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1.  Contribution of Germline Mutations in the RAD51B, RAD51C, and RAD51D Genes to Ovarian Cancer in the Population 
Journal of Clinical Oncology  2015;33(26):2901-2907.
Purpose
The aim of this study was to estimate the contribution of deleterious mutations in the RAD51B, RAD51C, and RAD51D genes to invasive epithelial ovarian cancer (EOC) in the population and in a screening trial of individuals at high risk of ovarian cancer.
Patients and Methods
The coding sequence and splice site boundaries of the three RAD51 genes were sequenced and analyzed in germline DNA from a case-control study of 3,429 patients with invasive EOC and 2,772 controls as well as in 2,000 unaffected women who were BRCA1/BRCA2 negative from the United Kingdom Familial Ovarian Cancer Screening Study (UK_FOCSS) after quality-control analysis.
Results
In the case-control study, we identified predicted deleterious mutations in 28 EOC cases (0.82%) compared with three controls (0.11%; P < .001). Mutations in EOC cases were more frequent in RAD51C (14 occurrences, 0.41%) and RAD51D (12 occurrences, 0.35%) than in RAD51B (two occurrences, 0.06%). RAD51C mutations were associated with an odds ratio of 5.2 (95% CI, 1.1 to 24; P = .035), and RAD51D mutations conferred an odds ratio of 12 (95% CI, 1.5 to 90; P = .019). We identified 13 RAD51 mutations (0.65%) in unaffected UK_FOCSS participants (RAD51C, n = 7; RAD51D, n = 5; and RAD51B, n = 1), which was a significantly greater rate than in controls (P < .001); furthermore, RAD51 mutation carriers were more likely than noncarriers to have a family history of ovarian cancer (P < .001).
Conclusion
These results confirm that RAD51C and RAD51D are moderate ovarian cancer susceptibility genes and suggest that they confer levels of risk of EOC that may warrant their use alongside BRCA1 and BRCA2 in routine clinical genetic testing.
doi:10.1200/JCO.2015.61.2408
PMCID: PMC4554751  PMID: 26261251
2.  Ovarian cancer screening and mortality in the UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS): a randomised controlled trial 
Lancet (London, England)  2016;387(10022):945-956.
Summary
Background
Ovarian cancer has a poor prognosis, with just 40% of patients surviving 5 years. We designed this trial to establish the effect of early detection by screening on ovarian cancer mortality.
Methods
In this randomised controlled trial, we recruited postmenopausal women aged 50–74 years from 13 centres in National Health Service Trusts in England, Wales, and Northern Ireland. Exclusion criteria were previous bilateral oophorectomy or ovarian malignancy, increased risk of familial ovarian cancer, and active non-ovarian malignancy. The trial management system confirmed eligibility and randomly allocated participants in blocks of 32 using computer-generated random numbers to annual multimodal screening (MMS) with serum CA125 interpreted with use of the risk of ovarian cancer algorithm, annual transvaginal ultrasound screening (USS), or no screening, in a 1:1:2 ratio. The primary outcome was death due to ovarian cancer by Dec 31, 2014, comparing MMS and USS separately with no screening, ascertained by an outcomes committee masked to randomisation group. All analyses were by modified intention to screen, excluding the small number of women we discovered after randomisation to have a bilateral oophorectomy, have ovarian cancer, or had exited the registry before recruitment. Investigators and participants were aware of screening type. This trial is registered with ClinicalTrials.gov, number NCT00058032.
Findings
Between June 1, 2001, and Oct 21, 2005, we randomly allocated 202 638 women: 50 640 (25·0%) to MMS, 50 639 (25·0%) to USS, and 101 359 (50·0%) to no screening. 202 546 (>99·9%) women were eligible for analysis: 50 624 (>99·9%) women in the MMS group, 50 623 (>99·9%) in the USS group, and 101 299 (>99·9%) in the no screening group. Screening ended on Dec 31, 2011, and included 345 570 MMS and 327 775 USS annual screening episodes. At a median follow-up of 11·1 years (IQR 10·0–12·0), we diagnosed ovarian cancer in 1282 (0·6%) women: 338 (0·7%) in the MMS group, 314 (0·6%) in the USS group, and 630 (0·6%) in the no screening group. Of these women, 148 (0·29%) women in the MMS group, 154 (0·30%) in the USS group, and 347 (0·34%) in the no screening group had died of ovarian cancer. The primary analysis using a Cox proportional hazards model gave a mortality reduction over years 0–14 of 15% (95% CI −3 to 30; p=0·10) with MMS and 11% (−7 to 27; p=0·21) with USS. The Royston-Parmar flexible parametric model showed that in the MMS group, this mortality effect was made up of 8% (−20 to 31) in years 0–7 and 23% (1–46) in years 7–14, and in the USS group, of 2% (−27 to 26) in years 0–7 and 21% (−2 to 42) in years 7–14. A prespecified analysis of death from ovarian cancer of MMS versus no screening with exclusion of prevalent cases showed significantly different death rates (p=0·021), with an overall average mortality reduction of 20% (−2 to 40) and a reduction of 8% (−27 to 43) in years 0–7 and 28% (−3 to 49) in years 7–14 in favour of MMS.
