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(2006) 25, 5837–5845 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc REVIEW and expression of hereditary tumors associated with BRCA1, BRCA2 and CHEK2 gene

E Honrado1, A Osorio1, J Palacios2 and J Benitez1

1Human Genetics Group, Spanish National Centre (CNIO), Madrid, Spain and 2Breast and Gynecological Cancer Group, Spanish National Cancer Centre (CNIO), Madrid, Spain

Tumors arising in BRCA1 and BRCA2 carriers cancer risk in specific populations (Meijers-Heijboer appear to have specific pathological and et al., 2002; Vahteristo et al., 2002). BRCA1 and BRCA2 profiles, which show a high level of concordance. BRCA1 mutation analysis is the only genetic test that can be tumors are high-grade, negative for hormone receptors, offered to hereditary breast and families, have a high proliferation rate, and are positive for some but the criteria for selecting families who will benefit . BRCA2 tumors present a from this test remain quite unspecific. phenotype opposite to BRCA1 tumors but very similar to In the last few years, a great amount of data has sporadic tumors, except that BRCA2 overexpress some emerged showing that breast tumors from patients with DNA repair markers such as CHEK2, show high germ line mutations in the BRCA1 and BRCA2 genes cytoplasmic expression of RAD51, and are negative for are morphologically and genetically different from each HER-2 amplification and expression.Some of these other, as well as from hereditary tumors not associated characteristics have also been found in cDNA expression with BRCA1/2 mutations and sporadic cases. What studies, although more analysis are necessary in order to these data suggest is that heritable mutations in BRCA1 obtain new markers that can be associated with a germ and BRCA2 lead to different further genetic alterations line mutation in BRCA1 or BRCA2.In this way, some that are specifically involved in the development of each studies in normal tissues of BRCA1/2 carriers suggest of these types of breast tumor. Knowing these molecular that differences exist in the level of expression of some differences at the tumor level will allow us to meet genes when compared with noncarriers.Finally, IHC central challenges in hereditary manage- studies in tumors carrying a mutation in CHEK2 are rare ment, such as defining tumor markers that can be used and show contradictory results, probably due to the low as predictors of the presence of the different germ line number of these cases.However, they represent an alterations, or finding more specific drug targets. In the example showing how different mutations of the same present review, we will discuss the more relevant data gene may be associated with specific histological subtypes about the specific characteristics of BRCA1-and of cancer. BRCA2-associated tumors regarding their pathology Oncogene (2006) 25, 5837–5845. doi:10.1038/sj.onc.1209875 and their genetic expression profiles.

Keywords: immunohistochemistry; profile; microarrays

Morphology of BRCA1- and BRCA2-associated tumors

The largest series concerning the BRCA-associated Introduction histopathology has been reported by the Breast Cancer Linkage Consortium (BCLC) (Lakhani et al., 1997, BRCA1 and BRCA2 are the only known high pene- 1998, 2002). These studies have demonstrated that trance genes involved in breast and ovarian cancer cancer arising in carriers of BRCA1 and BRCA2 gene susceptibility (Miki et al., 1994; Wooster et al., 1995). mutations differs morphologically from sporadic breast Although a great proportion of the familial risk cases is of age-matched controls (Lakhani et al., 1997, not explained by germ line mutations in BRCA1/2,no 1998, 1999; Armes et al., 1998). other susceptibility genes exclusively associated with Invasive ductal carcinoma not otherwise specified is increased risk of breast cancer have been identified so the most common histological subtype in hereditary far, except CHEK2, whose variant allele 1100delC has breast cancer and in sporadic breast cancer, but been associated with a moderate increase in breast interestingly, there are subtypes more frequently asso- ciated with hereditary breast tumors. Thus, in the case of BRCA1 tumors a higher incidence of medullary Correspondence: Dr J Benitez, Human Genetics Group, Spanish