An integrative hypothesis about the origin and development of sporadic and familial breast cancer subtypes

Carcinogenesis vol.29 no.8 pp.1475 1482, 2008 doi:10.1093/carcin/bgn157 Advance Access publication July 1, 2008 REVIEW An integrative hypothesis about the origin and development of sporadic and familial breast cancer subtypes Lorenzo Melchor and Javier Benítez Human Genetics Group, Human Cancer Genetics Programme, Spanish National Cancer Centre (CNIO), Madrid E-28029, Spain To whom correspondence should be addressed. Tel: þ34 912246900; Fax: þ34 912246923; Email: lmelchor@cnio.es Do breast cancer tumours have a common cell origin? Do different breast cancer molecular phenotypes arise from distinct cell types? The studies we have performed during the last few years in familial breast tumours (BRCA1, BRCA2 and non-brca1/2) widen questions about the development of sporadic breast cancer to hereditary breast cancer. Array-comparative genomic hybridisation (CGH) studies show universal genomic aberrations in both familial and sporadic breast cancer subtypes that may be selected in the breast tumour development. The inactivation of BRCA1 seems to play a critical role in oestrogen receptor (ER)-negative cancer stem cells (CSCs), driving the tumour development mostly towards a basal-like or, in some cases, to a luminal B phenotype, but other carcinogenetic events are proposed to explain the remaining tumour subtypes. The existence of common genomic alterations in basal-like, ERBB2 and luminal B breast tumours may suggest a common cell origin or clonal selection of these tumour subtypes, arising from an ER-negative CSC or from a progenitor cell (PC). Finally, specific genomic aberrations in ER-positive tumours could provide cellular proliferation advantages when the cells are exposed to oestrogen. We propose a combination of the CSC hypothesis (for the carcinogenesis processes) and the clonal selection model (in terms of tumour development). We uphold that the basal-like-, ERBB2- and luminal B-sporadic and familial tumour subtypes have an ER-negative breast stem/pc origin, whereas luminal A tumours arise from an ER-positive PC, supporting a hierarchical breast carcinogenesis model, whereas crucial genomic imbalances are clonally selected during the tumour development. Introduction Abbreviations: CGH, comparative genomic hybridisation; CSC, cancer stem cell; ER, oestrogen receptor; IHC, immunohistochemical; LOH, loss of heterozygosity; PC, progenitor cell; SC, stem cell. It is known that normal mammary tissue comprises different cell populations. The undifferentiated cohort of multipotent cells includes breast stem cells (SCs), characterized by their capacity for selfrenewal and differentiation into cell lineages, and progenitor breast cells, an amplifying population derived from SCs with a limited lifespan and proliferation. At the end of these cell lineages, the differentiated cohort of breast cells involves myoepithelial, ductal epithelial and alveolar cells. In normal development, mammary SCs give rise to (i) two SCs (symmetric self-renewal), which produces SC expansion or to (ii) one identical SC and a committed progenitor cell (PC), which undergoes cellular differentiation (asymmetric self-renewal) (1). Breast cancer is initiated by carcinogenesis in a group of cells. The stochastic model of carcinogenesis proposes that firstly malignant transformation occurs by multiple mutations in a random single cell and secondly there is a subsequent clonal selection. In contrast, the hierarchical model of carcinogenesis or cancer stem cell (CSC) hypothesis upholds that the malignant transformation occurs in a subset of normal SCs and PCs, probably through the deregulation of self-renewal pathways (2). The identification of human breast cancerinitiating cells, which are able to generate breast tumours when injected in immunodeficient NOD/SCID mice, strongly supports the unique ability of a set of cells to generate cancer in a proper cell niche. In addition, different cell markers associated with SC, such as CD44þ/ CD24 cells, firstly identified by Al-Hajj et al. (3), ALDH1þ cells (4), CD133þ cells (5) or CD24low/CK14þ/SMAþ cells and CD24high/ Pro1 /Sca1 cells (6), all point out the possible existence of different SC entities in breast cancer. More studies combining the different cell markers proposed are needed for the isolation and characterisation of these SCs. Sporadic breast cancer has been subdivided by expression array analyses in different breast cancer molecular subtypes that have distinct clinical behaviours: basal-like, ERBB2, luminal A and B and normal breast-like subtypes. Basal-like tumours are the most undifferentiated breast cancer malignancies, characterized by the absence of the expression of hormonal receptors and ERBB2 and by a high expression of genes typical of the basal epithelial cell layer. ERBB2 tumours show an over-expression of ERBB2 and multiple genes from the 17q11 amplicon and a negativity for hormonal receptors and basal cell markers. Although