Interpretation
Although the mortality reduction was not significant in the primary analysis, we noted a significant mortality reduction with MMS when prevalent cases were excluded. We noted encouraging evidence of a mortality reduction in years 7–14, but further follow-up is needed before firm conclusions can be reached on the efficacy and cost-effectiveness of ovarian cancer screening.
Funding
Medical Research Council, Cancer Research UK, Department of Health, The Eve Appeal.
doi:10.1016/S0140-6736(15)01224-6
PMCID: PMC4779792  PMID: 26707054
3.  Germline Mutations in the BRIP1, BARD1, PALB2, and NBN Genes in Women With Ovarian Cancer 
Background:
Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy, responsible for 13 000 deaths per year in the United States. Risk prediction based on identifying germline mutations in ovarian cancer susceptibility genes could have a clinically significant impact on reducing disease mortality.
Methods:
Next generation sequencing was used to identify germline mutations in the coding regions of four candidate susceptibility genes—BRIP1, BARD1, PALB2 and NBN—in 3236 invasive EOC case patients and 3431 control patients of European origin, and in 2000 unaffected high-risk women from a clinical screening trial of ovarian cancer (UKFOCSS). For each gene, we estimated the prevalence and EOC risks and evaluated associations between germline variant status and clinical and epidemiological risk factor information. All statistical tests were two-sided.
Results:
We found an increased frequency of deleterious mutations in BRIP1 in case patients (0.9%) and in the UKFOCSS participants (0.6%) compared with control patients (0.09%) (P = 1 x 10–4 and 8 x 10–4, respectively), but no differences for BARD1 (P = .39), NBN1 (P = .61), or PALB2 (P = .08). There was also a difference in the frequency of rare missense variants in BRIP1 between case patients and control patients (P = 5.5 x 10–4). The relative risks associated with BRIP1 mutations were 11.22 for invasive EOC (95% confidence interval [CI] = 3.22 to 34.10, P = 1 x 10–4) and 14.09 for high-grade serous disease (95% CI = 4.04 to 45.02, P = 2 x 10–5). Segregation analysis in families estimated the average relative risks in BRIP1 mutation carriers compared with the general population to be 3.41 (95% CI = 2.12 to 5.54, P = 7×10–7).
Conclusions:
Deleterious germline mutations in BRIP1 are associated with a moderate increase in EOC risk. These data have clinical implications for risk prediction and prevention approaches for ovarian cancer and emphasize the critical need for risk estimates based on very large sample sizes before genes of moderate penetrance have clinical utility in cancer prevention.
doi:10.1093/jnci/djv214
PMCID: PMC4643629  PMID: 26315354
4.  Decreased serum thrombospondin-1 levels in pancreatic cancer patients up to 24 months prior to clinical diagnosis: association with diabetes mellitus 
Purpose
Identification of serum biomarkers enabling earlier diagnosis of pancreatic ductal adenocarcinoma (PDAC) could improve outcome. Serum protein profiles in patients with pre-clinical disease and at diagnosis were investigated.
Experimental Design
Serum from cases up to 4 years prior to PDAC diagnosis and controls (UKCTOCS,n=174) were studied, alongside samples from patients diagnosed with PDAC, chronic pancreatitis, benign biliary disease, type 2 diabetes mellitus and healthy subjects (n=298). iTRAQ enabled comparisons of pooled serum from a test set (n=150). Validation was undertaken using MRM and/or western blotting in all 472 human samples and samples from a KPC mouse model.
Results
iTRAQ identified thrombospondin-1 (TSP-1) as reduced preclinically and in diagnosed samples. MRM confirmed significant reduction in levels of TSP-1 up to 24 months prior to diagnosis. A combination of TSP-1 and CA19-9 gave an AUC of 0.86, significantly outperforming both markers alone (0.69 & 0.77 respectively; P<0.01). TSP-1 was also decreased in PDAC patients compared to healthy controls (P<0.05) and patients with benign biliary obstruction (P<0.01). Low levels of TSP-1 correlated with poorer survival, pre-clinically (P<0.05) and at clinical diagnosis (P<0.02). In PDAC patients, reduced TSP-1 levels were more frequently observed in those with confirmed diabetes mellitus (P<0.01). Significantly lower levels were also observed in PDAC patients with diabetes compared to individuals with type 2 DM (P=0.01).
Conclusions
Circulating TSP-1 levels decrease up to 24 months prior to diagnosis of PDAC and significantly enhance the diagnostic performance of CA19-9. The influence of diabetes mellitus on biomarker behaviour should be considered in future studies.
doi:10.1158/1078-0432.CCR-15-0879
PMCID: PMC4820087  PMID: 26573598
PDAC; Serum; TSP-1; Biomarker; Diabetes
5.  HOTAIR and its surrogate DNA methylation signature indicate carboplatin resistance in ovarian cancer 
Genome Medicine  2015;7:108.
