Differential gene expression identifies a transcriptional regulatory network involving ER-alpha and PITX1 in invasive epithelial ovarian cancer.

BACKGROUND: The heterogeneous subtypes and stages of epithelial ovarian cancer (EOC) differ in their biological features, invasiveness, and response to chemotherapy, but the transcriptional regulators causing their differences remain nebulous. METHODS: In this study, we compared high-grade serous ovarian cancers (HGSOCs) to low malignant potential or serous borderline tumors (SBTs). Our aim was to discover new regulatory factors causing distinct biological properties of HGSOCs and SBTs. RESULTS: In a discovery dataset, we identified 11 differentially expressed genes (DEGs) between SBTs and HGSOCs. Their expression correctly classified 95% of 267 validation samples. Two of the DEGs, TMEM30B and TSPAN1, were significantly associated with worse overall survival in patients with HGSOC. We also identified 17 DEGs that distinguished stage II vs. III HGSOC. In these two DEG promoter sets, we identified significant enrichment of predicted transcription factor binding sites, including those of RARA, FOXF1, BHLHE41, and PITX1. Using published ChIP-seq data acquired from multiple non-ovarian cell types, we showed additional regulatory factors, including AP2-gamma/TFAP2C, FOXA1, and BHLHE40, bound at the majority of DEG promoters. Several of the factors are known to cooperate with and predict the presence of nuclear hormone receptor estrogen receptor alpha (ER-alpha). We experimentally confirmed ER-alpha and PITX1 presence at the DEGs by performing ChIP-seq analysis using the ovarian cancer cell line PEO4. Finally, RNA-seq analysis identified recurrent gene fusion events in our EOC tumor set. Some of these fusions were significantly associated with survival in HGSOC patients; however, the fusion genes are not regulated by the transcription factors identified for the DEGs. CONCLUSIONS: These data implicate an estrogen-responsive regulatory network in the differential gene expression between ovarian cancer subtypes and stages, which includes PITX1. Importantly, the transcription factors associated with our DEG promoters are known to form the MegaTrans complex in breast cancer. This is the first study to implicate the MegaTrans complex in contributing to the distinct biological trajectories of malignant and indolent ovarian cancer subtypes.

Recurrent patterns of DNA methylation in the ZNF154, CASP8, and VHL promoters across a wide spectrum of human solid epithelial tumors and cancer cell lines.

The study of aberrant DNA methylation in cancer holds the key to the discovery of novel biological markers for diagnostics and can help to delineate important mechanisms of disease. We have identified 12 loci that are differentially methylated in serous ovarian cancers and endometrioid ovarian and endometrial cancers with respect to normal control samples. The strongest signal showed hypermethylation in tumors at a CpG island within the ZNF154 promoter. We show that hypermethylation of this locus is recurrent across solid human epithelial tumor samples for 15 of 16 distinct cancer types from TCGA. Furthermore, ZNF154 hypermethylation is strikingly present across a diverse panel of ENCODE cell lines, but only in those derived from tumor cells. By extending our analysis from the Illumina 27K Infinium platform to the 450K platform, to sequencing of PCR amplicons from bisulfite treated DNA, we demonstrate that hypermethylation extends across the breadth of the ZNF154 CpG island. We have also identified recurrent hypomethylation in two genomic regions associated with CASP8 and VHL. These three genes exhibit significant negative correlation between methylation and gene expression across many cancer types, as well as patterns of DNaseI hypersensitivity and histone marks that reflect different chromatin accessibility in cancer vs. normal cell lines. Our findings emphasize hypermethylation of ZNF154 as a biological marker of relevance for tumor identification. Epigenetic modifications affecting the promoters of ZNF154, CASP8, and VHL are shared across a vast array of tumor types and may therefore be important for understanding the genomic landscape of cancer.

Assessment of the Prognostic Value of Two Common Variants of BRCA1 and BRCA2 Genes in Ovarian Cancer Patients Treated with Cisplatin and Paclitaxel: A Gynecologic Oncology Group Study.

