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Objective Detection of cell free tumor-specific DNA methylation has been proposed as a potentially useful noninvasive mechanism to detect malignancies, including ovarian cancer, and to monitor response to treatment. However, there are few easily implemented quantitative approaches available for DNA methylation analysis. Our objectives were to develop an absolute quantitative method for detection of DNA methylation using RASSF1A , a known target of promoter methylation in ovarian cancer, and test the ability to detect RASSF1A methylation in tumors and serum specimens of women with ovarian cancer. Methods Bisulfite modified DNAs were subjected to real time PCR using nondiscriminatory PCR primers and a probe with sequence containing a single CpG site, theoretically able to capture the methylation status of that CpG for every allele within a given specimen. Input DNA was normalized to ACTB levels detected simultaneously by assay multiplexing. Methylation levels were established by comparison to results obtained from universally methylated DNA. Results The assay was able to detect one methylated RASSF1A allele in 100,000 unmethylated alleles. RASSF1A was methylated in 54 of 106 (51%) invasive serous ovarian cancers analyzed and methylation status was concordant in 20/20 matched preoperative serum–tumor pairs. Serial serum specimens taken over the course of treatment for 8 of 9 patients showed fluctuations in RASSF1A methylation concomitant with disease status. Conclusions This novel assay provides a real-time PCR-based method for absolute quantitation of DNA methylation. Our results support feasibility of monitoring RASSF1A methylation from serum samples taken over the course of treatment from women with ovarian cancer.

The objective of this study was to examine the clinicopathologic correlates of T-regulatory (T(reg)) cell infiltration in serous ovarian cancers and to define gene signatures associated with high T(reg)s.Tumor infiltrating T(reg) and cytotoxic T-cells (CTLs) were quantitated in 232 primary serous ovarian cancers by immunostaining for FOXP3 and CD8. Expression microarray analysis was performed in a subset of 48 advanced cancers with the highest and lowest numbers of infiltrating T(reg)s and a genomic signature was developed using binary regression. ANOVA analysis was performed to assess the most differentially expressed genes and these genes were further assessed using Ingenuity Pathway Analysis (IPA) software.High T(reg) infiltration in ovarian cancers was associated with high grade (p0.0001), advanced stage (p=0.004) and suboptimal debulking (p0.04), but not with survival. In contrast, high tumor infiltrating CD8 CTL infiltration was associated with favorable survival (median survival 48.7 vs. 34.6 months, p=0.01). A microarray-based genomic signature for high tumor-infiltrating T(reg) cells had a 77% predictive accuracy using leave-one-out cross-validation. ANOVA of microarray data revealed the antigen presentation pathway as the most differentially expressed canonical pathway (p0.00001) between cancers with high and low T(reg) cells.These data suggest that there may be an association between increased T(reg) cell infiltration in ovarian cancers and advanced stage. Increased T(reg) infiltration is characterized by a genomic signature enriched with several immunologic pathway genes. Therapeutic strategies that reduce tumor infiltrating T(reg) cells are under investigation and may prove useful in ovarian cancers with high numbers of these cells.

Objectives. MKK4 is a metastasis suppressor that is downregulated in some ovarian cancers. We sought to investigate whether promoter methylation, loss of heterozygosity, or changes in phosphorylation are involved in MKK4 dysregulation during ovarian carcinogenesis. Methods. Bisulfite sequencing was used to determine MKK4 promoter methylation. PCR analysis of tumor/normal DNA was performed to determine LOH at the MKK4 locus. Normal human ovarian surface epithelium (HOSE) and SKOV-3 cells were serum starved and treated with EGF, TGFβ, or wortmannin. Western blotting was performed using antibodies that detect total and phosphorylated MKK4. Results. No MKK4 promoter hypermethylation was detected in 21 ovarian cancers. LOH was detected at the MKK4 intragenic marker D17S969 in 35% of cases and at D17S1303 in 20%. MKK4 protein was detected in 97% of ovarian tumors. The inactivated phosphoserine 80 (ser-80) form comprised 62% of phosphorylated MKK4 protein in ovarian tumors. Treatment of HOSE or SKOV-3 cells with EGF induced a 1.7- to 4.2-fold increase in phosphorylation of ser-80 MKK4 without altering total MKK4 protein. TGFβ increased MKK4 ser-80 phosphorylation by 5.4-fold above baseline. The PI3K/Akt pathway inhibitor wortmannin decreased the amount of ser-80 MKK4 by 50%, and inhibited EGF stimulation of MKK4 ser-80 phosphorylation by 60%. Conclusions. LOH of MKK4 occurs in some ovarian cancers, but without loss of MKK4 protein. MKK4 expression does not appear to be downregulated by promoter methylation. Peptide growth factors induce MKK4 ser-80 phosphorylation, which downregulates its activity. PI3K/Akt pathway inhibitors can partially block ser-80 phosphorylation and this may have therapeutic implications.

