive signatures remains uncertain. However, some groups have focused their studies on the tumour microenvironment in breast cancer.109116 Comprehensive gene expression prole of each cell type has shown that changes at the transcriptional level occur in epithelial, myoepithelial and stromal cells, already evident at the in situ carcinoma stage.109 However, genetic changes detected by microarray-based comparative genomic hybridisation appear to be limited to cancer epithelial cells.109 The studies of epithelial-mesenchymal interactions have brought some interesting insights about cancer progression.112,114,116 As dierent groups have failed to detect signicant dierences in genetic alterations between ductal carcinoma in situ (DCIS) and invasive tumours,82 the progression of in situ carcinoma to invasive carcinoma may not be due to the intrinsic properties of the tumour epithelial cells but determined by complex interactions between epithelial cells and all the cell types that compose the tumour microenvironment.112,116 It has been hypothesised that progression to invasion may be promoted by broblasts and inhibited by myoepithelial cells.116

Based on the above concepts, it is perhaps not surprising that prognostic signatures derived from the proles of stromal cells have been developed. Using gene signatures of two broblastic tumours, desmoid-type bromatosis (DTF) and solitary brous tumour (SFT), West et al.115 could stratify a breast carcinoma dataset in at least two subgroups with prognostic value independent of clinicopathological risk factors, suggesting that there might be distinct broblastic reaction patterns in breast cancer. The rst cluster expressed DTF genes and was associated with a good prognosis, whereas the second was less homogeneous and composed of two subgroups enriched for SFT genes. These results have been conrmed in dierent datasets and, at the protein level, breast cancers with strong expression of one single gene (SPARC) of the DTF cluster have shown a trend for increased survival.110 However, it should be noted that a previous study demonstrated that SPARC expression in a large consecutive series of breast cancers was associated with poor prognosis.117 One could hypothesise that the DTF stromal reaction pattern would be similar to that seen in the desmoplastic stromal pattern typically found in tubular carcinomas; however, no correlation between molecular and morphological features has been described as yet. Using a dierent approach, Bergamaschi et al.111 grossly dissected 28 breast cancers, with some retention of extracellular matrix, and carried out an unsupervised hierarchical clustering analysis. This analysis revealed that tumours were classied into four dierent groups according to the expression of 278 selected extra-cellular matrix-related genes. Interestingly, in the validation set, the authors found a strong association between one of the groups (Group 1) and high grade, ER negativity, TP53 mutations and poor outcome. When the ve previously dened molecular subtypes4,5 were compared with the stromal signature, it was observed that 81% of all basal-like, 4% of luminal A and 23% of luminal B tumours fell into Group 1. The luminal A and B tumours falling into this group displayed a signicantly worse outcome than other luminal tumours, indicating that the extra-cellular matrix signature can give additional information and is partly independent of the intrinsic subgroups. Furthermore, the authors identied three genes out of the 278 previously selected that were associated with prognosis. Tumours harbouring high expression levels of MARCO and low levels of PUNC and SPARC displayed a signicantly worse outcome. More recently, Finak et al.113 have isolated tumour stroma and matched normal stroma from breast tumours and derived a 26-gene signature strongly associated with outcome called stroma-derived prognosis predictor (SDPP). SDPP predicted prognosis with greater accuracy than the 70-gene signature (MammaPrint), could also stratify several published whole-tumour derived datasets and was independent of grade, age, lymph node involvement, chemotherapy, hormonal therapy and both ER and HER2 status. The poor outcome cluster showed enrichment for markers of hypoxic and angiogenic response, whereas the good outcome cluster over-expressed immune response genes. Interestingly, an immune response gene expression signature has been shown to identify a good outcome subtype of ER negative patients.118 Surprisingly, this subtype was not related either to the extent of lymphocytic

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inltrate, medullary carcinomas or BRCA mutation status. The immune system seems to play an important role in a subgroup of ER negative patients who may benet from treatments targeting tumour cells via the immune response, such as vaccine therapies.113 Interestingly, a small percentage of basal-like carcinomas, a group still without reliable prognostic markers or signatures that reliably identify those with good or poor prognosis, was included in this subtype.118