National Cancer Centre (CNIO), Melchor Fernandez Almagro, 3, carcinoma (11%) has been reported than in BRCA2 28029 Madrid, Spain. mutation carriers (2%) and in sporadic cases (1%) E-mail: [email protected] (Lakhani et al., 2000). Pathology and gene expression of hereditary tumors E Honrado et al 5838 BRCA1 tumors, following the characteristics of as seen in sporadic breast tumors (Armes et al., 1999; medullary carcinomas, are generally characterized by Lakhani et al., 2002; Palacios et al., 2003; Robson et al., higher grade than BRCA2 or sporadic tumors. It has 2004; Eerola et al., 2005a; Oldenburg et al., 2006). been reported than more than 70% of BRCA1 tumors These differences in grade, ER and PR have been are grade 3, compared with 35% in sporadic cancer observed by some authors only in BRCA1 tumors (Lakhani et al., 1997, 2000; Armes et al., 1998; Lynch from carriers diagnosed before 50 years of age et al., 1998; Quenneville et al., 2002; Goffin et al., 2003; (Vaziri et al., 2001a; Eerola et al., 2005b). However, in Palacios et al., 2003; Eerola et al., 2005a). Other BRCA2 tumors there is an opposite phenomenon, interesting aspects described in BRCA1 tumors are the showing the same expression than controls in patients scarce or absent ductal in situ component accompanying younger than 50 years of age and a lower expression in the invasive ductal component (Lakhani et al., 1997), those aged 50 years or more than in age-matched and the disruption of the expected positive correlation non-BRCA1/2 tumors (Eerola et al., 2005b). These between breast tumor size and lymph node status findings may reflect differences in the tumor develop- (Foulkes et al., 2003a). These two aspects of BRCA1 ment among the older and the younger BRCA1 and tumors have been associated with the fast growth of BRCA2 carriers, and point out the importance of these tumors, often presented as interval cancers considering tumor characteristics in correlation with (Brekelmans et al., 2001). age (Eerola et al., 2005b). BRCA2 tumors are not clearly associated with a specific subtype, but invasive lobular, pleomorphic lobular, tubular and cribiform forms have been reported HER-2 more frequently in this group (Marcus et al., 1996, 1997; HER-2 is overexpressed in approximately 15–20% of Armes et al., 1998). However, other series have not sporadic cancers and it is important to asses the HER-2 found any statistically significant difference in the status in hereditary tumors, because the availability of histological type of BRCA2 with respect to controls the monoclonal antibody herceptin that is used for (Lakhani et al., 1997, 1998; Agnarsson et al., 1998). treatment. The differences in methodology and positiv- Concerning grade, BRCA2 tumors are intermediate ity threshold give conflicting results and hence some between BRCA1 and sporadic tumors, showing 80% authors do not find differences between sporadic and of grade 2–3 vs 70% in sporadic tumors (Lakhani et al., hereditary breast tumors (Robson et al., 1998; Armes 2000; Palacios et al., 2003; Eerola et al., 2005a). et al., 1999; Eerola et al., 2005a). However, using a strong complete membrane staining of >10% of cells (score 3 þ in the DAKO score system) as positive threshold, the number of positive cases in BRCA1 Immunohistochemistry of BRCA1- and tumors is very low (3%) (Lakhani et al., 2002), or BRCA2-associated tumors nonexistent (Grushko et al., 2002; Palacios et al., 2003). Concerning BRCA2, when the score 3 þ is considered as We currently have data about more than 30 immuno- positive, the percentage of cases expressing HER-2 in histochemical markers studied in BRCA1 and BRCA2 BRCA2 tumors is between 0 and 3% (Lakhani et al., tumors which have allowed establishing different 2002; Palacios et al., 2003), which is similar to BRCA1 profiles for each group of tumors. BRCA1 tumors are tumors. better characterized, because they have a much more By using fluorescent in situ hybridization (FISH) there BRCA2 specific immunohistochemical phenotype than is a very good correlation both for BRCA1 and BRCA2 tumors which share many characteristics with sporadic tumors between HER-2 overexpression and amplifica- tumors. tion. Three studies on BRCA1 and BRCA2 tumors have not found amplification of HER-2 (HER-2:CEP17 ratio Hormone receptors X5.0) in correlation