PURPOSE: BRCA1/BRCA2 germline mutations appear to enhance the platinum-sensitivity, but little is known about the prognostic relevance of polymorphisms in BRCA1/BRCA2 in epithelial ovarian cancer (EOC). This study evaluated whether common variants of BRCA1/BRCA2 are associated with progression-free survival (PFS) and overall survival (OS) in patients with advanced stage sporadic EOC. EXPERIMENTAL DESIGN: The allelic frequency of BRCA1 (2612C > T, P871L-rs799917) and BRCA2 (114A > C, N372H-rs144848) were determined in normal blood DNA from women in Gynecologic Oncology Group protocol #172 phase III trial with optimally resected stage III EOC treated with intraperitoneal or intravenous cisplatin and paclitaxel (C + P). Associations between polymorphisms and PFS or OS were assessed. RESULTS: Two hundred and thirty-two women were included for analyses. African Americans (AA) had different distributions for the two polymorphisms from Caucasians and others. For non-AA patients, the genotype for BRCA1 P871L was distributed as 38% for CC, 49% for CT, and 13% for TT. Median PFS was estimated to be 31, 21, and 21 months, respectively. After adjusting for cell type, residual disease, and chemotherapy regimen, CT/TT genotypes were associated with a 1.40-fold increased risk of disease progression [95% confidence interval (CI) = 1.00-1.95, p = 0.049]. After removing seven patients with known BRCA1 germline mutations, the hazard ratio (HR) was 1.36 (95% CI = 0.97-1.91, p = 0.073). The association between BRCA1 P871L and OS was not significant (HR = 1.25, 95% CI = 0.88-1.76, p = 0.212). Genotype distribution of BRCA2 N372H among non-AA patients was 50, 44, and 6% for AA, AC, and CC, respectively and there is no evidence that this BRCA2 polymorphism was related to PFS or OS. CONCLUSION: Polymorphisms in BRCA1 P871L or in BRCA2 N372H were not associated with either PFS or OS in women with optimally resected, stage III EOC treated with cisplatin and paclitaxel.

Comparison of ERCC1/XPF genetic variation, mRNA and protein levels in women with advanced stage ovarian cancer treated with intraperitoneal platinum.

OBJECTIVE: Approximately 20% of patients receiving platinum-based chemotherapy for epithelial ovarian cancer (EOC) are refractory or develop early recurrence. Identifying these patients early could reduce treatment-associated morbidity and allow quicker transfer to more effective therapies. Much attention has focused on ERCC1 as a potential predictor of response to therapy because of its essential role in the repair of platinum-induced DNA damage. The purpose of this study was to accurately measure protein levels of ERCC1 and its essential binding partner XPF from patients with EOC treated with platinum-based therapy and determine if protein levels correlate with mRNA levels, patient genotypes or clinical outcomes. METHODS: ERCC1 and XPF mRNA and protein levels were measured in frozen EOC specimens from 41 patients receiving intraperitoneal platinum-based chemotherapy using reverse transcription polymerase chain reaction and western blots. Genotypes of common nucleotide polymorphisms were also analyzed. Patient outcomes included progression free (PFS) and overall survival (OS). RESULTS: Expression of ERCC1 and XPF were tightly correlated with one another at both the mRNA and protein level. However, the mRNA and protein levels of ERCC1 were not positively correlated. Likewise, none of the SNPs analyzed correlated with ERCC1 or XPF protein levels. There was an inverse correlation between mRNA levels and patient outcomes. CONCLUSION: Neither genotype nor mRNA levels are predictive of protein expression. Despite this, low ERCC1 mRNA significantly correlated with improved PFS and OS.

BRCA1/2 mutations and expression: response to platinum chemotherapy in patients with advanced stage epithelial ovarian cancer.

OBJECTIVE: Our objective was to determine the rate of BRCA1/2 deficiency in platinum-sensitive and platinum-resistant tumors from a cohort of unselect patients with advanced epithelial ovarian cancer (EOH). METHODS: BRCA1/2 mutation analysis was performed in 29 patients with platinum-sensitive EOC and 24 patients with platinum-resistant disease. Germline DNA was analyzed in mutation carriers when normal tissue was available. BRCA expression was ascertained by quantitative rt-PCR. Associations between BRCA mutation status and expression levels and parameters of platinum response were analyzed. RESULTS: Fifteen of 53 (28.3%) EOC tumors had BRCA1/2 mutations. Twelve mutations were in BRCA1, while 3 involved BRCA2. Of the 12 mutation-carriers with normal tissue available for DNA analyses, 33.3% of the mutations were found to be somatic. Three mutations were novel. The majority of BRCA mutations (73%) were identified in patients with platinum-sensitive disease. In total, 38% of platinum-sensitive tumors were found to have a BRCA mutation, compared to 17% of the platinum-resistant patients. A statistical trend toward platinum-sensitive disease was seen in BRCA mutation carriers (p=0.079). Nineteen (36%) study patients had some form of BRCA deficiency, and these patients were less likely to have platinum-resistant tumors (OR=0.29; p value=0.048). CONCLUSIONS: BRCA mutations occurred more frequently in platinum-sensitive EOC than platinum-resistant disease. The high overall frequency of BRCA deficiency in EOC underscores the importance of tumor profiling as therapies targeting the DNA repair pathway are being investigated.