both luminal A and luminal B subtypes are positive tumours for the expression of luminal cell markers and mostly oestrogen receptor (ER) positive (some luminal B tumours do not express ER), they show a discriminative expression of certain proteins (such as TOPO II, proliferating cell nuclear antigen, cell cycle proteins, etc.) and differential clinical outcomes (7,8). The proportions of these breast cancer subtypes in sporadic breast cancer are similar among different studies (Table I). In these recent years, defenders of the CSC hypothesis have proposed that the type of carcinogenetic event and the target cell could be the underlying causes for the breast cancer heterogeneity: carcinogenetic events in an ERnegative SC/PC would give rise to basal-like, ERBB2 or luminal B breast cancer subtypes, whereas in an ER-positive PC, they would produce a luminal A phenotype (14,15). On the other hand, familial breast cancer arises from patients who have mutations in either the two known breast cancer susceptibility genes (BRCA1 or BRCA2) or putative unknown genes that could explain the familial inheritance pattern (non-brca1/2 or BRCAX familial cases). In contrast to the idea of homogeneous BRCA1/2-associated cancers in mutation carriers, we have seen the same pattern of molecular heterogeneity in familial and sporadic breast cancer by immunohistochemical (IHC) analyses, although BRCA1- and BRCAXassociated tumours were mainly correlated with the basal-like and the luminal A phenotype, respectively (Table I) (12). This shared pattern of heterogeneity points out the possible existence of a common set of carcinogenetic pathways in both familial and sporadic breast cancer, where the role of BRCA1 could be critical as previously proposed (16). Hypothesis Our findings in familial breast cancer, using IHC and genomic change profiling, propose the existence of different genetic pathways of carcinogenesis and tumour development, which are common to sporadic breast cancer and may lead to the distinct breast cancer subtypes (12). In this manuscript, we disclose an integrative hypothesis of CSC and clonal selection models to explain the arising of breast cancer subtypes extending the view to both familial and sporadic breast cancer (Figure 1). In this sense, the basal-like cancer subtype could be driven by the BRCA1 silencing in an ER-negative SC, supporting the critical role of BRCA1 as a mammary SC regulator. However, a low proportion of BRCA1-associated cancers does not display the basal-like phenotype; here we are proposing that it may be due to (i) the BRCA1 silencing occurring in a different CSC or (ii) the BRCA1 mutation type Ó The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org 1475

L.Melchor and J.Benítez Table I. Proportion of breast cancer subtype in sporadic, BRCA1-, BRCA2- and BRCAX-associated cancers according to previous studies Breast cancer class Breast cancer subtype n (%) Classification tool Reference Basal like ERBB2 Luminal B Luminal A Other a Sporadic 14 (16.5) 11 (12.9) 15 (17.6) 32 (37.7) 13 (15.3) Intrinsic gene list (8) 19 (15.6) 23 (18.9) 17 (13.9) 51 (41.8) 12 (9.8) Intrinsic gene list (76) 84 (26.7) 29 (9.2) 60 (19.0) 90 (28.6) 52 (16.5) Intrinsic gene list (77) 25 (23.6) 10 (9.4) 22 (20.8) 49 (46.2) IHC b (9) 8 (16.0) 8 (16.0) 10 (20.0) 18 (36.0) 6 (12.0) IHC b (10) BRCA1 16 (88.9) 2 (11.1) Intrinsic gene list (11) 11 (61.1) 4 (22.2) 1 (5.6) 2 (11.1) IHC b (12) BRCA2 2 (100) Intrinsic gene list (11) 3 (18.8) 6 (37.5) 2 (12.5) 5 (31.2) IHC b (12) BRCAX 7 (14.0) 9 (18.0) 7 (14.0) 18 (36.0) 9 (18.0) IHC b (10) 2 (7.1) 4 (14.3) 7 (25.0) 13 (46.5) 2 (7.1) IHC b (12) a Other breast cancer subtype column compiles normal breast-like, interferon-like and other unclassified groups described in these analyses. b The IHC markers used to profile breast tumour classes in these analyses differ: Callagy et al. (9) used a panel of 12 antibodies, whereas Honrado et al. (10) and Melchor et al. (12) used 25 antibodies and histological grade. Another IHC analysis profiling familial breast cancer was performed by Oldenburg et al. (13) using cytokeratins 5/6 and 19. The authors described four groups (basal, mixed, luminal and zero) whose relations with Sorlie s subtypes are not easily established and thus, their proportions are not shown in the table; but they also pointed out the heterogeneity found within each of the familial breast cancer classes (BRCA1-, BRCA2- and BRCAX-associated cancers) (13). differing from those of basal-like carcinomas. The ERBB2 breast cancer subtype could arise from ER-negative SC/PC. The ERBB2 amplification that is found in this tumour phenotype would be one of the earliest carcinogenetic events in ER-negative SC PC, and it may not be compatible with mutations in the BRCA1/2 genes. Luminal B tumours could derive from ER-negative PCs that have crucial proliferation advantages, probably due to the oncogene amplification and/or over-expression, and that are able to undergo differentiation processes displaying heterogeneous expression of