Different Array CGH profiles within hereditary breast cancer tumors associated to BRCA1 expression and overall survival

Breast cancer is the first cause of female death by neoplasm around the world. In Chile, mortality rate due to breast cancer is in first place with 15.5/100.000 women (DEIS, MINSAL 2011). As all cancers, it has been described that breast cancer is driven by several alterations in tumor suppressor genes and oncogenes. Within these alterations, somatic mutations [1], gene deletion or duplication, and promoter hypermethylation [2] are described as the most frequent mechanisms occurring in cancer, and contributing to neoplastic progression [3, 4]. Mutations or alterations in tumor suppressor genes such as gene or chromosomal deletions can be found at different frequencies between tumors, being possible to find a cancer driver alteration in a low proportion of tumors [4]. Several methodologies, as next generation sequencing and array-CGH, are being used in order to detect and identify these mutations and rearrangements. Comparative genomic hybridization (CGH) and, more recently, array-based CGH have been extensively used in the analysis of gains and losses in tumor DNA [5, 6]. Among the most common genomic alterations described in sporadic and hereditary breast tumors are losses at chromosomes 8p, 11q, 13q and 17p; and gains within chromosomes 1q, 8q, 17q and 20q [7‐12]. Through the years, several groups have intended to associate genomic alterations with different breast tumor characteristics. Regarding hereditary tumors, which are the focus of this study, the main findings relay on the association of genomic instability levels with the presence of BRCA1/2 abnormalities [8, 13, 14] or with immunohistochemical phenotypes [15]. In this sense, tumors with BRCA1/2 mutations, BRCA1 promoter hypermethylation/loss of expression, and “basal like” phenotype are shown to have higher instability. These findings are in coherence with BRCA1 and BRCA2 nuclear role in DNA repair, and support their relevance, not only for cancer predisposition, but also for cancer progression. These studies add important and valuable information to the field, nevertheless the complexity and genetic heterogeneity of breast cancer, and the genetic heterogeneity of worldwide populations, support the need of further studies expanding in the analysis of hereditary tumors.

Loss of BRCA1 expression has been described to be associated frequently to LOH [16] and promoter hypermethylation [13, 16, 17] in sporadic and hereditary cases. Few somatic mutations have been found recently for these genes. More recently, miRNA regulation of BRCA1 mRNA stability appears as a new mechanism contributing to BRCA1 silencing [18‐20]. Interestingly, little has been done investigating genomic profiles in breast cancer tumors in association with BRCA1 expression. These studies have been mainly directed to triple negative sporadic breast cancer tumors [13, 21, 22].

The aim of the present work is to evaluate the genomic profiles of a Chilean subset of hereditary breast cancer tumors by array-CGH, highlighting the different alterations found in tumors with loss of BRCA1 expression, and in tumors with germline BRCA mutations. In addition, we identified hereditary tumors clusters in groups with different levels of genomic instability, and significant differences in overall survival. We identified particular genomic alterations in BRCA1 not expressing tumors relevant to functions associated with BRCA1/2 mutated tumors.

We analyzed 47 hereditary breast cancer tumors by array-CGH and found different alterations in relation to BRCA1 and BRCA2 mutation status, and to BRCA1 protein expression.

Tumor features and receptors status are specified in Table 1. Figure 1 shows a graphical representation of all probes involved in gained or lost regions across all chromosomes, and the number of tumors carrying such alterations; we observed that compared to gains, a greater number of deletions are present in unique tumors revealing heterogeneity at this level.

We analyzed through array CGH the genomic profile of 47 biopsies, from hereditary breast cancer patients, 40 from BRCAX patients, 3 from BRCA1 and 4 from BRCA2 mutation carriers (Table 1). To our knowledge this is the first study on genomic alterations, gene functions and molecular pathways involved in hereditary breast cancer tumors, in a Latin American population. The relevance of this study is based on the influence of Genetics and Environment as two key factors in cancer progression.