Background
Understanding carboplatin resistance in ovarian cancer is critical for the improvement of patients’ lives. Multipotent mesenchymal stem cells or an aggravated epithelial to mesenchymal transition phenotype of a cancer are integrally involved in pathways conferring chemo-resistance. Long non-coding RNA HOTAIR (HOX transcript antisense intergenic RNA) is involved in mesenchymal stem cell fate and cancer biology.
Methods
We analyzed HOTAIR expression and associated surrogate DNA methylation (DNAme) in 134 primary ovarian cancer cases (63 received carboplatin, 55 received cisplatin and 16 no chemotherapy). We validated our findings by HOTAIR expression and DNAme analysis in a multicentre setting of five additional sets, encompassing 946 ovarian cancers. Chemo-sensitivity has been assessed in cell culture experiments.
Results
HOTAIR expression was significantly associated with poor survival in carboplatin-treated patients with adjusted hazard ratios for death of 3.64 (95 % confidence interval [CI] 1.78–7.42; P < 0.001) in the discovery and 1.63 (95 % CI 1.04–2.56; P = 0.032) in the validation set. This effect was not seen in patients who did not receive carboplatin (0.97 [95 % CI 0.52–1.80; P = 0.932]). HOTAIR expression or its surrogate DNAme signature predicted poor outcome in all additional sets of carboplatin-treated ovarian cancer patients while HOTAIR expressors responded preferentially to cisplatin (multivariate interaction P = 0.008).
Conclusions
Non-coding RNA HOTAIR or its more stable DNAme surrogate may indicate the presence of a subset of cells which confer resistance to carboplatin and can serve as (1) a marker to personalise treatment and (2) a novel target to overcome carboplatin resistance.
Electronic supplementary material
The online version of this article (doi:10.1186/s13073-015-0233-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s13073-015-0233-4
PMCID: PMC4619324  PMID: 26497652
6.  Results of Annual Screening in Phase I of the United Kingdom Familial Ovarian Cancer Screening Study Highlight the Need for Strict Adherence to Screening Schedule 
Journal of Clinical Oncology  2012;31(1):49-57.
Purpose
To establish the performance characteristics of annual transvaginal ultrasound and serum CA125 screening for women at high risk of ovarian/fallopian tube cancer (OC/FTC) and to investigate the impact of delayed screening interval and surgical intervention.
Patients and Methods
Between May 6, 2002, and January 5, 2008, 3,563 women at an estimated ≥ 10% lifetime risk of OC/FTC were recruited and screened by 37 centers in the United Kingdom. Participants were observed prospectively by centers, questionnaire, and national cancer registries.
Results
Sensitivity for detection of incident OC/FTC at 1 year after last annual screen was 81.3% (95% CI, 54.3% to 96.0%) if occult cancers were classified as false negatives and 87.5% (95% CI, 61.7% to 98.5%) if they were classified as true positives. Positive and negative predictive values of incident screening were 25.5% (95% CI, 14.3 to 40.0) and 99.9% (95% CI, 99.8 to 100) respectively. Four (30.8%) of 13 incident screen-detected OC/FTCs were stage I or II. Compared with women screened in the year before diagnosis, those not screened in the year before diagnosis were more likely to have ≥ stage IIIc disease (85.7% v 26.1%; P = .009). Screening interval was delayed by a median of 88 days before detection of incident OC/FTC. Median interval from detection screen to surgical intervention was 79 days in prevalent and incident OC/FTC.
Conclusion
These results in the high-risk population highlight the need for strict adherence to screening schedule. Screening more frequently than annually with prompt surgical intervention seems to offer a better chance of early-stage detection.
doi:10.1200/JCO.2011.39.7638
PMCID: PMC3530690  PMID: 23213100
7.  Osteoprotegerin (OPG), The Endogenous Inhibitor of Receptor Activator of NF-κB Ligand (RANKL), is Dysregulated in BRCA Mutation Carriers 
EBioMedicine  2015;2(10):1331-1339.
Breast cancer development in BRCA1/2 mutation carriers is a net consequence of cell-autonomous and cell nonautonomous factors which may serve as excellent targets for cancer prevention. In light of our previous data we sought to investigate the consequences of the BRCA-mutation carrier state on RANKL/osteoprotegerin (OPG) signalling.
We analysed serum levels of RANKL, OPG, RANKL/OPG complex, oestradiol (E2), and progesterone (P) during menstrual cycle progression in 391 BRCA1/2-mutation carriers and 782 noncarriers. These studies were complemented by analyses of RANKL and OPG in the serum and mammary tissues of female cynomolgus macaques (n = 88) and serum RANKL and OPG in postmenopausal women (n = 150).
BRCA-mutation carriers had lower mean values of free serum OPG in particular in BRCA1-mutation carriers (p = 0.018) compared with controls. Among BRCA1/2 mutation carriers, lower OPG levels were associated with germline mutation locations known to confer an increased breast cancer risk (p = 0.003). P is associated with low OPG levels in serum and tissue, particularly in BRCA-mutation carriers (rho = − 0.216; p = 0.002). Serum OPG levels were inversely correlated (rho = − 0.545, p < 0.001) with mammary epithelial proliferation measured by Ki67 expression and increased (p = 0.01) in postmenopause.