ER. Finally, luminal A cancers could arise from carcinogenesis in ER-positive PCs. This hypothesis may be supported by the genomic aberration pattern of breast cancer subtypes that would include (i) common genomic aberrations in all breast cancer subtypes, which could represent the required genomic imbalances for the breast cancer development; (ii) specific genomic alterations in tumours that putatively derive from ER-negative SC PCs (basal-like, ERBB2 and luminal B), which could support their common initial tumour environment and (iii) specific genomic alterations in ER-positive tumours, which could reveal genomic imbalances providing cellular proliferation advantages in that tumour cell niche. The reader will find the basis of each of these proposed breast cancer subtype pathways in the following paragraphs. Support for the hypothesis Breast CSCs and BRCA1: the basal-like phenotype pathway. BRCA1- associated tumours are characterized by a high histological grade; an absence of the expression of steroid receptors, ERBB2 and BCL2 and an over-expression of basal cytokeratins (CK5/6), P53 and epidermal growth factor receptor (EGFR) (18,19). These features are also characteristic to basal-like carcinomas (7). From a genomic perspective, the pattern of genomic aberrations in BRCA1-associated cancers and sporadic basal-like carcinomas is quite similar: gains at 3q and losses at 4p, 4q, 5q and 13q (20 25). This is probably because most of BRCA1- associated cancers are classified as basal-like carcinomas, when using expression profiling (11) and IHC analyses (26,27) (Table I). Many studies have hypothesized about the close relationship between BRCA1-associated tumours and basal-like carcinomas (28,29). Following this trend, it has been proposed and recently demonstrated that BRCA1 acts as a SC regulator needed for the luminal differentiation. Thus, its absence would generate the undifferentiated features that characterize the basal-like phenotype (16,30). Although most of BRCA1-associated tumours present a basal-like phenotype, this molecular subtype can also be found in sporadic, BRCA2- and BRCAX-associated cancers with a frequency of 10 20% (Table I). In our studies, BRCAX-associated breast cancers with a basal-like subtype presented a double inactivation of BRCA1: gene 1476 promoter hypermethylation and loss of heterozygosity (LOH) (10). In sporadic breast cancer, Turner et al. (31) reported a frequent BRCA1 promoter hypermethylation in metaplastic carcinomas, a specific subtype of basal-like carcinomas, and an overall low level of BRCA1 expression in basal-like cancers due to mechanisms different from the BRCA1 promoter hypermethylation, such as a high expression of ID4, a suppressor of BRCA1. In addition, our array-comparative genomic hybridisation (CGH) study in familial breast cancer, which was classified according to IHC subtypes, revealed similar genomic aberrations in sporadic and familial basal-like carcinomas, the latter mainly composed of BRCA1-associated tumours (12) (Figure 2). These findings seem to corroborate the critical role of BRCA1 in the development of basal-like carcinomas (Figure 1A). Yehiely et al. (28) extended the role of BRCA1 as a SC regulator as proposed earlier (16), by explaining the transformation of a basal-like mammary SC to a basal-like cancer cell. The carcinogenetic event in basal-like carcinomas may be either (i) the LOH as a second hit in BRCA1 mutation carriers or (ii) the different above-mentioned downregulation processes of BRCA1 in the other tumour classes, both in a primitive ER-negative SC (Figure 1A). This primary event could involve (i) the arrest of the luminal differentiation process, which determines the undifferentiated phenotype of basal-like carcinomas and, thus, its unique expression of basal cytokeratins (CK5/6); indeed, this critical role of BRCA1 in regulating the differentiation of the ERnegative SC has been recently corroborated by Liu et al. (30); (ii) an increment of the telomerase activity since BRCA1 suppresses telomerase expression and (iii) DNA repair defects that generate an inactivation or loss of ataxia telangiectasia mutated (ATM) (32) and an increase in the genomic instability, which may be the reason for the greater number of genomic gains and losses in sporadic and familial basal-like cancers (Figure 2). In the tumour development, subsequent changes, such as an over-expression of cyclin E (to inactivate retinoblastoma), EGFR (to activate phosphatidylinositol 3 (PI3) kinase pathway) and TP53 (probably due to mutations in the gene) would take place. Data from expression and IHC analyses support this model since the over-expression of cyclin E, EGFR and TP53 are characteristic features in both basal-like carcinomas (33,34) and BRCA1-associated tumours (35 37). The down-regulation of phosphatase and tensin homolog gene (PTEN) may also occur in basal-like tumours because of the specific recurrent genetic aberrations in its chromosomal region (10q23.3) (38). All these tumour events are depicted in Figure 1B. In addition, there may be another pattern of heterogeneity within basal-like carcinomas as recently presented by Kreike et al. The authors clustered 97 triple-negative tumours, that were correlated with basal-like cancers, into five groups according to the differential

