About 10% of ovarian cancers arise in women with germline mutations in BRCA1
, and most of these cases are high-grade serous cancers as are most advanced stage sporadic cases. Invasive serous ovarian cancers have a propensity to present at an advanced stage and are responsible for most ovarian cancer deaths. Less than 5% of invasive serous ovarian cancers are confined to the ovary at diagnosis. Most early stage serous ovarian cancers are low malignant potential borderline tumors. Genetic analyses have suggested that borderline serous tumors are not precursors to high-grade invasive cancers. Mutations of the K-ras
genes are frequent in borderline tumors, but rarely occur in invasive cases (19
). Conversely, invasive cancers are characterized by frequent TP53
), but the frequency of these mutations is similar in early and advanced stage invasive serous cases (21
). Microarray studies also have highlighted global differences in gene expression between borderline and invasive serous ovarian tumors (6
Several groups have used microarrays as a tool to predict outcome of patients with advanced ovarian cancer (9
). We compared gene expression in patients who represent the extremes with respect to outcome - namely those who survived either less than 3 years or greater than 7 years. The observation that no single gene was more than 3-fold differentially expressed validates the rationale for examining patterns of gene expression that may reflect underlying tumor biology. Spentzos et al., used microarrays to develop a 115 gene model that classified ovarian cancers into two groups that exhibited significantly different survival (13
). The results are consistent with ours and together suggest that clinical differences in outcome are reflected in global patterns of gene expression that can be appreciated using microarrays.
In view of the potential for false-discovery, we previously confirmed that expression of the genes that comprise our predictive model for survival held prognostic value in the independent set of tumors used in the Spentzos study that were analyzed at another institution using a different microarray platform (12
). In the present study, we have demonstrated an 81% predictive accuracy for long versus short-term survival in a new set of 42 advanced stage serous cancers for which the microarray analysis was performed at another institution. These findings not only validate the predictive model, but also demonstrate that they can be replicated when samples are collected and subjected to independent microarray analysis (in a laboratory at another institution). These predictions were essentially driven by three of the seven gene probes in the original model that maintained significance in the new evaluation set of cancers. Once again, the MAL
gene had the highest fold difference between short and long-term survivors (3 fold), and was more than 20 fold higher in short-term survivors than in early stage cases. The other two significant probe sets correspond to L3MBTL
(U133A: 216076_at) and DGCR2
The availability of a specific monoclonal antibody that can detect the MAL protein in situ allowed us to further investigate expression of this gene with respect to ovarian cancer outcome. Staining a series of cancers lends further support for the expression of this gene as a prognostic indicator. From the set of specimens for which expression array data was available, we observed excellent concordance between protein and mRNA levels (r2=0.66). By staining a larger set of advanced serous cancers unselected for disease outcome, we found that high-level expression of the MAL protein is associated with shorter survival. We also show that benign fallopian tube epithelium have abundant expression of the MAL protein while ovarian surface epithelial cells have no detectable protein by IHC. These results further validate the expression array analysis and provide an interesting gene target for additional mechanistic studies.
In our initial study we found that patterns of gene expression in advanced stage serous cancers from long-term survivors, which represent only a small fraction of advanced stage serous cases, were shared by 11 early stage serous cancers (12
). In the present study, we were able to validate this finding using an independent set of cancers obtained from three tumor banks. We found that all but one of the 39 invasive early stage serous cancers and 75% of borderline cases were predicted to be long-term survivors using the seven gene model. These data provide compelling evidence that the favorable clinical outcome of both long-term survivors with advanced stage disease and early stage cases is attributable to a shared underlying biology. Conversely, a more virulent biology likely underlies the majority of ovarian cancers that are detected at an advanced stage and have worse survival.
Whether these differences in biology are related to the natural disease course or intrinsic response to therapy cannot be discriminated with these data. Virtually all of the advanced cancers in this study were treated in a similar fashion, providing no “control” arm. While some of the early stage cancers were treated with chemotherapy, we have incomplete data both on treatment and outcome from this cohort. At least for the MAL protein, we have independent data from breast cancer indicating that expression of this gene is a prognostic factor related to chemotherapeutic treatment (Horne et al., Molecular Cancer Research in press). In the current study, high MAL expression does correlate with reduced clinical response after surgery (t-test, p= 0.04) further supporting this possibility.
