High levels of genomic aberrations in serous ovarian cancers are associated with better survival.

Genomic instability and copy number alterations in cancer are generally associated with poor prognosis; however, recent studies have suggested that extreme levels of genomic aberrations may be beneficial for the survival outcome for patients with specific tumour types. We investigated the extent of genomic instability in predominantly high-grade serous ovarian cancers (SOC) using two independent datasets, generated in Norway (n = 74) and Australia (n = 70), respectively. Genomic instability was quantified by the Total Aberration Index (TAI), a measure of the abundance and genomic size of copy number changes in a tumour. In the Norwegian cohort, patients with TAI above the median revealed significantly prolonged overall survival (p<0.001) and progression-free survival (p<0.05). In the Australian cohort, patients with above median TAI showed prolonged overall survival (p<0.05) and moderately, but not significantly, prolonged progression-free survival. Results were confirmed by univariate and multivariate Cox regression analyses with TAI as a continuous variable. Our results provide further evidence supporting an association between high level of genomic instability and prolonged survival of high-grade SOC patients, possibly as disturbed genome integrity may lead to increased sensitivity to chemotherapeutic agents.

Lysophosphatidic acid-induced transcriptional profile represents serous epithelial ovarian carcinoma and worsened prognosis.

BACKGROUND: Lysophosphatidic acid (LPA) governs a number of physiologic and pathophysiological processes. Malignant ascites fluid is rich in LPA, and LPA receptors are aberrantly expressed by ovarian cancer cells, implicating LPA in the initiation and progression of ovarian cancer. However, there is an absence of systematic data critically analyzing the transcriptional changes induced by LPA in ovarian cancer. METHODOLOGY AND PRINCIPAL FINDINGS: In this study, gene expression profiling was used to examine LPA-mediated transcription by exogenously adding LPA to human epithelial ovarian cancer cells for 24 h to mimic long-term stimulation in the tumor microenvironment. The resultant transcriptional profile comprised a 39-gene signature that closely correlated to serous epithelial ovarian carcinoma. Hierarchical clustering of ovarian cancer patient specimens demonstrated that the signature is associated with worsened prognosis. Patients with LPA-signature-positive ovarian tumors have reduced disease-specific and progression-free survival times. They have a higher frequency of stage IIIc serous carcinoma and a greater proportion is deceased. Among the 39-gene signature, a group of seven genes associated with cell adhesion recapitulated the results. Out of those seven, claudin-1, an adhesion molecule and phenotypic epithelial marker, is the only independent biomarker of serous epithelial ovarian carcinoma. Knockdown of claudin-1 expression in ovarian cancer cells reduces LPA-mediated cellular adhesion, enhances suspended cells and reduces LPA-mediated migration. CONCLUSIONS: The data suggest that transcriptional events mediated by LPA in the tumor microenvironment influence tumor progression through modulation of cell adhesion molecules like claudin-1 and, for the first time, report an LPA-mediated expression signature in ovarian cancer that predicts a worse prognosis.

Gene expression programs in response to hypoxia: cell type specificity and prognostic significance in human cancers.

BACKGROUND: Inadequate oxygen (hypoxia) triggers a multifaceted cellular response that has important roles in normal physiology and in many human diseases. A transcription factor, hypoxia-inducible factor (HIF), plays a central role in the hypoxia response; its activity is regulated by the oxygen-dependent degradation of the HIF-1alpha protein. Despite the ubiquity and importance of hypoxia responses, little is known about the variation in the global transcriptional response to hypoxia among different cell types or how this variation might relate to tissue- and cell-specific diseases. METHODS AND FINDINGS: We analyzed the temporal changes in global transcript levels in response to hypoxia in primary renal proximal tubule epithelial cells, breast epithelial cells, smooth muscle cells, and endothelial cells with DNA microarrays. The extent of the transcriptional response to hypoxia was greatest in the renal tubule cells. This heightened response was associated with a uniquely high level of HIF-1alpha RNA in renal cells, and it could be diminished by reducing HIF-1alpha expression via RNA interference. A gene-expression signature of the hypoxia response, derived from our studies of cultured mammary and renal tubular epithelial cells, showed coordinated variation in several human cancers, and was a strong predictor of clinical outcomes in breast and ovarian cancers. In an analysis of a large, published gene-expression dataset from breast cancers, we found that the prognostic information in the hypoxia signature was virtually independent of that provided by the previously reported wound signature and more predictive of outcomes than any of the clinical parameters in current use. CONCLUSIONS: The transcriptional response to hypoxia varies among human cells. Some of this variation is traceable to variation in expression of the HIF1A gene. A gene-expression signature of the cellular response to hypoxia is associated with a significantly poorer prognosis in breast and ovarian cancer.