We found several chromosomal alterations with low frequency in hereditary breast cancer tumors, revealing high inter-tumor heterogeneity at the genomic level. As stated in results, the higher frequency of deletions or gains was 22.5 % among BRCAX tumors. Within the identified alterations in BRCAX tumors, several regions have been previously identified in similar studies for non-BRCA1/2 familial cancer, such as loss in 11q and 16q, and gains in 1q and 8q [14, 55].

In relation to tumors with BRCA1/2 germline mutations, frequency of recurrent alterations rises to 75 % within BRCA2 tumors, and 66 % within BRCA1 tumors. In addition to the most recurrent alterations, our work describes the presence of genomic alterations present only in the BRCA1/2 mutated tumors. Previous reports have described common alterations in BRCA1 and BRCA2 tumors [8, 13, 14, 55], that are also present in sporadic or familial BRCAX tumors, although in a lower frequency. Within the regions described in the literature, loss of 4q, 3p, 12q in BRCA1 tumors, and loss of 11q and 13q for BRCA2 are recurrent. In our tumors all the previous alterations were found, being loss of 4q and 11q present only in our BRCA mutated tumors. Among the regions described as altered for BRCA1/2 tumors in our study we found several genes that have been previously associated with relevant cellular processes such as DNA repair, cell growth and apoptosis.

Clustering of hereditary tumors using genomic alterations revealed that the tumors of the Yellow cluster have significant poor overall survival compared to the rest of the groups (Fig. 2b). In this relation, DARC and DAG1 genes, contained in the frequent genomic losses in the Yellow cluster, have been previously associated to poor survival. DAG1 encodes α-Dystroglycan, a highly relevant glycoprotein that binds to laminin maintaining the correct organization of epithelial tissues [56]. On the other hand, DARC as a chemokine receptor has a major role in inflammation, a process commonly present during invasion of tumor cells. In this sense, the loss of expression of these two genes associated to a poor prognosis, maybe due to a higher incidence of metastasis in these patients [52‐54]. In addition, as described in results the Yellow cluster present frequent a loss of PLEKHO1 and GDF11 genes, regulators of PI3K/AKT and EGF signaling, respectively. These two pathways have been extensively cited as highly activated in triple negative breast cancer tumors, which are well known for having a poor overall survival with respect to other breast cancer subtypes [57]. The contribution of the activation of PI3K/AKT and EGF pathways to poor survival has been related to the lower response and/or resistance to chemotherapy observed in patients [58, 59]. Finally, we also found loss of CD63 (member of the tetraspanin family), an event previously associated to advanced stages of melanoma [60]. The involvement of CD63 in cancer metastasis and its loss in tumors described in this study, is in concordance with a poorer overall survival of patients in the yellow cluster. The Blue cluster have also interesting features, since losses found in this group involve genes related to the processing and presentation of immunogenic peptides, which are frequently downregulated in different types of cancer (Cluster analysis section in Results). Downregulation of these genes affect peptide characteristics and their transport to the endoplasmic reticulum for its binding by MHC class I proteins. In this regard, tumors presenting these deletions will have a possibility for treatment with specific immunotherapy.

We found significant differences in the number of alterations between clusters, having the Green cluster the lower instability compared with Blue, Yellow and Purple clusters. A previous work by Stefansson et al [13] analyzed 29 tumors defined as “with BRCA alterations” (BRCA1/2 mutation or BRCA1 hypermethylation/loss of expression) compared to 38 sporadic tumors without any BRCA alteration. These authors described 4 clusters of tumors, three of which present a high instability, like in our study. Among those three clusters, two were enriched in BRCA altered tumors presenting mainly big size losses. This is consistent with our results, since the Yellow cluster (6 tumors) having high genomic instability and characterized mainly by losses, is enriched in BRCA1 and BRCA2 mutated tumors (3/6 tumors). In addition to this concordance with Stefansson’s results, regarding hereditary BRCA1 or 2 deficient tumors, we added to the knowledge the fact that this instable BRCA-enriched cluster has a poor overall survival, as mentioned in the previous paragraph. Our results in hereditary tumors are also consistent with Fridlyand et al [11], who described three groups of sporadic breast cancer tumors with differences in CNA number and type, and with survival.