These findings have implications for screening as an approach to decreasing ovarian cancer mortality. They suggest that serous cancers presently diagnosed at an early stage are a small subset with the most favorable biology rather than being representative of the full spectrum of the disease. The underlying biology of these cancers may confer slower growth and a longer interval of time during which these cancers exist prior to the development of disseminated metastasis. Conversely, the observation that only one early stage invasive serous ovarian cancer exhibited patterns of gene expression predictive of short-term survival in advanced disease suggests that the most highly lethal serous ovarian cancers may not be easily amenable to early detection. To be effective in reducing mortality due to high-grade serous ovarian cancer, screening approaches must have the ability to detect cancers that have a less favorable biology. This represents a considerable challenge, as these cancers likely are more rapidly progressive. Screening will have little impact on ovarian cancer mortality if it merely identifies cancers already destined to have a favorable outcome when diagnosed clinically. The utility of computerized tomography screening to reduce lung cancer mortality has been questioned on similar grounds.
In addition to defining gene expression patterns that predict outcome, our group has also developed genomic signatures associated with overexpression of specific oncogenes (15
). These signatures were developed by transfection and overexpression of specific oncogenes in normal mammary epithelial cells. The level of expression of these oncogenic pathway signatures in cell lines has been shown to be predictive of response to biological therapies that target these pathways (15
). In principle, these signatures could be used to direct the use of biological therapies that target these pathways specifically (25
In this paper we examined the pathway signatures in the spectrum of serous ovarian cancers from borderline tumors to early stage invasive cases and advanced stage cases with poor and favorable outcomes. Expression of the myc
pathway signatures, which reflect cell cycle progression, was higher in invasive cancers of all stages that in borderline tumors. Likewise, in another study that used microarrays to examine gene expression in serous ovarian cancers, Bonome et al. found that expression of cellular proliferation genes was higher in invasive cancers relative to borderline tumors, whereas invasive cancers had activation of pathways involved in metastasis and chromosomal instability (6
). These findings are also consistent with older studies that have shown that proliferation rates are higher in invasive cancers relative to borderline tumors (26
). The β-catenin
and pI3K pathway signatures also were notably higher in invasive cancers compared to borderline cases, and likely reflect differences in the molecular pathogenesis of these tumor types.
Expression of the ras
pathway signature was much higher in borderline tumors relative to early and advanced invasive cancers. This is consistent with the prior observation that activating mutations in codon 12 of K-ras
occur in a significant fraction of serous borderline tumors, but rarely in invasive cancers (19
). These findings parallel our prior microarray studies of lung cancer (15
). High expression of the ras
pathway signature in lung cancers was noted to be characteristic of adenocarcinomas, which also frequently have K-ras
). In contrast, the average ras
pathway signature was strikingly lower in squamous lung cancers, which do not have K-ras
mutations. Interestingly, activation of the ras
pathway has a more favorable clinical outcome in both ovarian (borderline) and lung (adenocarcinoma) cancers. However, the level of ras
pathway activation was not a predictor of outcome among invasive serous ovarian cancers.
Previously we reported that the src
signatures were associated with poor outcome in advanced serous ovarian cancers (15
), and that the src
pathway was particularly predictive of poor survival in patients who had an incomplete response to primary platinum-based chemotherapy (16
). In addition, our group (15
) and others (27
) have shown that signatures of src
pathway activation are predictive of response to biological therapies directed at this pathway. In the present study, both the src
pathway signatures were more highly expressed in advanced stage ovarian cancers that were short-term survivors relative to more favorable subgroups. This provides further evidence of the more virulent biology underlying ovarian cancers with high expression of these pathways. The mean pI3k pathway expression, but not AKT or p63, was also higher in invasive cancers than in borderline tumors.
In summary, both the model for long versus short survival in advanced ovarian cancer and the oncogenic pathway signatures suggest that the early and advanced stage serous ovarian cancers are fundamentally different at a biological level. These data suggest that the excellent clinical outcome of early stage cases is not attributable to fortuitous diagnosis prior to metastatic dissemination. Rather clinically detectable early stage cancers are diagnosed before they spread because they are inherently less virulent.