MOLECULAR GENETICS OF BREAST CANCER: FROM GENOMIC ARCHITECTURE TO BREAST CANCER GENETIC PATHWAYS
The concept that breast cancer encompasses a plethora of entities with distinctive biological characteristics and clinical behaviour is also underpinned at the molecular genetic level by a complex array of genetic alterations that aect the function and control of individual genes and cellular processes.1,2 Not only expression proling analysis, but also the study of cancer genetics has had a profound impact on our understanding of the evolutionary pathways and causative factors in the initiation, development and progression of breast cancer. Microarray-based comparative genomic hybridisation (aCGH) enables researchers to perform rapid and high resolution screening of genomes and a detailed analysis of the global copy number changes.119 While expression proling analysis looks at the transcriptional level, aCGH

scrutinises the genome-wide pattern of genetic alterations. Reliable results using DNA extracted from formalin-xed, paran-embedded samples can be achieved, in particular if BAC arrays are used. aCGH studies in breast cancer have shown that copy number changes are associated with dierent clinico-pathological features to the geneexpression subtypes previously described4,5 and to dierent outcomes.120,121 Three patterns of genomic changes have been recently described, probably related to dierent mechanisms of DNA repair defects found in breast cancers (Fig. 2).121 The rst pattern is called simplex and is dened by broad segments of gains and losses, usually comprising entire chromosomes or chromosome arms. Tumours with this pattern often display gains of 1q and losses of the whole 16q arm. This pattern accounts for 60% of diploid tumours and is related to low histological grade and luminal A phenotype.122 The remaining tumours fall into a category called complex. Tumours with a complex genomic architecture have been shown to have shorter survival in retrospective analysis and to comprise at least two distinct patterns: sawtooth (25%) and restorm or amplier (5%).121,122 The sawtooth pattern is characterised by many segments of gains and losses of varying sizes, often alternating; copy number changes aect almost the entire genome, however loci with high level amplication are usually not seen. The restorm pattern resembles the simplex, but shows at least one localised region of clustered, narrow peaks of amplication, with each cluster conned to

FIG. 2 Schematic illustration of the genomic patterns in breast cancer, as described by Hicks et al.,121 and their association with molecular characteristics, histological features and clinical variables. At the top representative genome plots of DNA copy number and chromosome position of the three distinct genomic proles. Log2 ratios are plotted on the Y axis against each clone according to genomic location on the X axis. The centromere is represented by a vertical dotted line. BACs categorised as displaying genomic gains or amplication are plotted in green and those categorised as genomic losses in red. *Chin et al.122 **Basal-like carcinomas do not display as many high level amplications as HER2 and luminal B carcinomas and they are predominantly of sawtooth pattern; HER2 carcinomas are typically of complex-amplier/restorm pattern.

