[Show abstract][Hide abstract] ABSTRACT: High-grade ovarian serous carcinomas (HGSC) are characterized by TP53 mutations and non-random patterns of chromosomal anomalies, where the nature of the TP53 mutation may correlate with clinical outcome. However, the frequency of common somatic genomic events occurring in HGSCs from demographically defined populations has not been explored. Whole genome SNP array, and TP53 mutation, gene and protein expression analyses were assessed in 87 confirmed HGSC samples with clinical correlates from French Canadians, a population exhibiting strong founder effects, and results were compared with independent reports describing similar analyses from unselected populations. TP53 mutations were identified in 91% of HGSCs. Anomalies observed in more than 50% of TP53 mutation-positive HGSCs involved gains of 3q, 8q and 20q, and losses of 4q, 5q, 6q, 8p, 13q, 16q, 17p, 17q, 22q and Xp. Nearly 400 regions of non-overlapping amplification or deletion were identified, where 178 amplifications and 98 deletions involved known genes. The subgroup expressing mutant p53 protein exhibited significantly prolonged overall and disease-free survival as compared with the p53 protein null subgroup. Interestingly, a comparative analysis of genomic landscapes revealed a significant enrichment of gains involving 1q, 8q, and 12p intervals in the subgroup expressing mutant p53 protein as compared with the p53 protein null subgroup. Although the findings show that the frequency of TP53 mutations and the genomic landscapes observed in French Canadian samples were similar to those reported for samples from unselected populations, there were differences in the magnitude of global gains/losses of specific chromosomal arms and in the spectrum of amplifications and deletions involving focal regions in individual samples. The findings from our comparative genomic analyses also support the notion that there may be biological differences between HGSCs that could be related to the nature of the TP53 mutation.
[Show abstract][Hide abstract] ABSTRACT: Ovarian carcinomas exhibit extensive heterogeneity, and their etiology remains unknown. Histological and genetic evidence has led to the proposal that low grade ovarian serous carcinomas (LGOSC) have a different etiology than high grade carcinomas (HGOSC), arising from serous tumours of low malignant potential (LMP). Common regions of chromosome (chr) 3 loss have been observed in all types of serous ovarian tumours, including benign, suggesting that these regions contain genes important in the development of all ovarian serous carcinomas. A high-density genome-wide genotyping bead array technology, which assayed >600,000 markers, was applied to a panel of serous benign and LMP tumours and a small set of LGOSC, to characterize somatic events associated with the most indolent forms of ovarian disease. The genomic patterns inferred were related to TP53, KRAS and BRAF mutations. An increasing frequency of genomic anomalies was observed with pathology of disease: 3/22 (13.6%) benign cases, 40/53 (75.5%) LMP cases and 10/11 (90.9%) LGOSC cases. Low frequencies of chr3 anomalies occurred in all tumour types. Runs of homozygosity were most commonly observed on chr3, with the 3p12-p11 candidate tumour suppressor region the most frequently homozygous region in the genome. An LMP harboured a homozygous deletion on chr6 which created a GOPC-ROS1 fusion gene, previously reported as oncogenic in other cancer types. Somatic TP53, KRAS and BRAF mutations were not observed in benign tumours. KRAS-mutation positive LMP cases displayed significantly more chromosomal aberrations than BRAF-mutation positive or KRAS and BRAF mutation negative cases. Gain of 12p, which harbours the KRAS gene, was particularly evident. A pathology review reclassified all TP53-mutation positive LGOSC cases, some of which acquired a HGOSC status. Taken together, our results support the view that LGOSC could arise from serous benign and LMP tumours, but does not exclude the possibility that HGOSC may derive from LMP tumours.
[Show abstract][Hide abstract] ABSTRACT: As gene expression signatures may serve as biomarkers, there is a need to develop technologies based on mRNA expression patterns that are adaptable for translational research. Xceed Molecular has recently developed a Ziplex technology, that can assay for gene expression of a discrete number of genes as a focused array. The present study has evaluated the reproducibility of the Ziplex system as applied to ovarian cancer research of genes shown to exhibit distinct expression profiles initially assessed by Affymetrix GeneChip analyses.
The new chemiluminescence-based Ziplex gene expression array technology was evaluated for the expression of 93 genes selected based on their Affymetrix GeneChip profiles as applied to ovarian cancer research. Probe design was based on the Affymetrix target sequence that favors the 3' UTR of transcripts in order to maximize reproducibility across platforms. Gene expression analysis was performed using the Ziplex Automated Workstation. Statistical analyses were performed to evaluate reproducibility of both the magnitude of expression and differences between normal and tumor samples by correlation analyses, fold change differences and statistical significance testing.