The P–RANKL/OPG system is dysregulated in BRCA-mutation carriers. These and previously published data provide a strong rationale for further investigation of antiprogestogens or an anti-RANKL antibody such as denosumab for breast cancer prevention.
Highlights
•Osteoprotegerin (OPG) is the endogenous inhibitor of Receptor Activator of NF-κB Ligand (RANKL)•RANKL has been shown to be crucially important in progesterone-mediated breast carcinogenesis•Serum OPG is regulated by progesterone and low in BRCA1/2 mutation carriers•Low serum OPG is associated with increased proliferation in the mammary gland•Antiprogestogens or anti-RANKL antibody (denosumab) may be new strategies for breast cancer prevention in BRCA1/2 carriers.
Preventing deadly cancers is a high priority of 21st century medicine. To find the most promising cancer causing targets which can be modulated using chemo-preventive drugs remains the biggest challenge. In this paper we show that OPG, which is the physiological antagonist of RANKL (a factor known to be crucially involved in breast carcinogenesis), is low in women who have inherited a gene mutation (i.e. in BRCA1 or 2) which puts them at extremely high risk of developing breast cancer. An antibody which mimics OPG might be a very attractive option in preventing breast cancer.
doi:10.1016/j.ebiom.2015.08.037
PMCID: PMC4634624  PMID: 26629528
Breast cancer; BRCA1 and BRCA2 mutations; RANKL; OPG; Carcinogenesis; Cancer prevention
8.  Serum CA19-9 is significantly up-regulated up to 2 years prior to diagnosis with pancreatic cancer: implications for early disease detection 
Purpose
Biomarkers for the early detection of pancreatic cancer are urgently needed. The primary objective of this study was to evaluate whether increased levels of serum CA19-9, CA125, CEACAM1 and REG3A are present prior to clinical presentation of pancreatic cancer and to assess the performance of combined markers for early detection and prognosis.
Experimental Design
This nested case control study within UKCTOCS included 118 single- and 143 serial-serum samples from 154 post-menopausal women who were subsequently diagnosed with pancreatic cancer and 304 matched non-cancer controls. Samples were split randomly into independent training and test sets. CA19-9, CA125, CEACAM1 and REG3A were measured using ELISA and/or CLIA. Performance of markers to detect cancers at different times prior to diagnosis and for prognosis was evaluated.
Results
At 95% specificity, CA19-9 (>37 U/mL) had a sensitivity of 68% up to 1 year, and 53% up to 2 yrs before diagnosis. Combining CA19-9 and CA125 improved sensitivity as CA125 was elevated (>30 U/mL) in ~20% of CA19-9-negative cases. CEACAM1 and REG3A were late markers adding little in combined models. Average lead times of 20-23 months were estimated for test-positive cases. Pre-diagnostic levels of CA19-9 and CA125 were associated with poor overall survival (HR 2.69 and 3.15, respectively).
Conclusions
CA19-9 and CA125 have encouraging sensitivity for detecting pre-clinical pancreatic cancer and both markers can be used as prognostic tools. This work challenges the prevailing view that CA19-9 is up-regulated late in the course of pancreatic cancer development.
doi:10.1158/1078-0432.CCR-14-0365
PMCID: PMC4181906  PMID: 24938522
pancreatic cancer; preclinical serum biomarkers; UKCTOCS; CA19-9; CA125; CEACAM1; REG3A
9.  Serum CA19-9 is significantly up-regulated up to 2 years prior to diagnosis with pancreatic cancer: implications for early disease detection 
Purpose
Biomarkers for the early detection of pancreatic cancer are urgently needed. The primary objective of this study was to evaluate whether increased levels of serum CA19-9, CA125, CEACAM1 and REG3A are present prior to clinical presentation of pancreatic cancer and to assess the performance of combined markers for early detection and prognosis.
Experimental Design
This nested case control study within UKCTOCS included 118 single- and 143 serial-serum samples from 154 post-menopausal women who were subsequently diagnosed with pancreatic cancer and 304 matched non-cancer controls. Samples were split randomly into independent training and test sets. CA19-9, CA125, CEACAM1 and REG3A were measured using ELISA and/or CLIA. Performance of markers to detect cancers at different times prior to diagnosis and for prognosis was evaluated.
Results
At 95% specificity, CA19-9 (>37 U/mL) had a sensitivity of 68% up to 1 year, and 53% up to 2 yrs before diagnosis. Combining CA19-9 and CA125 improved sensitivity as CA125 was elevated (>30 U/mL) in ~20% of CA19-9-negative cases. CEACAM1 and REG3A were late markers adding little in combined models. Average lead times of 20–23 months were estimated for test-positive cases. Pre-diagnostic levels of CA19-9 and CA125 were associated with poor overall survival (HR 2.69 and 3.15, respectively).