with its absence or low expression The more relevant characteristic of BRCA1 tumors is (Grushko et al., 2002; Palacios et al., 2003; Adem et al., the lack of expression of ER. Between 63 and 90% of 2004). There are several explanations for the lack of BRCA1 carcinomas have been reported to be ER HER-2 overexpression and amplification in high-grade negative in different series (Lynch et al., 1998; Osin tumors as are BRCA1 tumors. There could be a et al., 1998; Armes et al., 1999; Eisinger et al., 1999; codeletion of BRCA1 and HER-2 loci when the BRCA1 Noguchi et al., 1999; Osin and Lakhani, 1999; Lakhani locus is lost as a second inactivation hit (Johannsson et al., 2002; Palacios et al., 2003; Robson et al., 2004; et al., 1997). Changes in chromatin conformation Foulkes et al., 2004b; Eerola et al., 2005a; Oldenburg subsequent to BRCA1 inactivation or alterations in the et al., 2006). This percentage is higher than that found in nonhomologous end joining repair pathway (Stark, age- and grade-matched sporadic counterparts (Palacios 1993) are other feasible explanations, although it is not et al., 2003; Foulkes et al., 2004b). Similar to ER, PR is so clear in the case of BRCA2 tumors. An alternative more frequently negative in BRCA1 than in BRCA2 or hypothesis could be that BRCA1/2 tumors evolved sporadic tumors. through genetic pathways where gains of HER2 would In contrast to BRCA1 tumors, 60–90% of BRCA2 not confer a survival advantage for neoplastic cells carcinomas have been reported to be ER positive and, therefore, would not be maintained during and 40–80% PR positive, which is a similar proportion tumorogenesis.

Oncogene Pathology and gene expression of hereditary tumors E Honrado et al 5839 Cell cycle Apoptosis BRCA1 tumors show a higher proliferative index The presence of low levels of BCL2 in tumors is often assessed by Ki-67 than sporadic tumors (Armes et al., associated with high levels of proliferation, apoptosis 1999; Palacios et al., 2003), and in agreement with these and cell death (van Slooten et al., 1998). BRCA1 tumors findings, BRCA1 tumors overexpress that are more likely to exhibit low levels of the antiapoptotic promote cell cycle progression such as , A or BCL2 and the proapoptotic protein BAX than B1 (Foulkes et al., 2004a; Chappuis et al., 2005; Palacios sporadic tumors (Freneaux et al., 2000; Palacios et al., et al., 2005). Interestingly, expression is 2003), and at the same time overexpress the proapopto- repressed in BRCA1 tumors when compared with tic protein caspase 3 and the antiapoptotic protein sporadic tumors, which can be explained by the fact survivin (Palacios et al., 2005). BAX can be activated by that cyclin D1 is known to be upregulated by estrogen or directly by BRCA1 (Zhang et al., 1998), and and progesterone (Gillett et al., 1998); besides, cyclin D1 consequently mutations in this gene are associated with stimulates transcriptional activity of the ER gene low BAX expression. These results suggest that BRCA1- (Neuman et al., 1997), which is mainly absent in BRCA1 deficient cells survive through the balance between tumors. On the other hand, BRCA1 tumors under- proapoptotic and antiapoptotic factors. express proteins related to the inhibition of the cyclin- The overexpression of numerous markers related with CDK complexes, such as , p27 and (Foulkes protecting cells from undergoing apoptosis such as et al., 2004a; Palacios et al., 2005). Regarding p27, Skp2 BCL2 and NFKB, and expression of the proapoptotic is the -protein ligase necessary for p27 ubiqui- marker BAX has been reported in BRCA2 tumors tination (Carrano et al., 1999), and consequently Skp2 is (Armes et al., 1999; Freneaux et al., 2000; Palacios et al., overexpressed in BRCA1 tumors (Foulkes et al., 2004a; 2003, 2005). BCL2 in sporadic tumors has been Palacios et al., 2005). BRCA1 can activate p21 by a p53- associated with ER and PR positivity, and a low Ki-67 independent pathway (Zhang et al., 1998) and defects proliferation index (Linke et al., 2006; Ruiz et al., 2006), have been observed in the ability to activate p21 in which are all typical characteristics of BRCA2 tumors. BRCA1 mutants (Somasundaram et al., 1997). In BRCA2 tumors, the expression of proteins related DNA repair proteins to cell cycle is similar to that observed in sporadic As the main