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a single chromosome arm. Interestingly, complex patterns, in particular the restorm pattern, were associated with a worse outcome. Historically, breast cancer progression pathways were seen as a multistep model, similar to that described by Vogelstein et al.123 for colorectal cancer, where normal breast epithelial cells would transform and progress through a series of morphologically identiable precursors (i.e., normal hyperplasia of usual type atypical ductal hyperplasia low grade DCIS high grade DCIS invasive carcinoma).2 However, in the last few years it has become clear that breast cancer molecular pathways are much more complex than previously appreciated.1,2,124127 In fact, breast cancer molecular pathways encompass a network of inter-related multi-step pathways, which can be broadly classied into two groups/arms, based on histological grade.1,2 The low grade arm encompasses most of the precursors pathologists are aware of, including columnar cell changes, atypical ductal hyperplasia, lobular neoplasia (ALH/LCIS), and their invasive counterparts. These lesions are characterised by low nuclear grade, consistent positivity for hormone receptors and lack of HER2 expression, and quite simple diploid/near diploid karyotypes, with few recurrent changes: deletions of 16q and gains of 1q and 16p.1,2 On the other hand, the high grade arm encompasses high grade DCIS and invasive high grade invasive ductal carcinoma. These tumours frequently lack hormone receptors and express HER2 or basal markers. At the genetic level, tumours of the high grade arm are aneuploid, have complex karyotypes, with a plethora of unbalanced changes mapping to several chromosomal arms.1,2 Interestingly, 510% of high grade breast cancers harbour deletions of 16q and even when they do, the mechanisms leading to 16q deletions are dierent from those described in low grade breast cancer.128 Taken together, these lines of evidence support the idea that progression from low to high grade breast cancer is an exceedingly rare biological phenomenon. This is further supported by the fact that recurrences of DCIS have similar histological grade and molecular genetic features when compared with those of index cases.129 Another line of evidence stems from expression array analysis of matched atypical ductal hyperplasia/at epithelial atypia, DCIS and invasive breast carcinomas.1,2 As mentioned above, Ma et al. have demonstrated that lesions of similar histological grade rather than progression stage cluster together, suggesting that dierent histological grades are associated with distinct gene expression signatures. Last but not least, this is further corroborated by a recent observation that a mixture of grade I, grade II and grade III DCIS is seen in only 9.2% of cases.130 Results of molecular genetic studies, on the other hand, have blurred the boundaries between low grade ductal carcinomas and lobular neoplasia.1,2 Both show remarkably similar immunohistochemical and molecular genetic proles, the main dierence being the target gene of 16q losses.1,2,131 Whilst in ductal lesions the target gene remains to be identied,132134 in lobular carcinomas it has been proven to be the CDH1 gene.135,136 This gene encodes E-cadherin, an adhesion molecule that mediates homophylic-homotypic adhesions. This gene is reported to be inactivated in lobular neoplasia and invasive lobular carcinomas through a combination of genetic and epigenetic mechanisms.137,138 Moreover, identical CDH1 gene

mutations have been described in matched lobular neoplasia and invasive lobular cancer,136 providing strong evidence to suggest that at least some examples of lobular neoplasia are non-obligate precursors of invasive lobular carcinoma. Given the consistent down-regulation of Ecadherin in lobular lesions, anti-E-cadherin antibodies have proven to be useful in the diagnosis of lesions with indeterminate phenotype and in the characterisation of the pleomorphic variant of lobular carcinoma.139,140 Our group and others have demonstrated that pleomorphic LCIS and invasive pleomorphic lobular carcinoma are variants of classic lobular cancer, harbouring similar molecular genetic changes, including deletions of 16q141 144 and E-cadherin down-regulation.141144 However, pleomorphic LCIS (PLCIS) and invasive pleomorphic lobular carcinomas (iPLC) harbour additional genetic hits mapping to key oncogenes, such as MYC and HER2, which may account for the high nuclear grade and the more aggressive clinical behaviour reported.143,144 Given that PLCIS and iPLC are genetically advanced lesions, which are remarkably similar at the morphological, immunohistochemical and molecular genetic levels, PLCIS should be considered a direct non-obligate precursor of iPLC143,144 and managed less conservatively than classic lobular neoplasia. Molecular genetic analysis has also helped clarify the role of some putative breast cancer precursors. Some lesions once considered part of breast cancer progression pathways (e.g., hyperplasia of usual type) have been shown to harbour rare and fairly random chromosomal changes and their role as precursors have been called into question.1,2,145147 On the other hand, other lesions such as columnar cell changes/at epithelial atypia,148 have proven to be clonal and neoplastic, and to harbour molecular genetic changes similar to those found in low grade ductal carcinomas, including 16q deletions. In addition, apocrine lesions have also been shown to harbour recurrent unbalanced chromosomal changes;149 however, their actual role in breast cancer molecular pathways is yet to be determined. Interestingly, a recent morphological study of low grade breast lesions has given further support for the concept of a low grade breast neoplasia family, which encompasses columnar cell changes/at epithelial atypia, atypical ductal hyperplasia, low grade DCIS, lobular neoplasia and their respective invasive counterparts: tubular/tubularcribriform breast carcinomas, grade I invasive ductal carcinomas, invasive lobular carcinomas and tubulolobular carcinomas.150 These tumours seem to have a typical luminal A phenotype and, as mentioned above, are characterised by positivity for hormone receptors, deletions of 16q and have a rather indolent clinical course.2,7,15,46,148 Based on these lines of evidence it seems reasonable to consider at epithelial atypia, low grade ductal and lobular lesions as part of the same group of lesions. Furthermore, a recent expression proling analysis of special types of breast cancer has demonstrated the existence of a group of ER positive cancers encompassing classic lobular carcinomas and tubular carcinomas.131 On the other hand, there are few similarities between low grade ER positive and high grade ER negative ductal carcinomas in terms of their morphological, immunohistochemical and molecular features, suggesting that they constitute distinct entities.