Conclusions
CA19-9 and CA125 have encouraging sensitivity for detecting pre-clinical pancreatic cancer and both markers can be used as prognostic tools. This work challenges the prevailing view that CA19-9 is up-regulated late in the course of pancreatic cancer development.
doi:10.1158/1078-0432.CCR-14-0365
PMCID: PMC4181906  PMID: 24938522
pancreatic cancer; preclinical serum biomarkers; UKCTOCS; CA19-9; CA125; CEACAM1; REG3A
10.  Common alleles in candidate susceptibility genes associated with risk and development of epithelial ovarian cancer 
Common germline genetic variation in the population is associated with susceptibility to epithelial ovarian cancer. Microcell-mediated chromosome transfer and expression microarray analysis identified nine genes associated with functional suppression of tumorogenicity in ovarian cancer cell lines; AIFM2, AKTIP, AXIN2, CASP5, FILIP1L, RBBP8, RGC32, RUVBL1 and STAG3. Sixty-three tagging single nucleotide polymorphisms (tSNPs) in these genes were genotyped in 1,799 invasive ovarian cancer cases and 3,045 controls to look for associations with disease risk. Two SNPs in RUVBL1, rs13063604 and rs7650365, were associated with increased risk of serous ovarian cancer [HetOR = 1.42 (1.15–1.74) and the HomOR = 1.63 (1.10–1.42), p-trend = 0.0002] and [HetOR = 0.97 (0.80–1.17), HomOR = 0.74 (0.58–0.93), p-trend = 0.009], respectively. We genotyped rs13063604 and rs7650365 in an additional 4,590 cases and 6,031 controls from ten sites from the United States, Europe and Australia; however, neither SNP was significant in Stage 2. We also evaluated the potential role of tSNPs in these nine genes in ovarian cancer development by testing for allele-specific loss of heterozygosity (LOH) in 286 primary ovarian tumours. We found frequent LOH for tSNPs in AXIN2, AKTIP and RGC32 (64, 46 and 34%, respectively) and one SNP, rs1637001, in STAG3 showed significant allele-specific LOH with loss of the common allele in 94% of informative tumours (p = 0.015). Array comparative genomic hybridisation indicated that this nonrandom allelic imbalance was due to amplification of the rare allele. In conclusion, we show evidence for the involvement of a common allele of STAG3 in the development of epithelial ovarian cancer.
doi:10.1002/ijc.25554
PMCID: PMC3098608  PMID: 20635389
risk of ovarian cancer; polymorphism; association studies
11.  Genetic and cellular mechanisms of the formation of Esophageal Atresia and Tracheoesophageal Fistula 
Foregut separation involves dynamic changes in the activities of signaling pathways and transcription factors. Recent mouse genetic studies demonstrate that some of these pathways interact with each other to form a complex network, leading to a unique dorsal-ventral patterning in the early foregut. In this review we will discuss how this unique dorsal-ventral patterning is set prior to the foregut separation and how disruption of this patterning affects the separation process. We will further discuss the roles of downstream targets of these pathways in regulating separation at cellular and molecular levels. Understanding the mechanism of normal separation process will provide us insights into the pathobiology of a relatively common birth defect Esophageal Atresia (EA) with/without Tracheo-esophageal Fistula (TEF).
doi:10.1111/dote.12055
PMCID: PMC4321969  PMID: 23679023
foregut separation; EA/TEF; Sox2; Bmp
12.  A BRCA1-mutation associated DNA methylation signature in blood cells predicts sporadic breast cancer incidence and survival 
Genome Medicine  2014;6(6):47.
Background
BRCA1 mutation carriers have an 85% risk of developing breast cancer but the risk of developing non-hereditary breast cancer is difficult to assess. Our objective is to test whether a DNA methylation (DNAme) signature derived from BRCA1 mutation carriers is able to predict non-hereditary breast cancer.
Methods
In a case/control setting (72 BRCA1 mutation carriers and 72 BRCA1/2 wild type controls) blood cell DNA samples were profiled on the Illumina 27 k methylation array. Using the Elastic Net classification algorithm, a BRCA1-mutation DNAme signature was derived and tested in two cohorts: (1) The NSHD (19 breast cancers developed within 12 years after sample donation and 77 controls) and (2) the UKCTOCS trial (119 oestrogen receptor positive breast cancers developed within 5 years after sample donation and 122 controls).
Results
We found that our blood-based BRCA1-mutation DNAme signature applied to blood cell DNA from women in the NSHD resulted in a receiver operating characteristics (ROC) area under the curve (AUC) of 0.65 (95% CI 0.51 to 0.78, P = 0.02) which did not validate in buccal cells from the same individuals. Applying the signature in blood DNA from UKCTOCS volunteers resulted in AUC of 0.57 (95% CI 0.50 to 0.64; P = 0.03) and is independent of family history or any other known risk factors. Importantly the BRCA1-mutation DNAme signature was able to predict breast cancer mortality (AUC = 0.67; 95% CI 0.51 to 0.83; P = 0.02). We also found that the 1,074 CpGs which are hypermethylated in BRCA1 mutation carriers are significantly enriched for stem cell polycomb group target genes (P <10-20).