function of BRCA1 and BRCA2 genes is tumors with respect to both cyclins promoting cell cycle related to the response to DNA damage, a series of such progression (cyclins D, E, A and B1) and cyclin-CDK proteins was analysed in both types of tumors; it was complex inhibitors (p16, p27 and p21) (Palacios et al., found that BRCA1 tumors overexpress CHEK2 and 2005), which are overexpressed compared with BRCA1 PCNA and underexpress with respect to tumors. sporadic tumors, and do not show differences in the In relation to the low expression of cyclin D1 in expression of other proteins such as RAD51, ATM or BRCA1 tumors, it has been published absence of XRCC3 (Honrado et al., 2005). CCND1 amplification in BRCA1 and basal-like breast In BRCA2 tumors, overexpression of CHEK2 protein carcinomas (Vaziri et al., 2001b; Reis-Filho et al., has been observed as well but, in contrast to BRCA1 2006a), while BRCA2 tumors frequently showed tumors, there is no reduction of RAD50 expression CCND1 amplification (Palacios et al., 2003). compared with sporadic tumors. Interestingly, the nuclear expression of RAD51 in BRCA2 tumors is very low compared with BRCA1 and sporadic tumors (10 vs p53 35%), while cytoplasmic RAD51 staining is observed In BRCA1 tumors, expression of p53 has been reported more frequently in BRCA2 tumors with respect to to be positive more frequently than in sporadic tumors BRCA1 and sporadic tumors (50 vs 15%) (Honrado (Crook et al., 1998; Lynch et al., 1998; Phillips et al., et al., 2005). This may be explained by the fact that 1999; Lakhani et al., 2002; Palacios et al., 2003; Eerola BRCA2 integrity is necessary to transport RAD51 to the et al., 2005a). Mutations in the p53 gene are encountered nucleus, where it directs the reparation of double-strand in 20% of sporadic breast cancers, as contrasted by breaks of the DNA by . It is 30–77% found in BRCA1 tumors (Crook et al., 1998; important to note that this is one of the few markers Armes et al., 1999; Phillips et al., 1999). In the case of that help to distinguish BRCA2 positive from sporadic BRCA2, the results are more contradictory, but a high tumors. Using the immunohistochemical expression of proportion of p53 mutations have also been reported RAD51 and CHEK2 can be identified a BRCA2 tumor (29–63%). This is in accordance with the hypothesis that with a probability of 81%. downregulation of p53 is an important event to overcome cell cycle arrest and promote tumorigenesis in BRCA1- and BRCA2-deficient tumors (Xu et al., Basal epithelial markers 2001; Ongusaha et al., 2003; Cheung et al., 2004). Over the last years, numerous reports have been Interestingly, the localization of the mutations in the published about the basal-like phenotype and its coding region of p53 seems to be different in both association with BRCA1 tumors (Perou et al., 2000; BRCA1 and BRCA2 tumors when compared with Sorlie et al., 2001, 2003; Sotiriou et al., 2003; Foulkes sporadic tumors (Crook et al., 1998; Gretarsdottir et al., 2003b, 2004a, 2004; Arnes et al., 2005; Jacquemier et al., 1998; Greenblatt et al., 2001). et al., 2005). This basal-like phenotype is characterized

Oncogene Pathology and gene expression of hereditary tumors E Honrado et al 5840 by the expression of markers typical of the normal Furthermore, 45–100% of BRCA1 and BRCA2 basal/myoepithelium such as cytokeratins 5/6, 14, 17, mutation carriers that had developed breast cancer EGFR, p-cadherin, osteonectin, fascin, caveolin-1 showed LOH of the BRCA1 or BRCA2 gene, respec- which are more frequently positive in BRCA1 tumors tively, in normal breast tissue – even in the normal tissue (Palacios et al., 2003; van der Groep et al., 2004; Arnes of the contralateral breast (Lakhani et al., 1999; Cavalli et al., 2005; Lakhani et al., 2005; Pinilla et al., 2006; et al., 2004; Larson et al., 2005). This LOH observed in Rodriguez-Pinilla et al., 2006). Recently it has been the normal epithelium was more frequent in patients reported that two proteins, nerve with a breast tumor than in patients undergoing NGFR/p75NTR (Reis-Filho et al., 2006b) and the small reduction mammoplasty (Larson et al., 2005). It has heat shock protein a-basic-crystallin (ab-crystallin) been proposed to analyse LOH at the BRCA1 locus in (Moyano et al., 2006) that were commonly expressed DNA from ductal