Differential analysis of ovarian and endometrial cancers identifies a methylator phenotype.

Despite improved outcomes in the past 30 years, less than half of all women diagnosed with epithelial ovarian cancer live five years beyond their diagnosis. Although typically treated as a single disease, epithelial ovarian cancer includes several distinct histological subtypes, such as papillary serous and endometrioid carcinomas. To address whether the morphological differences seen in these carcinomas represent distinct characteristics at the molecular level we analyzed DNA methylation patterns in 11 papillary serous tumors, 9 endometrioid ovarian tumors, 4 normal fallopian tube samples and 6 normal endometrial tissues, plus 8 normal fallopian tube and 4 serous samples from TCGA. For comparison within the endometrioid subtype we added 6 primary uterine endometrioid tumors and 5 endometrioid metastases from uterus to ovary. Data was obtained from 27,578 CpG dinucleotides occurring in or near promoter regions of 14,495 genes. We identified 36 locations with significant increases or decreases in methylation in comparisons of serous tumors and normal fallopian tube samples. Moreover, unsupervised clustering techniques applied to all samples showed three major profiles comprising mostly normal samples, serous tumors, and endometrioid tumors including ovarian, uterine and metastatic origins. The clustering analysis identified 60 differentially methylated sites between the serous group and the normal group. An unrelated set of 25 serous tumors validated the reproducibility of the methylation patterns. In contrast, >1,000 genes were differentially methylated between endometrioid tumors and normal samples. This finding is consistent with a generalized regulatory disruption caused by a methylator phenotype. Through DNA methylation analyses we have identified genes with known roles in ovarian carcinoma etiology, whereas pathway analyses provided biological insight to the role of novel genes. Our finding of differences between serous and endometrioid ovarian tumors indicates that intervention strategies could be developed to specifically address subtypes of epithelial ovarian cancer.

Common variants in ABCB1, ABCC2 and ABCG2 genes and clinical outcomes among women with advanced stage ovarian cancer treated with platinum and taxane-based chemotherapy: a Gynecologic Oncology Group study.

PURPOSE: Efflux transporters of the ATP-binding cassette (ABC) family are major determinants of chemoresistance in tumor cells. This study examined associations between functional variants in ABCB1, ABCC2 and ABCG2 genes and clinical outcomes in patients with epithelial ovarian/primary peritoneal cancer (EOC/PPC) following platinum and taxane-based chemotherapy. METHODS: Sequenom iPLEXTMGOLD Assay and MALDI-TOF platform were used to genotype the non-synonymous G2677T/A (rs2032582; encoding Ala893Ser/Thr) and synonymous C3435T (rs1045642; encoding Ile1145Ile) variants in ABCB1, the non-synonymous G1249A variant in ABCC2 (rs2273697; encoding Val417Ile), and the non-synonymous C421A variant in ABCG2 (rs2231142; encoding Q141K, Gln141Lys) in normal DNA from up to 511 women in Gynecologic Oncology Group (GOG) phase III trials, GOG-172 or GOG-182. Progression-free survival (PFS) and overall survival (OS) were analyzed in relation to genetic polymorphisms using Kaplan-Meier and Cox proportional hazards model. RESULTS: The C421A variant (CA+AA versus CC) in ABCG2 was associated with a 6-month longer median PFS (22.7 versus 16.8 months, p=0.041). In multivariate analysis, patients with variant genotypes were at a reduced risk of disease progression (hazard ratio [HR]=0.75, 95% confidence interval [CI]=0.59-0.96, p=0.022). The association between C421A and OS was not statistically significant (HR=0.88, 95% CI=0.67-1.15, p=0.356). None of the other variants measured in either ABCB1 or ABCC2 was associated with PFS or OS. CONCLUSION: The C421A variant in ABCG2, previously shown to be associated with enhanced protein degradation and drug sensitivity, was associated with longer PFS in advanced stage EOC/PPC patents treated with platinum+taxane-based chemotherapy. This finding requires further validation.

Noninvasive assessment of cell proliferation in ovarian cancer using [18F] 3'deoxy-3-fluorothymidine positron emission tomography/computed tomography imaging.