Origin and tumour development of sporadic and familial breast cancer subtypes Fig. 1. Integrative model of carcinogenesis and tumour development in mammary SCs. The upper section shows the normal breast development from SCs to early and committed PCs. The ER and cytokeratins status are also shown (15,17). (A) Different driver carcinogenetic events occurring in distinct SCs/PCs are represented in the coloured boxes (red for the basal-like subtype pathway, blue for the ERBB2 subtype pathway, purple for the luminal B subtype pathway and green for the luminal A subtype pathway). Breast cancer classes are also depicted next to the carcinogenetic events; the yellow boxes represent the most frequent pathway that the breast cancer class develops according to the proportions shown in Table I. Crossed out boxes of BRCA1/2 mutation carriers in ERBB2 phenotype pathway represent their inability to undergo this genomic pathway. The universal genomic changes are shown on each pathway, and so are those initial genomic events in ER-negative CSC PC and ER-positive PC. The CSCs driving each of the breast cancer subtype pathways are represented at the end of this section. (B) Set of phenomena that occur during the tumour development of each breast cancer subtype pathway (the colours are the same as the ones in the carcinogenetic event boxes) such as differentiation processes (first boxes in the pathways), acquisition of genomic aberrations (second boxes in the pathways) and other tumourogenic events. The genomic instability is represented by arrows at the bottom, whose thickness represents the level of instability. expression of basal keratins, apocrine signalling-related genes, interferon-regulated genes and immunoglobulin genes (39). Although not all triple-negative tumours are basal-like cancers and vice versa, as discussed in the letters to Kreike et al. (39), these findings may reveal additional heterogeneity within basal-like carcinomas. The silencing of BRCA1 appears to be the key event in the basal-like carcinogenesis, but one could expect a heterogeneous inactivation of BRCA1 and an influence of the niche environment in the carcinogenesis and tumour development of this subtype. Therefore, this breast cancer subtype and the role of BRCA1 are highly interesting to be further studied as recently published (30). The ERBB2 phenotype pathway: the incompatibility of amplification/ over-expression and BRCA1/2 mutation. Over-expression of ERBB2 occurs in 15 20% of sporadic and BRCAX-associated cancers (10), whereas it is very low or inexistent in BRCA1/2-associated tumours (0 3%) (36,37,40). The correlation between the over-expression and the gene amplification has been demonstrated in many studies. In this sense, no FISH amplification of ERBB2 (in correlation with its absence or low expression) has been reported in BRCA1- or BRCA2- associated tumours (36,40,41). Why do BRCA1/2-associated cancers not amplify and/or overexpress ERBB2? Although there is a hypothesis about the physical co-deletion of ERBB2 and BRCA1 loci (as a second hit in BRCA1 mutation carriers) (42), this does not explain the lack of the ERBB2 over-expression in BRCA2-associated cancers. Recently, we postulated that the ERBB2 over-expression in cancer cells that have altered BRCA1/2 genes does not give a survival advantage and, thus, does not 1477

L.Melchor and J.Benítez Fig. 2. Genomic aberration patterns of sporadic and familial breast cancer subtypes adapted from two previous studies (12,25). d means universal genomic aberrations in all breast cancer subtypes, U underlines regions commonly affected in those subtypes putatively originated from ER-negative CSC/PC and represents the genomic aberrations recurrently present in ER-positive tumours. proliferate (19). Defects in the DNA repair system may not be compatible with the proliferation stress signal of the ERBB2 tyrosine kinase, and therefore, ERBB2 tumour cells that suffer significant genomic changes would not survive. The low genomic instability of ERBB2-related tumours, when compared with the other tumour subtypes, supports this hypothesis (12,24,25). The ERBB2 amplification/over-expression has been proposed as a carcinogenetic event in ER-negative SC PC (14) (Figure 1A). Given that BRCA1 is not altered, the ERBB2 CSC undergoes luminal differentiation, expressing luminal cytokeratins (i.e. CK8/18) and decreasing the expression of basal cytokeratins (i.e. CK5) (Figure 1B). 