Minimizing off-target effects by using diced siRNAs for RNA interference.

Microarray studies have shown that individual synthetic small interfering RNAs (siRNAs) can have substantial off-target effects. Pools of siRNAs, produced by incubation of dsRNAs with recombinant Dicer or RNase III, can also be used to silence genes. Here we show that diced siRNA pools are highly complex, containing hundreds of different individual siRNAs. This high complexity could either compound the problem of off-target effects, since the number of potentially problematic siRNAs is high, or it could diminish the problem, since the concentration of any individual problematic siRNA is low. We therefore compared the off-target effects of diced siRNAs to chemically synthesized siRNAs. In agreement with previous reports, we found that two chemically synthesized siRNAs targeted against p38alpha MAPK (MAPK14) induced off-target changes in the abundance of hundreds of mRNAs. In contrast, three diced siRNA pools against p38alpha MAPK had almost no off-target effects. The off-target effects of a synthetic siRNA were reduced when the siRNA was diluted 3-fold in a diced pool and completely alleviated when it was diluted 30- or 300-fold, suggesting that when problematic siRNAs are present within a diced pool, their absolute concentration is too low to result in significant off-target effects. These data rationalize the observed high specificity of RNA interference in C. elegans and D. melanogaster, where gene suppression is mediated by endogenously-generated diced siRNA pools, and provide a strategy for improving the specificity of RNA interference experiments and screens in mammalian cells.

Genetic and epigenetic modeling of the origins of multidrug-resistant cells in a human sarcoma cell line.

The origin of drug-resistant cells in human cancers has been a fundamental problem of cancer pharmacology. Two major contrasting hypotheses (genetics versus epigenetics) have been proposed to elucidate the mechanisms of acquired drug resistance. In this study, we answer these fundamental questions through investigation of the genetic and epigenetic pathways that control the origin of ABCB1 (MDR1) gene activation with acquired multidrug resistance in drug-sensitive human sarcoma (MES-SA cells). The genetic and epigenetic bases of this selected activation involve the initiation of transcription at a site 112 kb upstream of the ABCB1 proximal promoter (P1) in the drug-resistant cells. This activation was associated with a chromatin-remodeling process characterized by an increase in acetylated histone H3 within a 968-bp region 5' of the ABCB1 upstream promoter. These alterations provide both genetic and epigenetic susceptibility for ABCB1 expression in drug-resistant cells. Complete activation of the ABCB1 gene through the coding region was proposed by interactions of selected trans-alterations or epigenetic changes on the ABCB1 proximal promoter, which occurred during initial drug exposure. Thus, our data provide evidence for a major genomic alteration that changes the chromatin structure of the ABCB1 upstream promoter via acetylation of histone H3 initiating ABCB1 activation, further elucidating the genetic and epigenetic bases that determine chemotherapeutic response in drug-resistant derivatives of MES-SA cells.

Variation in gene expression patterns in effusions and primary tumors from serous ovarian cancer patients.

BACKGROUND: While numerous studies have characterized primary ovarian tumors, little information is available regarding expression patterns of metastatic sites of this cancer. To define sets of genes that distinguish primary and metastatic ovarian tumors, we used cDNA microarrays to characterize global gene expression patterns in 38 effusions (28 peritoneal, 10 pleural) and 8 corresponding primary ovarian tumors, and searched for associations between expression patterns and clinical parameters. RESULTS: We observed multidimensional variation in expression patterns among the cancers. Coordinate variation in expression of genes from two chromosomal regions, 8q and 19q, was seen in subsets of the cancers indicating possible amplifications in these regions. A set of 112 unique genes of known function was differentially expressed between primary tumors and effusions using supervised analysis. Relatively few differences were seen between effusions isolated from the pleural and peritoneal cavities or between effusions from patients diagnosed with stage III and stage IV cancers. A set of 84 unique genes was identified that distinguished high from lower grade ovarian cancers. The results were corroborated using immunocytochemistry, mRNA in situ hybridization, and immunoblotting. CONCLUSION: The extensive variation in expression patterns observed underscores the molecular heterogeneity of ovarian cancer, but suggests a similar molecular profile for ovarian carcinoma cells in serosal cavities.