INTRODUCTION: Positron emission tomography (PET)/computed tomography (CT) imaging of suspected new and recurrent ovarian carcinoma was performed to assess the relationship between [(18)F] 3'deoxy-3'fluorothymidine ((18)FLT) uptake and histopathological tissue markers of cellular proliferation (Ki67) and thymidine kinase-1 (TK-1) expression. METHODS: Six subjects were included in this pilot study. Subjects were injected with 5 mCi of (18)FLT prior to a planned surgery and then scanned on a GE Discovery-ST PET/CT scanner within an hour of injection. Regions of interest in tumor and control tissue were identified on the diagnostic CT scans and marked for later surgical biopsy. Surgery was performed within 2 days after the scan. At the time of surgery, the regions of interest identified on PET/CT were available to guide the surgeon to the tumor biopsy sites. Tissue from normal ovarian tissue control regions was also sampled. (18)FLT uptake in tumor and control tissue regions was calculated by measuring the maximum standardized uptake values (SUV(max)). The excised tumor and normal ovarian tissue control tissues were analyzed by immunohistochemical staining for Ki67 and CD34. TK-1 messenger RNA expression was measured by real-time polymerase chain reaction. RESULTS: (18)FLT uptake (SUV(max)) was higher in malignant (mean 4.85/range 1.7-8.8) compared to benign (1.65/range 1.4-1.9) and normal ovarian control tissue (1.12/range 0.6-1.5). Mitotic index, as determined by Ki67 staining, was higher in malignant (18.89/range 11.97-27.19) compared to benign (0.59/range 0.23-0.95) and control tissue (0.45/range 0.06-1.20). TK-1 expression was also higher in malignant (35.52/range 5.21-106.62) compared to benign (8.71/range 4.74-12.67) and control tissue (9.79/range 0.85-39.46). An increasing trend between (18)FLT uptake and Ki67 mitotic index is seen in malignant tissue CD 34 staining between malignant, benign and control tissues was not qualitatively different. CONCLUSION: An increasing trend between (18)FLT uptake and Ki67 mitotic index is seen in malignant tissue. Additional studies will determine whether (18)FLT PET/CT is specific enough to distinguish between cancerous and noncancerous cells and to assess its role in ovarian carcinoma patient management.

Single nucleotide polypmorphisms in ERCC1 are associated with disease progression, and survival in patients with advanced stage ovarian and primary peritoneal carcinoma; a Gynecologic Oncology Group study.

OBJECTIVE: This study evaluated common polymorphisms in excision repair cross-complementation group 1 (ERCC1) involved in repair of platinum-induced DNA damage in advanced-stage, epithelial ovarian/peritoneal/tubal cancer (EOC/PPC/FTC) patients treated with intravenous carboplatin- and paclitaxel-based chemotherapy. METHODS: Pyrosequencing was performed to examine single nucleotide polymorphisms (SNPs) in codon 118 and C8092A in ERCC1 in leukocyte DNA from the Gynecologic Oncology Group phase III protocol, GOG-182. Kaplan-Meier method and adjusted Cox regression modeling were used to examine associations between ERCC1 polymorphisms and progression-free survival (PFS) and overall survival (OS). RESULTS: The genotype distribution at codon 118 (n=278) in ERCC1 for CC, CT, and TT was 23%, 45% and 32%, and the median OS was 32, 47 and 43 months, respectively. Patients with the CT+TT versus CC genotype in codon 118 in ERCC1 were at a reduced risk of death (hazard ratio [HR]=0.68, 95% confidence interval [CI]=0.49-0.95, p=0.025). The genotype distribution for C8092A in ERCC1 (N=280) was 50%, 42% and 8%, and the median OS was 45, 40 or 30 months for CC, CA and AA, respectively. Women with the CA+AA versus CC genotype in C8092A in ERCC1 had a trend suggesting an increased risk of death (HR=1.29, 95% CI=0.97-1.72, p=0.077). CONCLUSIONS: The polymorphism in codon 118 in the DNA repair gene ERCC1 was an independent predictor for better survival in EOC/PPC/FTC patients treated with intravenous carboplatin- and paclitaxel-based chemotherapy. The relationship between the C8092A polymorphisms in ERCC1 and survival was modest with an effect size that was not always statistically significant.

Pharmacologic and phenotypic study of docetaxel in patients with ovarian or primary peritoneal cancer.