lavage and nipple aspirate fluid and in basal-like tumors are predictors of good prognosis to use it as a risk marker (Isaacs et al., 2004), since there and poor survival, respectively, in breast cancer patients is no good correlation between cellular atypia and breast independently of other prognostic markers. Finally, cancer development (Mitchell et al., 2005). there is a group of other markers more frequently In summary, a great amount of information about expressed in BRCA1 tumors that are not basal/ BRCA1 and BRCA2 tumors is now available that can be myoepithelial markers but that have been proposed as very useful to distinguish these two types of tumors from markers associated with the basal-like phenotype: cyclin each other, and from sporadic breast tumors (Table 1). E, p53, Skp2 and negativity for p27 (Signoretti et al., BRCA1 tumors are high grade and, compared to 2002; Nielsen et al., 2004; Palacios et al., 2004; Foulkes sporadic tumors, negative for estrogen and progesterone et al., 2004a; Lakhani et al., 2005). receptors, the apoptotic proteins BAX and BCL2, the The importance of this association between BRCA1 cell cycle-related proteins cyclin D1, p16, p21, p27, and the basal-like phenotype is the correlation with poor the DNA repair protein RAD50 and HER-2, and prognostics and the possibility of specific treatments like positive for p53, Ki-67, Skp2, cyclins E, A, and B1, anti-EGFR agents. In this way, a higher frequency of PCNA, and CHEK2. It is very important to note that EGFR mutations in BRCA1 and BRCA2 tumors (45%) BRCA1 tumors show also a basal-like phenotype than in sporadic ones (15%) has been published, characterized by overexpression of cytokeratins (5/6, although the mutations are located in exon 20 in 14, 17) and other markers such as EGFR, p-cadherin, contrast to those reported in gefitinif-sensitive non- osteonectin, fascin and caveolin-1 (Table 1). In addition, small-cell lung carcinoma (NSCLC) that are located in if a medullary carcinoma is observed in a patient with exons 18–21 (Weber et al., 2005). The effect of EGFR familial antecedents of breast/ovarian cancer, it is highly mutations in BRCA1/2 tumors, in anti-EGFR cancer probable that the case is attributable to a BRCA1 germ , is still unknown. line mutation. On the other hand, BRCA2 tumors present an opposite phenotype to BRCA1 tumors (Figure 1). They Premalignant lesions are grade 2/3 and when compared to sporadic tumors, An important field to be explored concerns the early they show a similar expression of hormone receptors, morphological, immunohistochemical and molecular cell cycle- and apoptosis-related proteins, and the group changes that occur in normal breast tissue from women of markers associated with the basal-like phenotype. at hereditary risk: this is of interest for genetic Only three markers can be defined as BRCA2 specific counseling and the management of BRCA1 and BRCA2 with respect to sporadic tumors: overexpression of mutation carriers. CHEK2, negativity for HER-2, and lack of nuclear High-risk lesions have been found with higher expression but high cytoplasmic expression of RAD51 prevalence in prophylactically removed from (Table 1). women at hereditary risk including BRCA1/2 mutation carriers, than in breasts from women with sporadic Table 1 Immunophenotype of BRCA1 and BRCA2 tumors, compared tumors. Atypical lobular and ductal hyperplasias with sporadic tumors were the more frequent lesions, followed by lobular Antibodies BRCA1 BRCA2 Sporadic and ductal carcinoma in situ (DCIS) (Hoogerbrugge et al., 2003; Kauff et al., 2003). In a recent report ER, PR À ++ (Hoogerbrugge et al., 2003), high-risk histopathology BCL-2, BAX À ++ Cyclin D1 À ++ lesions were found in almost all (14out of 15 women) p16, p27, p21 À ++ of women older than age 40 years at high genetic risk RAD50 À ++ of breast cancer with no demonstrable BRCA1/2 RAD51 (cytoplasm) À + À mutation but only 50% in BRCA1/2 mutation HER-2 ÀÀ+ carriers. This difference was associated to the higher CHEK2 + + À RAD51 (nucleus) + À + proportion and the protective effect of oophorectomy in p53, Ki-67 + ÀÀ carriers. The high prevalence of DCIS and other Cyclins (E, A, B1) + ÀÀ high-risk lesions in prophylactically removed breasts Skp2 + ÀÀ supports the relevance of prophylactic in CK5/6, 14, 17 + ÀÀ EGFR, p-cadherin + these women. ÀÀ