1478 The luminal B phenotype pathway: proliferation advantages. The luminal B breast cancer subtype is characterized by a high/medium grade, a variable expression of hormonal receptors, an expression of luminal cytokeratins (CK8/18) and an up-regulation of the cell cycle (e.g. Cyclin E1) and the cell growth (e.g. TOPO II) promoters (8). Genomic analyses in luminal B-sporadic and familial breast cancer subtypes showed more high-level DNA amplifications than in the other subtypes (12,24,25). This cancer phenotype is also associated with a poorer prognosis than luminal A tumours in terms of disease relapse (8) and has been found in a small proportion in all breast cancer classes (Table I). The CSC hypothesis proposed that luminal B tumours derive from primitive ER-negative CSC or PC, which could undergo luminal differentiation and display a variable expression of ER (Figure 1A B) (14,15). Patients who develop ER-positive tumours are frequently treated with a hormonal therapy. However, this treatment would only produce transient remissions in luminal B patients since the targets of carcinogenesis are ER-negative SCs (15). This hypothesis is one possible explanation for the high disease relapse present in luminal B tumours. Nevertheless, there are questions that need to be addressed. What determines a luminal B phenotype? From all those carcinogenetic events occurred in an ER-negative SC to become CSC, we think that the crucial one for the development of luminal B carcinomas could be the amplification and/or over-expression of cell cycle promoters. It may be a similar critical process to the amplification and/or over-expression of ERBB2 in ERBB2 tumours (Figure 1A). Initial amplifications could happen at 8p11 p12, 8q21 q24 or 20q13, given their high frequencies in luminal B tumours (12,24,25). This phenomenon would give an acute proliferation advantage to ERnegative CSC, which then undergoes differentiation (luminal features and ER expression). In the tumour development, those cancer clones with a higher proliferation activity survive by developing new amplifications (Figure 1B) since tumours with enough genomic instability to have an amplification may have an increased probability to arise multiple amplifications (43). The presence of amplifications in luminal B tumours also correlates with its worst prognosis (25). What is the difference between those BRCA1 mutation carriers who develop basal-like tumours and those who develop luminal B carcinomas? We have seen that BRCA1-associated luminal B tumours also have LOH as a second event (data not shown); thus, these patients have inactivated both BRCA1 alleles. These tumours are supposed to arise from ER-negative SCs (15) or ER-negative PC (14,44); thus, it is plausible that the BRCA1 silencing in ER-negative SCs gives rise to basal-like cancers, whereas in ER-negative PC arises luminal B malignancies. Here, we are offering another suggestion: the different type of BRCA1 mutation. Certain BRCA1 mutations give rise to (i) aberrant BRCA1 products if they cause an amino acid change (missense mutations) or (ii) truncated forms if they produce a stop codon that does not trigger the non-sense-mediated decay system (45 47). These aberrant BRCA1 products could (i) have a limited function enabling the cell to differentiate or (ii) on the contrary, act as a dominant negative and lead to a more aggressive phenotype than those mutations that cause a complete absence of the protein (48). In any case, not all mutations may necessarily have the same phenotypic effect. We have observed in our tumour series that the 185delAG mutation in BRCA1, which produces a truncated messenger RNA that avoids non-sense-mediated decay system (46), appears preferentially in BRCA1-associated cancers displaying the basal-like phenotype (12/ 13 185delAG mutation carriers displayed basal-like cancers) (data not shown). However, more BRCA1 samples (basal like and non-basal like) are needed to address these questions. Carcinogenesis in an ER-positive progenitor SC: the luminal A pathway. Luminal A cancers are present mostly in sporadic and BRCAX-associated cancers, whereas they are described in a minority of BRCA1- and BRCA2-associated cancers (Table I). Although Sorlie et al. (11) related BRCA2-associated cancers to the luminal A subtype in an expression analysis, only two samples were analysed, thus the result was not conclusive. The luminal A phenotype is the least aggressive breast cancer molecular subtype (8) and is also related to

Origin and tumour development of sporadic and familial breast cancer subtypes a low genomic instability according to the studies on sporadic and familial breast cancer subtypes (Figure 2). In their CSC hypothesis, Dontu et al. (15) postulated that carcinogenesis taking place in ER-positive PCs gives rise to ER-positive breast cancers (Figure 1A), which express luminal markers, are composed of more differentiated cells and respond to anti-oestrogen treatment. SCs are subjected to the accumulation of multiple mutations by their long-lived nature. Therefore, the chance to accumulate a second hit in the wild-type allele of the BRCA1/2 genes could be higher in SCs than in PCs. This is a possible explanation for the association of BRCA1/2 cancers with other phenotypes rather than for the luminal A subtype. When the second hit is produced