PURPOSE: The objectives of this study were to determine whether the midazolam clearance predicted docetaxel pharmacokinetics, CA-125 change, and response and to assess the impact of cytochrome P450 (CYP) 3A5 and ATP-binding cassette, subfamily B, member 1 (ABCB1) genotypes on docetaxel pharmacokinetics and pharmacodynamics in ovarian or primary peritoneal cancer patients. METHODS: Thirty-four patients with advanced ovarian and primary peritoneal cancer were administered docetaxel at 75 mg/m(2) as a 1-h infusion in combination with carboplatin IV over 30 min at a target AUC of 5 mg/ml min. Cycles were repeated every 21 days for 6 cycles. Midazolam was administered at 2 mg as a 30-min IV infusion the day prior to cycle one of docetaxel administration. Pharmacokinetic studies of docetaxel and CYP3A5 and ABCB1 genotype studies were performed. RESULTS: There was an inverse relationship between midazolam clearance (CL) and CA-125 level after cycle 6 where a higher midazolam CL was associated with a CA-125 <10 U/ml (P = 0.007) and CA-125 <15 U/ml (P = 0.048). The CA-125 categories were associated with response achieved (complete response/partial response) (CR/PR), stable disease (SD), and progressive disease (PD) at the end of therapy (P = 0.0173). Docetaxel CL was not related to midazolam CL or genotype. Docetaxel exposure and genotypes were not related to toxicity or response (P > 0.05). CONCLUSIONS: The midazolam CL predicted CA-125 levels and response that was independent of other factors including docetaxel pharmacokinetics. Future studies need to evaluate the mechanism for the relationship between midazolam CL and response in patients with ovarian cancer.

Cigarette exposure induces changes in maternal vascular function in a pregnant mouse model.

Smoking is associated with multiple adverse pregnancy outcomes, including fetal growth restriction. The objective of this study was to determine whether cigarette smoke exposure during pregnancy in a mouse model affects the functional properties of maternal uterine, mesenteric, and renal arteries as a possible mechanism for growth restriction. C57Bl/CJ mice were exposed to whole body sidestream smoke for 4 h/day. Smoke particle exposure was increased from day 4 of gestation until late pregnancy (day 16-19), with mean total suspended particle levels of 63 mg/m(3), representative of moderate-to-heavy smoking in humans. Uterine, mesenteric, and renal arteries from late-pregnant and virgin mice were isolated and studied in a pressure-arteriograph system (n = 23). Plasma cotinine was measured by ELISA. Fetal weights were significantly reduced in smoke-exposed compared with control fetuses (0.88 +/- 0.1 vs. 1.0 +/- 0.08 g, P < 0.02), while litter sizes were not different. Endothelium-mediated relaxation responses to methacholine were significantly impaired in both the uterine and mesenteric vasculature of pregnant mice exposed to cigarette smoke during gestation. This difference was not apparent in isolated renal arteries from pregnant mice exposed to cigarette smoke; however, relaxation was significantly reduced in renal arteries from smoke-exposed virgin mice. In conclusion, we found that passive cigarette smoke exposure is associated with impaired vascular relaxation of uterine and mesenteric arteries in pregnant mice. Functional maternal vascular perturbations during pregnancy, specifically impaired peripheral and uterine vasodilation, may contribute to a mechanism by which smoking results in fetal growth restriction.

Relationship between ERCC1 polymorphisms, disease progression, and survival in the Gynecologic Oncology Group Phase III Trial of intraperitoneal versus intravenous cisplatin and paclitaxel for stage III epithelial ovarian cancer.

PURPOSE: We hypothesized that common polymorphisms in excision repair cross-complementation group 1 (ERCC1), involved in nucleotide excision repair of platinum-induced damage, would be associated with progression-free survival (PFS) and overall survival (OS) in women with optimally resected, stage III epithelial ovarian cancer (EOC) treated with cisplatin and paclitaxel (C+P). PATIENTS AND METHODS: Single nucleotide polymorphism analysis was carried out by direct pyrosequencing at two sites (codon 118 and C8092A) in ERCC1 in leukocyte DNA from women who participated in the Gynecologic Oncology Group (GOG) phase III protocol-172 and were randomly assigned to intraperitoneal or intravenous C+P. RESULTS: ERCC1 genotyping was performed in 233 of the 429 women who participated in GOG-172. The genotype distribution at codon 118 was 17% with C/C, 43% with C/T, and 40% with T/T, and the genotype distribution at C8092A was 56% with C/C, 37% with C/A, and 7% with A/A. Adjusted Cox regression analysis revealed that the codon 118 polymorphism in ERCC1 was not significantly associated with disease progression or death. Women with the C8092A C/A or A/A genotypes compared with the C/C genotype had an increased risk of disease progression (hazard ratio [HR] = 1.44; 95% CI, 1.06 to 1.94; P = .018) and death (HR = 1.50; 95% CI, 1.07 to 2.09; P = .018). Median PFS and OS were 6 and 17 months shorter for women with the C8092A C/A or A/A genotypes versus the C/C genotype, respectively. CONCLUSION: Although the ERCC1 codon 118 polymorphism does not seem to be associated with clinical outcome, the C8092A polymorphism was an independent predictor of PFS and OS in women with optimally resected EOC.