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BREAST CANCER SPECIAL TYPES: GENOTYPIC-PHENOTYPIC CORRELATION

Although histological grade has been extensively analysed from a molecular perspective, histological type has not received the same attention. It should be noted, however, that an obvious example of genotypic-phenotypic correlation in breast cancer is the secretory carcinoma of the breast. This tumour, which has an indolent clinical behaviour and not uncommonly aects prepubertal patients, has been shown to consistently harbour a t(13;15) chromosomal translocation,151 involving the genes ETV6 and NTRK3.151154 Subsequent studies have shown that this translocation in breast cancer is specic to secretory breast carcinomas and that even tumours once thought to be variants of this special type (i.e., acinic cell carcinomas of the breast) do not harbour this translocation.155 As mentioned above, there are several lines of evidence to suggest a genotypic-phenotypic correlation between BRCA1 germline mutations and specic morphological features of breast cancer. When compared with sporadic controls, BRCA1 tumours have been shown to be enriched for medullary cancers, to be of histological grade III, have higher mitotic counts, pushing borders and brisk lymphocytic inltrate.156158 At the immunohistochemical level, BRCA1 cancers have been shown to more often lack hormone receptors and HER2 when compared with controls157159 and to preferentially be of basal-like phenotype.158,160,161 The link between BRCA1 mutations and the phenotypic characteristics listed above is further conrmed by two engineered mouse models developed by two independent groups, where Brca1 and Trp53 were inactivated in the epithelial cells of the mouse mammary gland.27,28,158 Tumours developing in these animals were characterised by the same constellation of morphological features and immunohistochemical prole as described for human BRCA1 tumours. A recent detailed expression proling analysis of special types of breast cancer demonstrated that some of the histological types of breast cancer have a distinctive transcriptome.131 One of the best examples of this phenomenon is invasive micropapillary carcinoma, which displayed a luminal phenotype, but consistently formed a distinct cluster in hierarchical clustering analysis.131 These results are in accord with our ndings that demonstrate that micropapillary carcinomas of the breast have an immunohistochemical phenotype and a pattern of genetic aberrations that are consistent with a luminal B phenotype.162 On the other hand, Weigelt et al.131 demonstrated that other special types of breast cancer, such as apocrine carcinoma, may not constitute a distinct entity. From a pathologists standpoint, the study of the molecular features of special types of breast cancer may reveal not only the basis for histological type, but also help identify novel therapeutic targets for these tumours, given that each subtype has been proven to be more homogeneous at the molecular level than invasive ductal carcinomas of no special type.131

tailored therapies, a paradigm shift from morphological and prognostic classication systems to predictive models is of paramount importance. It is anticipated that by combining morphological, immunohistochemial and molecular techniques, a more biologically and clinically meaningful classication of breast cancer and its precursors will emerge. ACKNOWLEDGMENTS Jorge S. Reis-Filho and Felipe C. Geyer are funded by Breakthrough Breast Cancer. Caterina ` is part of the PhD programme Tecniche avanzate di Marchio localizzazione dei tumori umani (University of Turin) and is funded in part by AIRC (Milam, Regional Grant 1182) and Breakthrough Breast Cancer. The authors would like to thank the Microarray Laboratory of the Breakthrough Breast Cancer Research Centre for the courtesy of the microarray gene expression image.
Address for correspondence: Dr J. S. Reis-Filho, The Breakthrough Breast Cancer Research Centre, Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, United Kingdom. E-mail: Jorge.Reis-Filho@icr.ac.uk

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