Oncogene Pathology and gene expression of hereditary tumors E Honrado et al 5841

Figure 1 Unsupervised cluster analysis showing the typical immunohistochemical phenotype of BRCA1 (1) and BRCA2 (2) tumors.

CHEK2 tumors: a morphology and immunophenotype different mutations of the same gene may be associated with specific histological subtypes of cancer. A specific mutation in the CHEK2 gene has been found Concerning immunohistochemical characteristics, in 11. 4% of patients selected from families with at least results are contradictory. While de Bock et al. (2004) three cases of breast cancer diagnosed before the age of found positivity for ER and PR more frequently in 60 years (Oldenburg et al., 2003) and 2–8% (Vahteristo tumors of CHEK2 carriers than in tumors of non- et al., 2002; de Bock et al., 2004; Huzarski et al., 2005; carriers, Kilpivaara et al. (2005) found that CHEK2 Kilpivaara et al., 2005) in unselected breast cancer tumors tend to be more frequently ER positive but that patients from the North of Europe (Netherlands, there are no differences in PR expression. At the same Finland, Poland). However, in other countries, this time, Oldenburg et al. (2006), in a short series of nine frequency is much lower or null (Offit et al., 2003; tumors, found no differences between carriers and Caligo et al., 2004; Osorio et al., 2004; Jekimovs et al., noncarriers for ER and PR, but showed that CHEK2 2005; Kleibl et al., 2005; Rashid et al., 2005). In a large tumors were more often negative for luminal cytokeratin study by the CHEK2 Breast Cancer Case–Control 19 staining. In all the studies, CHEK2 protein expres- Consortium including 10 860 unelected patients with sion has been found to be absent or grossly reduced in breast cancer from 10 different studies and five the majority of tumors from carriers of the 100delC countries, they found a prevalence of 1.6% of CHEK2 germ line variant (Vahteristo et al., 2002; Oldenburg 1100delC (Consortium, 2004). As a result of the low et al., 2003; Kilpivaara et al., 2005; Oldenburg et al., frequency it is very difficult to obtain large series to be 2006). able to establish a specific phenotype. There are not As different mutations of the same gene may be many reports about the morphological phenotype of associated with specific histological subtypes of cancer tumors from CHEK2 mutation carriers, and there is no (Huzarski et al., 2005), it would be interesting to analyse coincidence about the results. While some authors have whether there is a specific immunophenotype associated reported that these tumors are of larger size and higher with the I157T mutation. grade than tumors from patients without a CHEK2 mutation (Kilpivaara et al., 2005), others find a lower grade and more unfavorable prognostics associated with CHEK2 tumors (de Bock et al., 2004). De Bock et al. Gene expression profiling of BRCA1- and (2004) reported a higher frequency of contralateral BRCA2-associated tumors breast cancer and a shorter period of distant metastasis- free and disease-free survival in CHEK2-associated The introduction of DNA microarray technology has tumors than in tumors from noncarriers. As far as the meant a real revolution in cancer research. The histological subtype is concerned, no correlation has possibility of analysing the levels of expression of been found with the germ line CHEK2 1100delC variant thousands of genes in a single experiment allows the that is the most frequent in the North of Europe. molecular dissection of tumors, as well as finding more However, it has been published that in Poland, where specific outcome and drug response predictors. Several there are two other founder variants with higher key publications in this field have focused on sporadic prevalence than 1100delC, the I157T mutation is asso- breast cancer (Perou et al., 2000; van de Vijver et al., ciated with the lobular carcinoma subtype (Huzarski 2002; van ‘t Veer et al., 2002; Chang et al., 2003; Sorlie et al., 2005). This is the first report demonstrating how et al., 2003) and show how molecular signatures are able