High-resolution gene copy number and expression profiling of human chromosome 22 in ovarian carcinomas.

Previous low-resolution studies of chromosome 22 in ovarian carcinoma have suggested its involvement in the development of the disease. We report a high-resolution analysis of DNA copy number and gene expression of 22q in 18 ovarian carcinomas using a 22q-specific genomic microarray. We identified aberrations in 67% of the studied tumors, which displayed 3 distinct gene copy number profiles. The majority of the cases (11 of 18) demonstrated heterozygous terminal deletions of various sizes, the smallest of which was 3.5 Mb. The second profile, detected in 3 tumors, revealed the coexistence of heterozygous deletions and different patterns of low-copy-number gain that involved the proximal half of 22q. The latter finding has not been reported previously in ovarian carcinoma. One case displayed a continuous deletion encompassing the entire 22q, consistent with monosomy 22. Furthermore, we compared the results with the available data on these tumors by using cDNA microarrays to define the degree of correlation between abnormalities at the DNA level and variation in mRNA expression. By a comparison with the expression data, we were able to identify 21 deleted genes showing low mRNA levels and 12 amplified genes displaying elevated gene expression, several of which play roles in cell cycle control and the induction of apoptosis. Our results indicated significant correlation between DNA copy number aberrations and variation in mRNA expression. We also identified several regions and candidate genes on 22q that should be studied further to determine their role in the development of ovarian cancer.

A method for detecting and correcting feature misidentification on expression microarrays.

BACKGROUND: Much of the microarray data published at Stanford is based on mouse and human arrays produced under controlled and monitored conditions at the Brown and Botstein laboratories and at the Stanford Functional Genomics Facility (SFGF). Nevertheless, as large datasets based on the Stanford Human array began to accumulate, a small but significant number of discrepancies were detected that required a serious attempt to track down the original source of error. Due to a controlled process environment, sufficient data was available to accurately track the entire process leading to up to the final expression data. In this paper, we describe our statistical methods to detect the inconsistencies in microarray data that arise from process errors, and discuss our technique to locate and fix these errors. RESULTS: To date, the Brown and Botstein laboratories and the Stanford Functional Genomics Facility have together produced 40,000 large-scale (10-50,000 feature) cDNA microarrays. By applying the heuristic described here, we have been able to check most of these arrays for misidentified features, and have been able to confidently apply fixes to the data where needed. Out of the 265 million features checked in our database, problems were detected and corrected on 1.3 million of them. CONCLUSION: Process errors in any genome scale high throughput production regime can lead to subsequent errors in data analysis. We show the value of tracking multi-step high throughput operations by using this knowledge to detect and correct misidentified data on gene expression microarrays.

Gene expression patterns in ovarian carcinomas.

We used DNA microarrays to characterize the global gene expression patterns in surface epithelial cancers of the ovary. We identified groups of genes that distinguished the clear cell subtype from other ovarian carcinomas, grade I and II from grade III serous papillary carcinomas, and ovarian from breast carcinomas. Six clear cell carcinomas were distinguished from 36 other ovarian carcinomas (predominantly serous papillary) based on their gene expression patterns. The differences may yield insights into the worse prognosis and therapeutic resistance associated with clear cell carcinomas. A comparison of the gene expression patterns in the ovarian cancers to published data of gene expression in breast cancers revealed a large number of differentially expressed genes. We identified a group of 62 genes that correctly classified all 125 breast and ovarian cancer specimens. Among the best discriminators more highly expressed in the ovarian carcinomas were PAX8 (paired box gene 8), mesothelin, and ephrin-B1 (EFNB1). Although estrogen receptor was expressed in both the ovarian and breast cancers, genes that are coregulated with the estrogen receptor in breast cancers, including GATA-3, LIV-1, and X-box binding protein 1, did not show a similar pattern of coexpression in the ovarian cancers.