Gross genomic alterations and gene expression profiles of high- grade serous carcinoma of the ovary with and without BRCA1 inactivation

In the western world, ovarian cancer is the leading cause of death among patients with gynecological cancers [1]. High-grade serous carcinoma accounts for 70% of all ovarian cancers, and a disproportionate number of deaths as these tumors are more likely to present with advanced stage disease [2]. Germ line mutations of BRCA1 or BRCA2 genes predispose primarily to high-grade serous carcinoma of the ovary and approximately 16% of high-grade serous carcinoma is associated with germ line BRCA gene mutation [3]. BRCA1 gene inactivation is caused either through mutation or epigenetic silencing by promoter hypermethylation, in contrast to BRCA 2 gene where promoter hypermethylation does not significantly contribute to loss of function [4].

Operating as tumor suppressor genes, the primary function of BRCA genes is to preserve the structural and numerical stability of chromosomes during cell division [5]. The proteins are expressed in the dividing cells and located in the nucleus. BRCA1, by forming a multi-protein complex [6], senses double strand DNA breaks and recruits molecules that repair the breaks by error-free homologous recombination [7, 8]. BRCA2, on the other hand, functions as a specific mediator of the interactions leading to homologous recombination [9]. In absence of functional BRCA1 or BRCA2, double stand DNA breaks are repaired by error-prone non-homologous end joining mechanism leading to further mutations and genomic instability [10]. According to the chromosomal instability model for the pathogenesis of BRCA-associated cancers, genetic alterations causing loss of cell-cycle checkpoints and chromosomal instability are crucial during oncogenesis [11, 12]. Chromosomal instability can be assessed by degrees of aneuploidy [13] and DNA ploidy related parameters [14]. Gross genomic alteration evidenced by aneuploidy is usually the result of chromosomal instability [15].

Earlier, by analyzing BRCA1 mutation, expression and promoter hypermethylation, we proposed a potential subclassification of high-grade serous adenocarcinomas into three groups: BRCA 1 loss through mutation, BRCA1 epigenetic loss and no BRCA loss [16]. Therapeutically, the subclassification might be useful for tumors susceptible to targeted treatment with inhibitors of poly (ADP-ribose) polymerase (PARP1) [17]. In order to determine associations between BRCA1 loss and gross genomic alteration, tumor proliferation rate and gene expression profile, we have evaluated DNA ploidy and S-phase fraction by high-resolution image cytometry and gene expression profile using oligonucleotide microarrays, in a cohort of high-grade serous carcinomas with defined BRCA1 and BRCA2 status.

We found that the gross genomic alteration and gene expression profiles were similar in high- grade serous carcinoma of the ovary with BRCA1 loss through mutation, BRCA1 epigenetic loss and no evidence of BRCA1 loss. There is mounting evidence that BRCA1 plays a critical role in maintaining the genomic stability of cells [23]. Mouse embryonic fibroblasts carrying targeted deletion of BRCA1 gene were defective in a G2-M check point leading to multiple spindle poles within a single cell resulting unequal segregation of chromosomes, abnormal nuclear division and aneuploidy [5]. The mechanism of the genetic instability is caused by the failure of homologous DNA recombination, one of the pathways for the repair of double-stranded DNA breaks during DNA replication. In this process, the damaged strand is repaired using intact, homologous sequence as a template [7, 8]. BRCA1 acts at the DNA damaged site as a recruiter of molecules that sense and repair DNA break and an effector of response to DNA damage during homologous recombination process [6]. In absence of BRCA1 function, the repair is through an alternate pathway, nonhomologous end joining, which is error-prone and mutagenic leading to genetic instability and aneuploidy [11].

In breast carcinoma, the total number of genomic changes, as determined by cytogenetics, was found to be almost two times higher in tumors with BRCA1 mutation than in control group [24]. In ovarian tumors, increased clonal chromosomal aberrations was observed in BRCA mutated tumors, compared to BRCA non-mutant tumors [25]. In that series, all BRCA positive tumors were serous carcinoma and the BRCA non-mutant tumors were of different histologic types. It is well known that the morphologically defined ovarian carcinomas are distinct diseases with different molecular events during oncogenesis [12], and it seems likely that this may have confounded the findings. In order to address this, we analyzed a series consisting of only high-grade serous carcinomas, excluding other subtypes (including genomically stable low-grade serous carcinomas) [19, 26]. Furthermore, we have separately evaluated the tumors with BRCA1 loss due to mutation and due to epigenetic silencing due to a reported difference in prognosis [27]. In this series of 28 cases, we have observed that there is no significant difference in the distribution of aneuploidy and tetraploidy in the three subgroups. This indicates that BRCA1 inactivation is not the only mechanism for the development of aneuploidy in high-grade serous carcinoma of the ovary. Importantly, none of these cases had BRCA2 mutations that could account for chromosomal instability. In addition, all aneuploid tumors had DNA index > 1.4 indicating genomic unstable tumors [13]. Therefore, the results indicate a second currently unknown mechanism that leads to aneuploidy in ovarian serous cancer.