Conclusions
A DNAme signature derived from BRCA1 carriers is able to predict breast cancer risk and death years in advance of diagnosis. Future studies may need to focus on DNAme profiles in epithelial cells in order to reach the AUC thresholds required of preventative measures or early detection strategies.
doi:10.1186/gm567
PMCID: PMC4110671  PMID: 25067956
13.  Role of DNA Methylation and Epigenetic Silencing of HAND2 in Endometrial Cancer Development 
PLoS Medicine  2013;10(11):e1001551.
TB filled in by Laureen
Please see later in the article for the Editors' Summary
Background
Endometrial cancer incidence is continuing to rise in the wake of the current ageing and obesity epidemics. Much of the risk for endometrial cancer development is influenced by the environment and lifestyle. Accumulating evidence suggests that the epigenome serves as the interface between the genome and the environment and that hypermethylation of stem cell polycomb group target genes is an epigenetic hallmark of cancer. The objective of this study was to determine the functional role of epigenetic factors in endometrial cancer development.
Methods and Findings
Epigenome-wide methylation analysis of >27,000 CpG sites in endometrial cancer tissue samples (n = 64) and control samples (n = 23) revealed that HAND2 (a gene encoding a transcription factor expressed in the endometrial stroma) is one of the most commonly hypermethylated and silenced genes in endometrial cancer. A novel integrative epigenome-transcriptome-interactome analysis further revealed that HAND2 is the hub of the most highly ranked differential methylation hotspot in endometrial cancer. These findings were validated using candidate gene methylation analysis in multiple clinical sample sets of tissue samples from a total of 272 additional women. Increased HAND2 methylation was a feature of premalignant endometrial lesions and was seen to parallel a decrease in RNA and protein levels. Furthermore, women with high endometrial HAND2 methylation in their premalignant lesions were less likely to respond to progesterone treatment. HAND2 methylation analysis of endometrial secretions collected using high vaginal swabs taken from women with postmenopausal bleeding specifically identified those patients with early stage endometrial cancer with both high sensitivity and high specificity (receiver operating characteristics area under the curve = 0.91 for stage 1A and 0.97 for higher than stage 1A). Finally, mice harbouring a Hand2 knock-out specifically in their endometrium were shown to develop precancerous endometrial lesions with increasing age, and these lesions also demonstrated a lack of PTEN expression.
Conclusions
HAND2 methylation is a common and crucial molecular alteration in endometrial cancer that could potentially be employed as a biomarker for early detection of endometrial cancer and as a predictor of treatment response. The true clinical utility of HAND2 DNA methylation, however, requires further validation in prospective studies.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Cancer, which is responsible for 13% of global deaths, can develop anywhere in the body, but all cancers are characterized by uncontrolled cell growth and reduced cellular differentiation (the process by which unspecialized cells such as “stem” cells become specialized during development, tissue repair, and normal cell turnover). Genetic alterations—changes in the sequence of nucleotides (DNA's building blocks) in specific genes—are required for this cellular transformation and subsequent cancer development (carcinogenesis). However, recent evidence suggests that epigenetic modifications—reversible, heritable changes in gene function that occur in the absence of nucleotide sequence changes—may also be involved in carcinogenesis. For example, the addition of methyl groups to a set of genes called stem cell polycomb group target genes (PCGTs; polycomb genes control the expression of their target genes by modifying their DNA or associated proteins) is one of the earliest molecular changes in human cancer development, and increasing evidence suggests that hypermethylation of PCGTs is an epigenetic hallmark of cancer.
Why Was This Study Done?
The methylation of PCGTs, which is triggered by age and by environmental factors that are associated with cancer development, reduces cellular differentiation and leads to the accumulation of undifferentiated cells that are susceptible to cancer development. It is unclear, however, whether epigenetic modifications have a causal role in carcinogenesis. Here, the researchers investigate the involvement of epigenetic factors in the development of endometrial (womb) cancer. The risk of endometrial cancer (which affects nearly 50,000 women annually in the United States) is largely determined by environmental and lifestyle factors. Specifically, the risk of this cancer is increased in women in whom estrogen (a hormone that drives cell proliferation in the endometrium) is functionally dominant over progesterone (a hormone that inhibits endometrial proliferation and causes cell differentiation); obese women and women who have taken estrogen-only hormone replacement therapies fall into this category. Thus, endometrial cancer is an ideal model in which to study whether epigenetic mechanisms underlie carcinogenesis.
What Did the Researchers Do and Find?
The researchers collected data on genome-wide DNA methylation at cytosine- and guanine-rich sites in endometrial cancers and normal endometrium and integrated this information with the human interactome and transcriptome (all the physical interactions between proteins and all the genes expressed, respectively, in a cell) using an algorithm called Functional Epigenetic Modules (FEM). This analysis identified HAND2 as the hub of the most highly ranked differential methylation hotspot in endometrial cancer. HAND2 is a progesterone-regulated stem cell PCGT. It encodes a transcription factor that is expressed in the endometrial stroma (the connective tissue that lies below the epithelial cells in which most endometrial cancers develop) and that suppresses the production of the growth factors that mediate the growth-inducing effects of estrogen on the endometrial epithelium. The researchers hypothesized, therefore, that epigenetic deregulation of HAND2 could be a key step in endometrial cancer development. In support of this hypothesis, the researchers report that HAND2 methylation was increased in premalignant endometrial lesions (cancer-prone, abnormal-looking tissue) compared to normal endometrium, and was associated with suppression of HAND2 expression. Moreover, a high level of endometrial HAND2 methylation in premalignant lesions predicted a poor response to progesterone treatment (which stops the growth of some endometrial cancers), and analysis of HAND2 methylation in endometrial secretions collected from women with postmenopausal bleeding (a symptom of endometrial cancer) accurately identified individuals with early stage endometrial cancer. Finally, mice in which the Hand2 gene was specifically deleted in the endometrium developed precancerous endometrial lesions with age.