Local invasion is a common pattern of spread in uterine cervical squamous cell carcinoma (CSCC). Although transforming growth factor-beta (TGF-β) facilitates invasion of various types of cancer cells, the role of the TGF-β pathway in CSCC is unclear. In this study, we analyzed the role of TGF-β signaling in the progression of CSCC.Immunohistochemistry was used to examine the expression of TGF-β pathway molecules in 67 CSCC samples with clinicopathological data. Activation of the TGF-β pathway was investigated following co-culture of CSCC cells and cervical cancer-associated fibroblasts (CCAFs).Clinicopathological analysis of CSCC samples revealed that prominent expression of TGF-β receptor-2 was more frequent in CSCC with lymphovascular space invasion (LVSI) than without LVSI (p0.01). Lymph node metastasis was more frequent in cases in which phosphorylated SMAD3 (pSMAD3) was localized exclusively at the boundary of tumor clusters (n = 9, p0.05). Recombinant TGF-β1 increased pSMAD3 expression and enhanced cellular invasion (p0.005) in CSCC cells, which was attenuated by an inhibitor of the TGF-β receptor (p0.005). Enhanced pSMAD3 expression and invasion was also observed when conditioned media from CSCC cells co-cultured with CCAFs were administered. Luciferase assays showed that this medium contained a large amount of active TGF-β. Along with TGF-β activation, thrombospondin-1 was upregulated in both CSCC cells and CCAFs, while thrombospondin-1 silencing in either CSCC cells or CCAFs repressed the activity of TGF-β. Thrombospondin-1 was prominently expressed in cases with pSMAD3 boundary staining (p0.05).These results suggest that interaction between CSCC cells and surrounding CCAFs activates TGF-β via thrombospondin-1 secretion to facilitate CSCC invasion.

Introduction Gene expression signatures have been identified for epithelial ovarian cancer survival (TCGA) and intrinsic subtypes (Tothill et al.). One obstacle to clinical translation is that these signatures were developed using frozen tissue, whereas usually only formalin-fixed, paraffin embedded (FFPE) tissue is available. The aim of this study was to determine if gene expression signatures can be translated to fixed archival tissues. Methods RNA extracted from FFPE sections from 240 primary ovarian cancers was analyzed by DASL on Illumina BeadChip arrays. Concordance of expression at the individual gene level was assessed by comparing array data from the same cancers (30 frozen samples analyzed on Affymetrix arrays versus FFPE DASL). Results The correlation between FFPE and frozen survival signature estimates was 0.774. The TCGA signature using DASL was predictive of survival in 106 advanced stage high grade serous ovarian cancers (median survival 33 versus 60months, estimated hazard ratio for death 2.30, P=0.0007). Similar to Tothill, we found using DASL that most high grade serous ovarian cancers (102/110, 93%) were assigned to subtypes 1, 2, 4 and 5, whereas most endometrioid, clear cell, mucinous and low grade serous cases (39/57, 68%) were assigned to subtypes 3 and 6 (P Conclusions Although individual probe estimates of microarrays may be weakly correlated between FFPE and frozen samples, combinations of probes have robust ability to predict survival and subtype. This suggests that it may be possible to use these signatures for prognostic and predictive purposes as we seek to individualize the treatment of ovarian cancer.