Comparison of cell cycle regulatory gene mRNA in three different types of human trophoblasts and effect of transforming growth factor.

AIM: Identifying the factors responsible for reducing the proliferation, syncytialization, and invasiveness of trophoblast tissues, as seen with preeclampsia, intrauterine growth restriction, and spontaneous miscarriage, is a current challenge in reproductive biology. These factors, transforming growth factor (TGF)-beta as an example, can work by altering trophoblast differentiation or proliferation. We therefore investigated and compared specific markers of trophoblast proliferation and differentiation in three commonly used trophoblast tissue cell models, and also investigated the influence of TGF-beta on these markers. METHODS: In this study, we isolated human trophoblasts from first trimester and term placentas, and additionally used human choriocarcinoma cells (JEG-3). Baseline values of human chorionic gonadotropin (hCG) secretion and relative mRNA levels of cell cycle regulators (cyclin E, p21, p27, and p57) were investigated for each cell type. We also investigated the influence of TGF-beta on these parameters. RESULTS: Quantitative and longitudinal production of hCG differed between the three cell types. Significantly different amounts of cyclin E, p21, p27, and p57 mRNA were demonstrated within each cell type, as well as between all the cell types, throughout the culture time period. Each trophoblast type demonstrated a reduction of hCG secretion in response to TGF-beta. TGF-beta did not show a consistent effect on the cell cycle mRNA of any of the cell types. CONCLUSION: We were able to characterize and compare the differential production of hCG, as well as the differential expression of cell cycle-associated mRNA of early trophoblasts, term trophoblasts, and choriocarcinoma cells. The production of hCG was altered by TGF-beta, although mRNA levels were not markedly altered by TGF-beta.

Use of gene expression profiles to stage concurrent endometrioid tumors of the endometrium and ovary.

OBJECTIVE: The objectives of this study were to determine whether there are distinct gene expression profiles for endometrial and ovarian endometrioid carcinomas and to create a statistical model using these profiles to predict their organ of origin. METHODS: Expression profiles of seven stage I, grades 1 and 2, endometrioid endometrial carcinomas and seven stage I ovarian endometrioid carcinomas were analyzed as the training set. The test set included seven advanced endometrial carcinomas and nine dual primary endometrial and ovarian primary tumors of endometrioid histology. Unsupervised hierarchical clustering, multidimensional scaling (MDS) and predictive analysis of microarrays (PAM) were used to analyze the data. RESULTS: We identified 163 differentially expressed (DE) genes between endometrial and ovarian tumors. Both unsupervised hierarchical clustering and multidimensional scaling showed clear separation of the two groups. Pathway analysis of the 163 DE genes revealed significant biological differences between the two groups, which included metabolic and biosynthetic pathways. Further classification analysis on the training data using predictive analysis of microarray generated a 119-gene predictive model with 100% cross-validation accuracy. When the 119-gene model was applied to the test set of 16 samples, we found concordance in 11/16 samples and discordance in 5/16 samples with the pathologic classification. CONCLUSION: We conclude that, although similar in histologic appearance, endometrioid carcinomas of the ovary and endometrium have distinct gene expression patterns. A larger test set is needed to prospectively evaluate this prediction model further.

Expression and activity of taxane-metabolizing enzymes in ovarian tumors.