in an ER-positive PC, a luminal A phenotype would arise in both BRCA1/2 mutation carriers (Figure 1A). Other carcinogenetic events have to be produced to let the carcinogenetic PC acquire self-renewal and other CSC features. Given that PCs are not under the BRCA1-driven differentiation regulation, these tumours may be able to differentiate and acquire the classical luminal features whether or not the cells have any mutation in the BRCA1 gene (Figure 1B). Another possible explanation for the less frequent luminal A BRCA1-associated cancers could be the putative highest aggressiveness and proliferation capacity of cancer cell clones, arising from BRCA1-altered ER-negative SC PC than BRCA1-altered ER-positive PC, providing a clonal SC advantage in the former ones, that may reverse the phenotype of a tumour from luminal A (if the ER-positive PC was the first CSC entity) to luminal B or basal like (if the ERnegative PC SC are transformed to CSCs). A recent analysis in mammary tumours from BRCA1 mutant mice pointed out the phenotype reversion from ER-positive tumours (at their early stages) to ERnegative tumours (at later stages of tumour development) (49). In addition, the intratumoural clone diversity displaying different cancer subtypes in the same tumour has been described in ductal carcinomas in situ, and the authors proposed that some cancers may even contain multiple SC-competitive clones (50). Interpretation of the genomic aberrations found in the breast cancer subtypes. The differences in the genomic aberrations between breast cancer phenotypes could be the evidence of a set of genetic pathways in the breast cancer progression. For example, two different genetic pathways in the breast cancer progression have been established using genomic, morphological and IHC features: low-grade tumours, where few genomic changes such as 16q are present, and high-grade tumours, where a higher genomic instability is described. These findings may conclude that most of the grade I breast tumours do not progress to grade III breast tumours (51,52). Therefore, we wanted to apply the array-cgh knowledge of the breast cancer subtypes to our integrative model in order to elucidate the different genetic pathways. Though each of the breast cancer subtypes displays a specific genomic aberration pattern (Figure 2), there are common aberrations that could point out common cell origins or proliferation advantages in concrete cell niches. Universal genomic aberrations in breast cancer. Data from array- CGH analyses in sporadic (25) and familial breast cancer subtypes (12) have shown strong similarities in the genomic aberration patterns in all the breast cancer subtypes. If we consider those genomic imbalances present in 50% of all breast tumour subtypes, the most common genomic aberration is a gain at 1q (Figure 2). This alteration has been described as an early common event in breast cancer in previous conventional CGH studies (23,53). More recently, it has also been characterized by using array-cgh platforms (54,55). Orsetti et al. described several minimal regions of gains at 1q, which contained genes showing a significant over-expression correlated with copy number gains. These genes belonged to pathways such as positive regulation of cell proliferation, transcriptional regulation or chromatin remodelling and cellular trafficking or basic cellular metabolism (54). The gain at 1q could be an event required for the deregulation of these pathways and, thus, to enable the breast carcinogenesis (Figure 1A). Therefore, this genomic event could be needed or clonally selected in each of the genetic pathways that give rise to the breast cancer subtypes. Other genomic aberrations, such as the gain at 8q22-qtel and the loss at 11q23, are relatively common to all breast cancer subtypes. Buerger et al. (53) described the gain at 8q as an early genomic event in all breast cancer genetic pathways, probably defining more pleomorphic tumours. However, it is not as frequent in the luminal A subtype as in the other tumour subtypes (12,24) perhaps due to its low histological grade. On the other hand, the loss at 11q23 is also recurrent in all familial breast cancer subtypes (12) although its frequency is,50% in ERBB2- and luminal A-sporadic breast cancer subtypes (Figure 2). This genomic region is affected frequently by LOH in breast cancer (56,57), where ATM is one of the postulated tumour suppressor genes that could be targeted (58). These closely common aberrations possibly play a crucial role in the overall development of a breast tumour. Our model upholds that these genomic imbalances may be clonally selected in the breast tumour development because of the cellular advantages they provide in the breast environment. They could also be critical to enable the tumour to arise (although not crucial to determine the final tumour phenotype, dictated by the CSC/PC and the driving carcinogenetic event). Another interpretation could support an initial common tumour stage among all the breast cancer