Oncogene Pathology and gene expression of hereditary tumors E Honrado et al 5842 to subclassify tumours in previously unknown classes ER-negative status determined by immunohistochemical and depending on their clinical outcome, although its staining, and all of them showing downregulation of the clinical utility still needs to be proven, specially in terms ER-a and ER target genes at the expression level. of a prospective validation. Much less is known about However, in a further supervised analysis, the authors hereditary breast cancer: there are only a few relevant found a signature of 100 genes that was able to publications about the molecular profiling of BRCA1 distinguish BRCA1-associated tumors from the other and BRCA2 tumors (Hedenfalk et al., 2001, 2003), and ER-negative sporadic cases (van ‘t Veer et al., 2002). none about tumors from patients with CHEK2 germ line These results show once again how molecular profiling is mutations. concordant with histopathological data, but able to go The study by Hedenfalk et al. (2001) is the only one more deeply into the tumor biology. focused specifically on BRCA1 and BRCA2 breast The same set of 18 BRCA1 tumors was included tumors. The authors defined gene expression signatures together with 97 sporadic tumors in the gene expression by using a cDNA microarray containing 6512 clones. analysis performed by Sorlie et al. (2003), who defined The study included a series of seven breast tumors from five subgroups of breast tumors: ER positive subdivided patients with germ line mutations in the BRCA1 gene, into luminal A and B, and ER negative subdivided into seven with mutations in BRCA2, and seven sporadic basal-like, ERBB2 þ and normal-like tumors. Interest- tumors were used as controls. They found 176 genes that ingly, all BRCA1-positive tumors were found to fall into significantly differed in their expression levels between the basal-like subgroup characterized by lack of expres- BRCA1- and BRCA2-associated tumors, and identified sion of ER and poor prognosis and showing high 51 genes whose differences were able to best distinguish expression of cytokeratins 5 and 17, annexin 8, CX3CL1 between the three types of tumors analysed (P ¼ 0.001). and TRIM29. This confirms the histopathological Among the genes found differentially expressed between findings about the basal-like phenotype in BRCA1- BRCA1 and BRCA2 tumors, a few are important to associated tumors (Foulkes et al., 2003b; Palacios et al., note because of their correlation with histopathological 2004; Lakhani et al., 2005; Rodriguez-Pinilla et al., data: cyclin D1 for instance, was overexpressed in 2006). BRCA2 with respect to BRCA1 tumors, as has also been All these studies show very interesting results in terms described to occur at the immunohistochemical level. of tumor classification based on expression profiling. On The same results were found for estrogen and proges- the one hand, gene expression signatures are able to terone receptor genes whose expression was highly distinguish quite accurately tumors with different germ correlated with the ER status of the tumors determined line alterations. These signatures show good correlation by immunohistochemical staining. However, it is with pathological features, but also open the possibility important to note that the tumors were still correctly of analysing differences in the expression levels of classified after removal of the ER-associated genes. thousands of genes at the same time; they thus constitute Interesting findings have been derived from this study, a step further to eventually identify the biological such as that genes involved in DNA repair, like MSH2, pathways specifically altered when a germ line mutation and in apoptosis induction, like PDCD5, were over- in BRCA1 or BRCA2 exists. As a result of the difficulty expressed in BRCA1-associated tumors, this could of obtaining large series of fresh BRCA1 and BRCA2 indicate a mechanism to over come BRCA1 deficiency tumor specimens, these results have not been validated. by over using other pathways. This is a crucial step to identify those genes that will Moreover, a class-prediction method was established allow a better understanding of the functions of BRCA1 by considering the nine and 11 genes whose expression and BRCA2. was specific for BRCA1 and BRCA2 tumors, respec- tively, when each was compared with the rest of the tumors. The predictor was able to correctly classify all BRCA1-positive tumors and all except one BRCA1- Gene expression profiling in normal