Although we found in our tumors, genomic alterations previously described in the literature, these are present in a low proportion of tumors. In addition, it comes to our attention that tumors of the Green cluster, gathering almost half of our hereditary tumors, have a low number of alterations. Latin American populations, like the one in this study, constitute an admixture of Spanish and Amerindian individuals, being genetically different from breast cancer cases frequently analyzed in similar studies. These ethnic differences in conjunction with environmental factors may lead into differences in the molecular mechanisms of cancer progression among populations.

In our study, we included different pathological subtypes such as ductal and lobular in situ and invasive carcinomas. According to our results, these carcinomas are distributed across all clusters, indicating that in situ diseases are as heterogeneous as, and behave similar to, the invasive tumors.

BRCA1 silencing in sporadic and hereditary tumors have been described in the last years to be a relevant mechanism associated to breast cancer progression in patients with no germline mutation [16, 17]. In our study, small groups of BRCA1 not expressing tumors share common genomic alterations though the majority of tumors do not have the same genes affected. Nonetheless, the relevant cellular processes highlighted for these tumors revealed that the affected genes, although different, involve the same molecular pathways. This observation is in agreement with previous reports describing core affected pathways in pancreatic cancer [61, 62]. In addition, we identified genomic alterations and cellular processes shared by BRCA1 mutated and BRCA1 not expressing tumors. This is in line with the fact that some tumors, lacking germline mutations in BRCA1 show a BRCAness phenotype, implying that they could have a cancer progression program similar to BRCA1 mutated tumors.

The results obtained for BRCA1 not expressing tumors suggest a more relevant contribution of BRCA1 functional deficiency to the general genomic instability of the tumors than to the development of specific alterations. As observed, none of the tumor clusters are characterized by a particular BRCA1 expression status, but they do carry common alterations (Fig. 2a). This evidence may reflect that the consequences of BRCA1 functional deficiency depend on the genetic background of the tumors, the mechanism of inactivation, or the moment at which this event occurs. Moreover, it is necessary to determine whether other alterations of BRCA1 function, such as cytoplasmic retention, somatic mutations or post-translational regulation by miRNAs may contribute to the particular genomic profiles observed in each cluster.

Array CGH have been used in recent years to get relevant information for clinical trials. Two prospective trials, SAFIR01 and MOSCATO, intend to destine patients to different targeted therapies depending on genomic gains and somatic mutations affecting relevant targets for therapy. In these studies, amplifications of low recurrence involving genes such as EGFR, FGFR and FGF ligands, AKT, PIK3CA and IGF1R are suitable markers for moderate or good antitumor response (stable disease or remission) to specific inhibitor for these pathways. In our study (data not shown), amplification of AKT, PIK3CA and FGF receptors and ligands were observed in BRCA1 not expressing tumors, opening a new therapeutic opportunity for tumors with a BRCAness phenotype. In this relation, it has already been demonstrated in triple negative breast cancer cell lines, that combining PI3K and EGFR inhibitors produces a better response than each inhibitor alone [63] becoming a promising strategy for BRCAness tumors treatment. In addition, a group of our tumors (Yellow cluster) exhibit deletions of PLEKHO1 and GDF11, which products regulate PI3K and EGF signaling pathways. Patients carrying this type of tumors, showing a poor overall survival, could be good candidates for the combined therapy mentioned before. These therapies may bring an alternative treatment to patients carrying BRCAness tumors, or could be used in combination with PARP inhibitors.

Our results support the fact that BRCA1 expression in tumors should be used as a marker for BRCAness and for selection of these patients for synthetic lethality approaches such as treatment with PARP inhibitors. In addition, the identification of specific alterations in breast tumors associated with poor survival, immune response or with a BRCAness phenotype will allow the use of a more personalized treatment in these patients.