What Do These Findings Mean?
These and other findings identify HAND2 methylation as a common, key molecular alteration in endometrial cancer. These findings need to be confirmed in more women, and studies are needed to determine the immediate molecular and cellular consequences of HAND2 silencing in endometrial stromal cells. Nevertheless, these results suggest that HAND2 methylation could potentially be used as a biomarker for the early detection of endometrial cancer and for predicting treatment response. More generally, these findings support the idea that methylation of HAND2 (and, by extension, the methylation of other PCGTs) is not a passive epigenetic feature of cancer but is functionally involved in cancer development, and provide a framework for identifying other genes that are epigenetically regulated and functionally important in carcinogenesis.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001551
The US National Cancer Institute provides information on all aspects of cancer and has detailed information about endometrial cancer for patients and professionals (in English and Spanish)
The not-for-profit organization American Cancer Society provides information on cancer and how it develops and specific information on endometrial cancer (in several languages)
The UK National Health Service Choices website includes an introduction to cancer, a page on endometrial cancer, and a personal story about endometrial cancer
The not-for-profit organization Cancer Research UK provides general information about cancer and specific information about endometrial cancer
Wikipedia has a page on cancer epigenetics (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
The Eve Appeal charity that supported this research provides useful information on gynecological cancers
doi:10.1371/journal.pmed.1001551
PMCID: PMC3825654  PMID: 24265601
14.  In vitro three-dimensional modeling of fallopian tube secretory epithelial cells 
BMC Cell Biology  2013;14:43.
Background
Fallopian tube secretory epithelial cells (FTSECs) have been implicated as a cell-of-origin for high-grade serous epithelial ovarian cancer. However, there are relatively few in vitro models of this tissue type available for use in studies of FTSEC biology and malignant transformation. In vitro three-dimensional (3D) cell culture models aim to recreate the architecture and geometry of tissues in vivo and restore the complex network of cell-cell/cell-matrix interactions that occur throughout the surface of the cell membrane.
Results
We have established and characterized 3D spheroid culture models of primary FTSECs. FTSEC spheroids contain central cores of hyaline matrix surrounded by mono- or multi-layer epithelial sheets. We found that 3D culturing alters the molecular characteristics of FTSECs compared to 2D cultures of the same cells. Gene expression profiling identified more than a thousand differentially expressed genes between 3D and 2D cultures of the same FTSEC lines. Pathways significantly under-represented in 3D FTSEC cultures were associated with cell cycle progression and DNA replication. This was also reflected in the reduced proliferative indices observed in 3D spheroids stained for the proliferation marker MIB1. Comparisons with gene expression profiles of fresh fallopian tube tissues revealed that 2D FTSEC cultures clustered with follicular phase tubal epithelium, whereas 3D FTSEC cultures clustered with luteal phase samples.
Conclusions
This 3D model of fallopian tube secretory epithelial cells will advance our ability to study the underlying biology and etiology of fallopian tube tissues and the pathogenesis of high-grade serous epithelial ovarian cancer.
doi:10.1186/1471-2121-14-43
PMCID: PMC3849984  PMID: 24070420
Fallopian tube secretory epithelial cells; Gene expression microarray; Three-dimensional in vitro models; Tissue microenvironment; Ovarian cancer
15.  Genetic and Cellular Mechanisms Regulating Anterior Foregut and Esophageal Development 
Developmental biology  2012;369(1):54-64.
Separation of the single anterior foregut tube into the esophagus and trachea involves cell proliferation and differentiation, as well as dynamic changes in cell-cell adhesion and migration. These biological processes are regulated and coordinated at multiple levels through the interplay of the epithelium and mesenchyme. Genetic studies and in vitro modeling have shed light on relevant regulatory networks that include a number of transcription factors and signaling pathways. These signaling molecules exhibit unique expression patterns and play specific functions in their respective territories before the separation process occurs. Disruption of regulatory networks inevitably leads to defective separation and malformation of the trachea and esophagus and results in the formation of a relatively common birth defect, esophageal atresia with or without tracheoesophageal fistula (EA/TEF). Significantly, some of the signaling pathways and transcription factors involved in anterior foregut separation continue to play important roles in the morphogenesis of the individual organs. In this review, we will focus on new findings related to these different developmental processes and discuss them in the context of developmental disorders (or birth defects) commonly seen in clinics.
doi:10.1016/j.ydbio.2012.06.016
PMCID: PMC3409292  PMID: 22750256
EA/TEF; Sox2; BMP; Esophageal Muscle; Myenteric System
17.  The Dynamics and Prognostic Potential of DNA Methylation Changes at Stem Cell Gene Loci in Women's Cancer 
PLoS Genetics  2012;8(2):e1002517.