subtypes, where these universal genomic aberrations could be accumulated earlier than the driving carcinogenetic events in the SC PC. Although we do not want to discard this idea, which seems to be opposed to the CSC model, it may fit in our hypothesis in the critical role of these aberrations for the tumour to arise. Genomic aberrations putatively occurring in ER-negative CSC. Those breast cancer subtypes that derive from ER-negative CSC (basal-like, ERBB2 and luminal B) could share genomic aberrations, which would not be as recurrent in luminal A tumours (derived from ER-positive CSCs). Following this pattern, the loss at 8p is the most common genomic aberration among these three breast cancer subtypes (both sporadic and familial) (Figure 2). The CSC hypothesis suggests that the deregulation of the selfrenewal pathways in normal mammary SCs is one of the sources for the malignancy of SC. The genetic pathways involved in the self-renewal are Notch, Sonic Hedgehog and Wnt signalling pathways (59). If tumours that derive from ER-negative CSC shared genomic aberrations, these altered regions would probably contain crucial genes that regulate the self-renewal process. Interestingly, when we checked the genes located in 8p, two genes at 8p11.21 were described as down-regulated in breast cancer and constitutively promoted the Wnt signalling pathway: SFRP1 (60 62) and DKK4 (63). Although most of these studies reported the promoter hypermethylation as the silencing mechanism, Veeck et al. (60) also described a frequent LOH at SFRP1, whereas Lo et al. (62) reported a SFRP1 down-regulation by hypermethylation in cell lines, which also presented losses at 8p in a recent array-cgh analysis (64). Additionally, SFRP1 links Hedgehog and Wnt signalling pathways (65,66). Therefore, the genomic loss of the region that contains these genes could promote the deregulation of Hedgehog and Wnt signalling pathways, and thus, the self-renewal process in ER-negative mammary SC to become CSC (Figure 1A). However, an SFRP1 over-expression has been described in basal-like tumours (67). Moreover, the search for a tumour suppressor gene in 8p has been extensive without obtaining any conclusive results, so this reduction to the SFRP1 and DKK4 roles might be very categorical. Genomic aberrations associated with an oestrogen-positive cell niche. Different studies have pointed out the genomic aberrations associated with ER status in sporadic (68,69) and familial breast cancers (70). Under the light of the genomic aberration patterns of each molecular breast cancer subtype, a heterogeneity within each ER status group is distinguished. Though basal-like and ERBB2 breast subtypes are both ER-negative tumours, they clearly have different 1479

L.Melchor and J.Benítez genomic aberration patterns; and the same is applied to ER-positive tumours (luminal A and B subtypes) (Figure 2). Indeed, the aberrations classically associated with ER-negative tumours can be specifically attributed to basal-like tumours (such as þ3q25-qtel, 4p, 4q22-qtel, 5q, etc.). This association is explained by an over-representation of basal-like tumours in the studied ER-negative tumour cohorts. On the other hand, genomic aberrations in luminal B tumours differed clearly from luminal A malignancies, probably due to the different carcinogenesis and tumour development. However, there are common genomic aberrations present in these two breast cancer subtypes: þ16p, 16q and an amplification at 11q13 (CCND1 locus) that are not as frequent in the other tumour subtypes. We think that these genomic aberrations might provide a proliferation advantage in an ER-positive cell niche. The loss at 16q seems to be an initial event in lobular and low-grade carcinomas, whereas it is considered a secondary event in high-grade carcinomas, probably due to a high overall genomic instability. This chromosomal aberration is, in fact, one of the few genomic changes in low-grade tumours (51). A recent array-cgh analysis of the rearrangements at 16q discriminated the aberrations occurred in lowand high-grade tumours (71). CDH1 is the putative tumour suppressor gene at 16q22.1, which has been related to lobular and low-grade tumours, but other tumour suppressor genes are supposed to be targeted in high-grade carcinomas (reviewed in ref. 72). As mentioned above, the origin of low- and high-grade breast invasive carcinomas is controversial: two different genetic pathways are supposed to take place in grade I and grade II III tumours (51 53), although a recent analysis describes a possible evolution from low-grade to high-grade carcinomas (50). In the same way, as luminal A tumours are preferentially low-grade carcinomas and luminal B tumours are grade II or III (9,12,67), a loss at 16q could be produced in these two tumour subtypes by different mechanisms. In luminal A tumours, it would be an early genomic event, whereas in luminal B cancers, it would be produced by the genomic instability. Given the high aberration frequency in both tumour subtypes, the loss at 