We observed lower S-phase in the group of tumors with BRCA1 loss through mutation compared to the other groups. The S-phase of BRCA mutant mouse embryonic fibroblast cells was significantly lower than the control cells as determined by flow cytometry[5]. However, ovarian carcinomas with BRCA germ line mutation had higher proliferation fraction than sporadic tumors as measured by Ki 67 [28].

BRCA1 and BRCA2 mutated ovarian tumors have different gene expression profiles, however, the gene expression profile of sporadic ovarian tumor overlaps with both [29]. In this study, we found essentially no differences in gene expression profile based on BRCA1 status. Only two differentially expressed genes, out of thousands examined, were identified in one of the pair-wise comparisons. This is in keeping with an earlier finding made by Tone et al on a smaller series of 13 high-grade serous carcinomas (of either ovarian or tubal origin), where highly overlapping gene expression profiles were observed between cases with known BRCA1/2 mutation and/or family history and cases with unknown familiar status [30]. These genes show no functional relationship to each other or to the genes known to be involved in BRCA function and this finding is most probably due to chance alone. While the relative small sample sizes (n = 8~9) of the different BRCA1-defined groups examined here may contributes to the paucity of consistent differences identified, it does represent the largest series examined to date and a larger number of differentially expressed genes can usually be identified between different tumor types with similar sample sizes [31, 32]. Therefore, the paucity of differences observed between these groups of serous carcinomas with different BRCA1 status is likely a reflection of intra-group non-uniformity and inter-group overlap in the gene expression patterns. In addition, we recently showed that these groups of ovarian carcinoma classified based on BRCA1 status also show near-identical miRNA expression profiles [33]. This absence of distinct patterns of mRNA or miRNA expression in groups with different BRCA1 status may reflect the rapid divergence in tumors once they acquire chromosomal instability, so that every individual tumor is sufficiently unique that clustering analysis identifies no patterns. As such, their gene expression profiles irrespective of BRCA1 status all show significant dysregulation/difference from that of the putative tissues of origin in normal ovarian surface epithelium and normal fallopian tube as demonstrated previously [30, 34]. This rapid divergence can also explain the dearth of differentially expressed genes on supervised (SAM) analysis, as some consistent abnormalities would have to occur within each group for there to be differences in gene expression. What this does indicate, however, is that the same abnormality, chromosomal instability, appears to be present in all groups of high-grade serous carcinoma analyzed, irrespective of BRCA1 status. While chromosomal instability can be accounted for in the BRCA1 mutant and BRCA1 epigenetically silenced groups, it will be important to identify the mechanism in the large group of tumors that lack BRCA1 or BRCA2 abnormalities and these may involve BRCA1/2-related mechanism(s) or non-BRCA1/2 related mechanism(s). PARP inhibitors have been shown to have activity in tumors with mutations of BRCA1[35]. PARP inhibitors target base excision repair mechanisms in the cell [36]. In cells that lack BRCA1 or BRCA2, homologous repair of double-stranded DNA is defective and the single strand breaks that cannot be repaired because of PARP inhibition are converted to double strand breaks in dividing cells; in the absence of BRCA proteins the double strand breaks are repaired by non-homologous mechanisms, such as non-homologous end joining, which is lethal to the cell [17, 37]. Thus PARP inhibition can specifically target cells lacking BRCA, while sparing normal cells. It remains to be seen whether PARP inhibitors will be active in high-grade serous carcinomas with either BRCA1 epigenetic silencing or no evidence of BRCA1 loss, although it is conceivable that such cells may lack homologous repair functions.

There was no relationship between gross genomic alteration, detected by high resolution DNA image analysis, and BRCA1 inactivation in high-grade serous carcinoma of the ovary. Gene expression profile analysis similarly revealed no significant differences between these groups. This raises two questions. What is the mechanism underlying genomic instability and development of aneuploidy in ovarian high-grade serous carcinomas that lack BRCA1 and BRCA2 abnormalities? Will these tumors, which do have genomic instability, be sensitive to therapy targeted at cells lacking in DNA repair capability, such as PARP inhibitors?