OBJECTIVE: Current firstline chemotherapy for ovarian cancer consists of carboplatin combined with either paclitaxel or docetaxel. Disposition of carboplatin is determined by renal clearance, while the taxanes are metabolized by cytochrome P450 (CYP450) enzymes. Although the majority of taxane metabolism occurs in the liver, recent data have shown that some solid tumors express CYP450 enzymes in the tumors themselves. The objective of this study was to determine whether ovarian tumors express genes regulating cellular efflux and subsequent metabolism, and whether any clinico-pathologic features correlated with expression. METHODS: Gene expression of CYP2C8, CYP3A4/A5 and the ABC transporter ABCB1 was determined in 56 primary epithelial ovarian tumors. Cells were grown from seven different tumors and exposed ex vivo to paclitaxel (PAC) and docetaxel (DOC) for up to 24 h. PAC and DOC concentrations were measured in the media by an LC-MS assay. RESULTS: Results from this analysis demonstrate that ovarian cancer cells do express functional taxane-metabolizing enzymes. Such expression appeared to enhance the ability of cancer cells to metabolize DOC. Specifically, the PK of DOC was correlated with the ratio of CYP4A5 to ABCB1 gene expression, thus representing a novel mechanism of chemotherapy resistance. There was no relationship between PAC PK parameters and gene expression. CONCLUSIONS: Knowledge of inter-individual variation in CYP450 enzyme and ABC transporter tumor expression and activity may influence the individualization of chemotherapy, by avoiding agents that are rapidly metabolized and selecting agents that are not.

Coordinate up-regulation of TMEM97 and cholesterol biosynthesis genes in normal ovarian surface epithelial cells treated with progesterone: implications for pathogenesis of ovarian cancer.

BACKGROUND: Ovarian cancer (OvCa) most often derives from ovarian surface epithelial (OSE) cells. Several lines of evidence strongly suggest that increased exposure to progesterone (P4) protects women against developing OvCa. However, the underlying mechanisms of this protection are incompletely understood. METHODS: To determine downstream gene targets of P4, we established short term in vitro cultures of non-neoplastic OSE cells from six subjects, exposed the cells to P4 (10-6 M) for five days and performed transcriptional profiling with oligonucleotide microarrays containing over 22,000 transcripts. RESULTS: We identified concordant but modest gene expression changes in cholesterol/lipid homeostasis genes in three of six samples (responders), whereas the other three samples (non-responders) showed no expressional response to P4. The most up-regulated gene was TMEM97 which encodes a transmembrane protein of unknown function (MAC30). Analyses of outlier transcripts, whose expression levels changed most significantly upon P4 exposure, uncovered coordinate up-regulation of 14 cholesterol biosynthesis enzymes, insulin-induced gene 1, low density lipoprotein receptor, ABCG1, endothelial lipase, stearoyl- CoA and fatty acid desaturases, long-chain fatty-acyl elongase, and down-regulation of steroidogenic acute regulatory protein and ABCC6. Highly correlated tissue-specific expression patterns of TMEM97 and the cholesterol biosynthesis genes were confirmed by analysis of the GNF Atlas 2 universal gene expression database. Real-time quantitative RT-PCR analyses revealed 2.4-fold suppression of the TMEM97 gene expression in short-term cultures of OvCa relative to the normal OSE cells. CONCLUSION: These findings suggest that a co-regulated transcript network of cholesterol/lipid homeostasis genes and TMEM97 are downstream targets of P4 in normal OSE cells and that TMEM97 plays a role in cholesterol and lipid metabolism. The P4-induced alterations in cholesterol and lipid metabolism in OSE cells might play a role in conferring protection against OvCa.

Enzymatic removal of asparagine-linked carbohydrate chains from heterodimer human chorionic gonadotrophin and effect on bioactivity.

The native form of human chorionic gonadotropin (hCG) is a heterodimer protein with two asparagine (Asn)-linked carbohydrate chains on each subunit. Removal of the Asn-linked carbohydrate chains from hCG has resulted in hCG variants with consistent antagonistic properties on isolated murine cells. Specific and direct enzymatic removal of these carbohydrate chains from native hCG with resultant antagonistic properties has not been reported. An antagonist to the hCG/luteinising hormone (LH) receptor could be used as an anticancer therapy, emergency contraceptive or for therapeutic resolution of ectopic pregnancies. Therefore, our aim was to use enzymes to specifically remove Asn-linked carbohydrate chains from hCG in the heterodimer form and analyse the resultant bioactivity. Native hCG was treated with endoglycosidases, carbohydrate removal was analysed with electrophoresis and the hCG variants were tested for altered bioactivity with human and murine cells. Endoglycosidases were able to cleave most of the Asn-linked carbohydrate chains from the native hCG. The deglycosylated hCG demonstrated a 75% reduction in bioactivity on a murine Leydig cell line and a 65% reduction in bioactivity on human granulosa cells. These results exemplify a simple and efficient method for creating deglycosylated hCG and provide the most direct evidence for the importance of Asn-linked carbohydrate chains in maintaining hCG bioactivity.