Expressions of 82 of 93 (88.2%) genes were highly correlated (p < 0.01) in a comparison of the two platforms. Overall, 75 of 93 (80.6%) genes exhibited consistent results in normal versus tumor tissue comparisons for both platforms (p < 0.001). The fold change differences were concordant for 87 of 93 (94%) genes, where there was agreement between the platforms regarding statistical significance for 71 (76%) of 87 genes. There was a strong agreement between the two platforms as shown by comparisons of log2 fold differences of gene expression between tumor versus normal samples (R = 0.93) and by Bland-Altman analysis, where greater than 90% of expression values fell within the 95% limits of agreement.
Overall concordance of gene expression patterns based on correlations, statistical significance between tumor and normal ovary data, and fold changes was consistent between the Ziplex and Affymetrix platforms. The reproducibility and ease-of-use of the technology suggests that the Ziplex array is a suitable platform for translational research.
Full-text · Article · Aug 2009 · Journal of Translational Medicine
[Show abstract][Hide abstract] ABSTRACT: Loss of heterozygosity (LOH) analyses of epithelial ovarian cancers (EOCs) previously identified a candidate tumor suppressor gene (TSG) locus within the chromosomal region 3p25.3-pter. Loss of heterozygosity analysis was performed to define the locus and identify candidates for further study. Loss of heterozygosity analysis of 124 malignant EOC samples of different histopathologic subtypes using 12 polymorphic microsatellite repeat markers identified a 330-kilobase minimal region of overlapping deletions at 3p26.3 that contained contactin 4 (CNTN4) as the only known TSG candidate. However, evaluation of the LOH patterns in the serous EOC samples, the most common subtype, enabled the identification of a second, broader region of LOH also included the cell adhesion molecule with homology to L1CAM (CHL1) and CNTN6 as candidates. Gene expression by reverse transcription polymerase chain reaction was not detectable in primary cultures of normal ovarian surface epithelial cells for any of these candidates. CNTN6 expression was also not detectable in serous EOC samples. In contrast, gene expression of CNTN4 and CHL1, particularly overexpression of CHL1, was observed in serous EOC samples. Mutation and gene expression analyses of well-defined EOC cell lines (OV-90, TOV-112D, TOV-21G, and TOV-81D) that differ in their tumorigenic potential and chromosome 3p26-pter genomic content revealed CNTN4 expression and a novel mutation only in the tumorigenic EOC cell line TOV-21G. This mutation was neither observed in controls (n = 105) nor detected by sequencing analysis of complementary DNA. Taken together, these results do not support the candidacy of CHL1, CNTN6, and CNTN4 as TSGs in the 3p26-pter region. However, the overexpression of CHL1, a member of the L1 cell adhesion molecule (L1CAM) family, warrants further investigation.
No preview · Article · Jun 2009 · International Journal of Gynecological Cancer
[Show abstract][Hide abstract] ABSTRACT: Cytogenetic, molecular genetic and functional analyses have implicated chromosome 3 genes in epithelial ovarian cancers (EOC). To further characterize their contribution to EOC, the Affymetrix U133A GeneChip(R) was used to perform transcriptome analyses of chromosome 3 genes in primary cultures of normal ovarian surface epithelial (NOSE) cells (n = 14), malignant serous epithelial ovarian tumors (TOV) (n = 17), and four EOC cell lines (TOV-81D, TOV-112D, TOV-21G, and OV-90). A two-way comparative analysis of 735 known genes and expressed sequences identified 278 differentially expressed genes, where 43 genes were differentially expressed in at least 50% of the TOV samples. Three genes, RIS1 (at 3p21.31), GBE1 (at 3p12.2), and HEG1 (at 3q21.2), were similarly underexpressed in all TOV samples. Deregulation of the expression of these genes was not associated with loss of heterozygosity (LOH) of the genetic loci harboring them. LOH analysis of the RIS1, GBE1, and HEG1 loci was observed at frequencies of 14.3%, 13.7%, and 9.2%, respectively, in a series of 66 malignant TOV samples of the serous subtype. Reduced expression levels of RIS1, GBE1, and HEG1 were observed only in the tumorigenic EOC cell lines (TOV-21G, TOV-112D, and OV-90) and did not correlate with LOH. These results combined suggest that RIS1, GBE1, and HEG1, unlike classical tumor suppressor genes, are not likely to be primary targets of inactivation. This study provides a comprehensive analysis of chromosome 3 gene expression in NOSE and in EOC samples and identifies chromosome 3 gene candidates for further study.
Full-text · Article · Jan 2008 · Molecular Carcinogenesis