tissues from negative tumors; the only sporadic tumor that was BRCA1- and BRCA2 carriers incorrectly classified as BRCA1 showed somatic hyper- of the BRCA1 promoter and a consequent It has been suggested that, due to their involvement in lack of expression of the gene, which now is known to DNA repair, heterozygosity for BRCA1 and BRCA2 mimic the behavior of a real BRCA1 tumor (Wei et al., mutations can already be associated with some 2005). The classification of tumors as BRCA2 positive or level of genetic instability (Warren et al., 2003; Kote- negative was not so accurate, confirming the idea that Jarai et al., 2004; Arnold et al., 2006). In the case this group does not have many specific characteristics of BRCA1, more cellular processes may be affected and is more similar to sporadic tumors, as demonstrated by haploinsufficiency, given that the gene is involved in by immunohistochemical studies. a wide variety of pathways such as chromatin remodel- In a further study by van ‘t Veer et al. (2002), the ing, regulation, authors analysed expression profiling in a set of 98 control, or maintenance of the inactive X breast tumors, including 18 from patients with BRCA1 (Scully and Livingston, 2000; Venkitaraman, 2002). germ line mutations. In a first unsupervised analysis, 16 Taking this into account, it can be hypothesized that BRCA1 tumors were clustered together with another 18 normal tissue from BRCA1 and BRCA2 mutation sporadic tumors, all of them characterized by their carriers could show differences in the level of expression

Oncogene Pathology and gene expression of hereditary tumors E Honrado et al 5843 of certain genes when compared with noncarriers. In a history only, which turns out unspecific given that recent study by Kote-Jarai et al. (2004), the authors were around 70% of the studied families are not linked to able to differentiate breast fibroblasts from BRCA1 either of these two genes. This, in addition to the large mutation carriers from those derived from reduction size of the genes, ends up with BRCA1 and BRCA2 mammoplasties, after inducing DNA damage by radia- analysis being one of the most expensive genetic tests. tion. By using a cDNA microarray, they identified 79 The identification of some predictors of BRCA status clones which could distinguish between both classes with will enormously improve and counseling. 85% accuracy; some of the clones found to be down- In this way, tumors arising in BRCA1 and BRCA2 regulated in BRCA1 carriers corresponded to DNA mutation carriers seem to have specific pathological and repair genes such as RAD51 and RAD23, suggesting that gene expression profiles, which show a great level of heterozygous mutations in BRCA1 are already affecting concordance. However, more validation studies are this process. Interestingly, the p27 gene was also needed for an eventual application of expression downregulated, as has also been found in BRCA1 profiling to the clinical practice. These studies will also tumors, suggesting that this must be a very early event in allow the identification of the biological pathways tumorigenesis. altered as a consequence of BRCA1 and BRCA2 Although these results need to be confirmed and disruption, and a deepening of the knowledge of these extended to other tissues such as peripheral blood, the special types of breast tumors. possibility of detecting gene expression alterations in Finally, the identification of the genes somatically normal tissues opens interesting perspectives in terms of altered in BRCA1- and BRCA2-associated tumors opens directing genetic testing and improving risk assessment. the possibility to identify new molecular drug targets specific for these hereditary cases, as has already been reported for other tumors. Conclusions and future perspectives Acknowledgements The identification of a discrete number of genes able to predict if a breast tumor is associated with a germ line This work was partially financed by grants from the Health mutation in BRCA1 or BRCA2 will be very useful for and Science Ministries, FIS04-2240 and SAF03-02497 respec- the genetic testing routine. The current criteria to select tively. EH is financed by the Spanish Association Against patients for BRCA1/2 analysis are based on family Cancer (AECC).

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