Clinical use of combined positron emission tomography and computed tomography (FDG-PET/CT) in recurrent ovarian cancer.

OBJECTIVE: The aim of this study was to evaluate the use of co-registered PET/CT using F-18 fluorodeoxyglucose (FDG) for surveillance and follow-up of ovarian cancer patients to detect recurrent disease. MATERIAL AND METHODS: A retrospective chart review was performed on 39 ovarian cancer patients who underwent a total of 59 FDG-PET/CT scans. The following information was obtained: clinical indication for FDG-PET/CT, the results of FDG-PET/CT particularly with regard to the additional diagnostic information, the localization of disease and subsequent clinical patient management. RESULTS: Twenty-four FDG-PET/CT were performed in 22 patients with previously negative or indeterminate CT scans but rising CA-125 levels providing a sensitivity of 90% for localizing disease. Nine FDG-PET/CT in 8 patients with clinical symptoms of recurrence but normal CA-125 levels detected all three patients who had recurrent disease confirmed within 6 months of follow-up. In addition, 4 FDG-PET/CT performed as routine follow-up with no clinical evidence of recurrent disease were true-negative in all cases. Fourteen FDG-PET/CT in 12 patients with recurrent disease already identified by conventional CT imaging were useful in guiding treatment decisions such as radiation therapy, surgery or chemotherapy by confirming the recurrence and more precisely localizing the site(s) of disease. Of note, FDG-PET/CT helped to avoid surgery in four patients who had additional disease detected in unresectable anatomic areas. A total of 51 FDG-PET/CT were performed in the patients described above with an overall sensitivity and specificity of 94.5% and 100%, respectively. Eight FDG-PET/CT scans in five patients performed for assessment of treatment response following chemotherapy or radiation were useful as the disease was not clearly visualized by conventional CT imaging at baseline. CONCLUSIONS: In our experience, FDG-PET/CT has the greatest utility in settings of suspected ovarian cancer recurrence, particularly in patients with rising CA-125 levels and negative conventional imaging. FDG-PET/CT was specifically helpful in optimizing the selection of patients for site-specific treatment, including radiation treatment planning, and aided in the selection of optimal surgical candidates. The co-registered metabolic-anatomic information from combined FDG-PET/CT holds promise in replacing the single imaging procedures.

The effects of cigarette smoking on circulating maternal leukocytes during pregnancy.

During pregnancy, cigarette smoke exposure, a common environmental insult, is damaging to both mother and fetus and is associated with pregnancy loss. The mechanism underlying the pathophysiology of injury is not understood. We hypothesized that smoking during pregnancy interferes with the normal physiological adaptation of the maternal immune system. We used flow cytometry to measure changes in the distribution of subsets of circulating leukocytes and adhesion molecule expression in a prospective cohort of 198 women who had 325 blood samples obtained throughout pregnancy. Smoking status was assessed by plasma cotinine concentration. Smoking increased the frequency of CD3(+) lymphocytes and decreased CD56(+) cells at 14-20 weeks gestation. Smoking also decreased the expression of CD54 on monocytes and CD62L on granulocytes throughout pregnancy. Our data demonstrate that smoking affects several immune parameters, especially early in pregnancy. These perturbations may play a role in pregnancy loss in women who smoke.

Preeclampsia activates circulating immune cells with engagement of the NF-kappaB pathway.

PROBLEM: Compelling evidence implicates peripheral immune activation in the pathophysiology of preeclampsia. Polymorphonuclear neutrophils appear to be the cells most strongly affected, with changes in expression of surface markers and release of granule enzymes. Here, we investigated activation in additional leukocyte populations among women with preeclampsia. METHOD: We used flow cytometry to evaluate changes in leukocyte markers in preeclampsia compared with uncomplicated pregnancy. To gain insights into intracellular pathways involved in leukocyte activation, we monitored the NF-kappaB signal transduction pathway. Plasma levels of interleukin-6 (IL-6) were also studied as an additional indication of cellular activation. RESULTS: Preeclampsia is associated with changes in L-selectin (CD62L) on neutrophils (P = 0.004), monocytes (P = 0.013), and T cells (P = 0.048) when compared with normal pregnancy. These changes include an increase in nuclear translocation of NF-kappaB and increased levels of IL-6 (P = 0.005). CONCLUSIONS: These findings are consistent with the presence of a generalized phenomenon of immune activation in preeclampsia.