16q may be crucial in the carcinogenesis and tumour development under an ER-positive cell niche. The clonal selection and low- and high-grade tumour evolution recently presented by Allred et al. (50) may not be incompatible with our interpretation because different CSC entities could coexist and those with the highest aggressiveness would predominate and revert the initial less aggressive phenotypes. Allred et al. associated intratumoral grade-diverse tumours with their P53 over-expression, a surrogate of TP53 mutations, which indirectly may drive to genetic instability. These lesions in ER-negative PCs could create more aggressive clones than in ER-positive PCs and, thus, reverse the phenotype as described in BRCA1 mammary tumours in mice (49). In addition, the amplification at 11q13 (CCND1 locus) is another common aberration present in luminal A and B cancer phenotypes. A strong correlation between the CCND1 amplification and the overexpression has been reported. The CCND1 over-expression has been associated with the ER expression and it is rarely present in basal-like carcinomas (73,74). Our analysis on familial breast cancer subtypes also showed that the CCND1 over-expression was associated with luminal tumours and inversely correlated to basal-like cancers (12). These findings suggest that CCND1 does not play an important role in the basal-like tumour development, whereas it seems important in the luminal tumour genetic pathways. The relevance of the BRCA genes and future steps Although the proportions of the breast cancer subtypes differ slightly, specially in BRCA1- and BRCA2-associated cancers (Table I), our hypothesis defends a common pattern of heterogeneity in sporadic and familial breast cancer. As presented in this manuscript, BRCA1 may play a critical role in the arising of basal-like cancers, and the alterations in BRCA1/2 may be incompatible with the amplification/ over-expression in ERBB2. Due to their involvement in the DNA repair pathways and in other cell processes, tumours with a BRCA1/2 alteration would also be expected to have a higher genetic instability. However, future studies should be done by matching tumour samples 1480 not only by grade, sex or age but also by breast cancer subtype. This approach would serve to elucidate the characteristic features that germ line mutations in the BRCA1/2 genes could provide to the tumour appearance or development, given that previous comparisons could have been blurred by their design (for example, a high proportion of basal-like BRCA1-associated cancers versus the gradeand sex-matched sporadic samples, which could have provided features characteristic to basal-like samples rather than to BRCA1- associated cancers and so on). Summary An integrative model of breast carcinogenesis and tumour development is proposed here. It includes concepts such as the BRCA1/2 genes and the shared genomic aberrations, and it is a comprehensive model of CSC hypothesis and clonal selection model (75). We defend that the target SC/PC and the occurrence of a set of driving carcinogenetic events are the underlying causes for the breast cancer subtype origins (Figure 1). These interpretations may be consistent with the possible existence of different CSC entities that compete in the tumour development, making heterogeneous tumours, or those tumours that change their phenotypes after treatment. The description of genomic aberrations, common to all breast cancer subtypes, may propose that those imbalances may be required for the breast tumour development in all genetic pathways (þ1q, 11q23, etc.). Those aberrations, specific for the ER-negative CSC population ( 8p) and for tumour cells confined to an ER-positive cell niche (þ16p), may point out critical changes that provide proliferative cellular advantages under specific cell niches. Finally, the role of the BRCA1/2 genes, besides the BRCA1 relationship with basal-like carcinogenesis and the apparent BRCA1/2 alteration incompatibilities with ERBB2 amplification, would be elucidated by matching tumour comparisons by the breast cancer subtype. Funding Spanish Ministry of Education and Science (FPU AP-2004-0448) to L.M.; Spanish Ministry of Education and Science (SAF 06-06149). Acknowledgements We thank Juan M.Rosa-Rosa, Sara Álvarez, María J.García and Ana Osorio for their scientific assistance in the manuscript redaction. We also thank Ellen Alacid for her English assistance. Conflict of Interest Statement: None declared. References 1. Liu,S. et al. (2005) Mammary stem cells, self-renewal pathways, and carcinogenesis. Breast Cancer Res., 7, 86 95. 2.Wicha,M.S. et al. (2006) Cancer stem cells: an old idea a paradigm shift. Cancer Res., 66, 1883 1890; discussion 1895 1886. 3. Al-Hajj,M. et al. (2003) Prospective identification of tumorigenic breast cancer cells. Proc. Natl Acad. Sci. USA, 100, 3983 3988. 4.Ginestier,C. et al. (2007) ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell, 1, 555 567. 5. Wright,M.H. et al. 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