Cell lines derived from human ovarian and endometrial cancers, and their immortalized non-malignant counterparts, are critical tools to investigate and characterize molecular mechanisms underlying gynecologic tumorigenesis, and facilitate development of novel therapeutics. To determine the extent of misidentification, contamination and redundancy, with evident consequences for the validity of research based upon these models, we undertook a systematic analysis and cataloging of endometrial and ovarian cell lines.Profiling of cell lines by analysis of DNA microsatellite short tandem repeats (STR), p53 nucleotide polymorphisms and microsatellite instability was performed.Fifty-one ovarian cancer lines were profiled with ten found to be redundant and five (A2008, OV2008, C13, SK-OV-4 and SK-OV-6) identified as cervical cancer cells. Ten endometrial cell lines were analyzed, with RL-92, HEC-1A, HEC-1B, HEC-50, KLE, and AN3CA all exhibiting unique, uncontaminated STR profiles. Multiple variants of Ishikawa and ECC-1 endometrial cancer cell lines were genotyped and analyzed by sequencing of mutations in the p53 gene. The profile of ECC-1 cells did not match the EnCa-101 tumor, from which it was reportedly derived, and all ECC-1 isolates were genotyped as Ishikawa cells, MCF-7 breast cancer cells, or a combination thereof. Two normal, immortalized endometrial epithelial cell lines, HES cells and the hTERT-EEC line, were identified as HeLa cervical carcinoma and MCF-7 breast cancer cells, respectively.Results demonstrate significant misidentification, duplication, and loss of integrity of endometrial and ovarian cancer cell lines. Authentication by STR DNA profiling is a simple and economical method to verify and validate studies undertaken with these models.

Objectives To elucidate the regulation of MASPIN expression in epithelial ovarian cancer (EOC) and associations with p53 status and MASPIN promoter methylation. Methods Seven EOC cell lines and 110 advanced stage EOC specimens were analyzed for MASPIN promoter methylation. The cell lines were treated with 5-azacytidine (5-azaC) and evaluated for MASPIN promoter methylation, protein, and mRNA expression. Wild-type (wt) p53 was transiently transfected into the mutant p53 (m p53 ) SKOV3 cells which were treated with 5-azaC. Phosphor imager analysis quantified the percent methylation of the MASPIN promoter. Results Of the 3 MASPIN-low m p53 cell lines 2 had greater than 5% MASPIN methylation whereas only 1 of 4 MASPIN-high wt p53 cell lines had greater than 5% MASPIN methylation. Despite the presence of aberrant MASPIN promoter methylation in SKOV3 cells, wt p53 -transfection alone resulted in a 3.3-fold increase in MASPIN mRNA. The combination of 5-azaC and wt p53 -transfection produced a 36% reduction in MASPIN promoter methylation and 4.5-fold increase in MASPIN transcription. Among the 110 ovarian cancer specimens analyzed for methylation of the MASPIN promoter, 81.8% were weakly methylated, 14.5% were heavily methylated and 3.6% were fully methylated. There was no relationship between promoter methylation and p53 status or MASPIN protein expression. However, MASPIN protein was 6 times more likely to be detected in cancer specimens that harbor a p53 mutation relative to cancer specimens with a wt p53 gene. Conclusion The regulation of MASPIN is a complex multifactorial process that may be controlled by both p53 -dependent and -independent epigenetic mechanisms.

Objectives It has been suggested that the 6A allele of the type I TGFβ receptor ( TGFβR1 ) polyalanine repeat tract polymorphism may increase susceptibility to various types of cancer including ovarian cancer. Methods The TGFβR1 polyalanine polymorphism was genotyped in 588 ovarian cancer cases and 614 controls from a population-based case-control study in North Carolina. Results Significant racial differences in the frequency of the 6A allele were observed between Caucasian (10.7%) and African-American (2.4%) controls ( P TGFβR1 polyalanine polymorphism was carried by 18% of all controls and 19% of cases, and there was no association with ovarian cancer risk (OR = 1.07, 95% CI 0.80–1.44). The odds ratio for 6A homozygotes was 1.81 (95% CI 0.655.06), but these comprised only 0.98% of controls and 1.70% of cases. Conclusions The 6A allele of the TGFβR1 polyalanine polymorphism does not appear to increase ovarian cancer risk. Larger studies would be needed to exclude the possibility that the small fraction of individuals who are 6A homozygotes have an increased risk of ovarian or other cancers.