Aberrant DNA methylation is an important cancer hallmark, yet the dynamics of DNA methylation changes in human carcinogenesis remain largely unexplored. Moreover, the role of DNA methylation for prediction of clinical outcome is still uncertain and confined to specific cancers. Here we perform the most comprehensive study of DNA methylation changes throughout human carcinogenesis, analysing 27,578 CpGs in each of 1,475 samples, ranging from normal cells in advance of non-invasive neoplastic transformation to non-invasive and invasive cancers and metastatic tissue. We demonstrate that hypermethylation at stem cell PolyComb Group Target genes (PCGTs) occurs in cytologically normal cells three years in advance of the first morphological neoplastic changes, while hypomethylation occurs preferentially at CpGs which are heavily Methylated in Embryonic Stem Cells (MESCs) and increases significantly with cancer invasion in both the epithelial and stromal tumour compartments. In contrast to PCGT hypermethylation, MESC hypomethylation progresses significantly from primary to metastatic cancer and defines a poor prognostic signature in four different gynaecological cancers. Finally, we associate expression of TET enzymes, which are involved in active DNA demethylation, to MESC hypomethylation in cancer. These findings have major implications for cancer and embryonic stem cell biology and establish the importance of systemic DNA hypomethylation for predicting prognosis in a wide range of different cancers.
Author Summary
DNA methylation is an important chemical modification of DNA that can affect and regulate the activity of genes in human tissue. Abnormal DNA methylation and its subsequent effects on gene activity are a hallmark of cancer, yet when precisely these DNA methylation changes occur and how they contribute to the development of cancer remains largely unexplored. In this work we measure the methylation state of DNA at over 14,000 genes in over 1,475 samples, including normal and benign cells, invasive cancers, and metastatic cancer tissue. Using cervical cancer as a model, we show that gain of abnormal methylation at genes typically un-methylated in stem cells can be detected up to 3 years in advance of the appearance of pre-cancerous cells, while those genes typically methylated in stem cells lose this methylation progressively throughout cancer development. Furthermore, we discover that this process of methylation loss during cancer progression is a marker of poor disease outcome common to all four major women-specific cancers: breast, ovarian, endometrial, and cervical cancers. Finally we demonstrate the relationship between loss of methylation and cancer-specific over-production of a specific protein known to play an active role in removing methylation from DNA. Taken together these findings highlight the complex nature of DNA methylation dynamics in cancer development as well as their potential exploitation for clinical gain.
doi:10.1371/journal.pgen.1002517
PMCID: PMC3276553  PMID: 22346766
18.  Microcell-Mediated Chromosome Transfer Identifies EPB41L3 as a Functional Suppressor of Epithelial Ovarian Cancers12 
Neoplasia (New York, N.Y.)  2010;12(7):579-589.
We used a functional complementation approach to identify tumor-suppressor genes and putative therapeutic targets for ovarian cancer. Microcell-mediated transfer of chromosome 18 in the ovarian cancer cell line TOV21G induced in vitro and in vivo neoplastic suppression. Gene expression microarray profiling in TOV21G+18 hybrids identified 14 candidate genes on chromosome 18 that were significantly overexpressed and therefore associated with neoplastic suppression. Further analysis of messenger RNA and protein expression for these genes in additional ovarian cancer cell lines indicated that EPB41L3 (erythrocyte membrane protein band 4.1-like 3, alternative names DAL-1 and 4.1B) was a candidate ovarian cancer-suppressor gene. Immunoblot analysis showed that EPB41L3 was activated in TOV21G+18 hybrids, expressed in normal ovarian epithelial cell lines, but was absent in 15 (78%) of 19 ovarian cancer cell lines. Using immunohistochemistry, 66% of 794 invasive ovarian tumors showed no EPB41L3 expression compared with only 24% of benign ovarian tumors and 0% of normal ovarian epithelial tissues. EPB41L3 was extensively methylated in ovarian cancer cell lines and primary ovarian tumors compared with normal tissues (P = .00004), suggesting this may be the mechanism of gene inactivation in ovarian cancers. Constitutive reexpression of EPB41L3 in a three-dimensional multicellular spheroid model of ovarian cancer caused significant growth suppression and induced apoptosis. Transmission and scanning electron microscopy demonstrated many similarities between EPB41L3-expressing cells and chromosome 18 donor-recipient hybrids, suggesting that EPB41L3 is the gene responsible for neoplastic suppression after chromosome 18 transfer. Finally, an inducible model of EPB41L3 expression in three-dimensional spheroids confirmed that reexpression of EPB41L3 induces extensive apoptotic cell death in ovarian cancers.
PMCID: PMC2907584  PMID: 20651987
19.  Association between invasive ovarian cancer susceptibility and 11 best candidate SNPs from breast cancer genome-wide association study 
Human Molecular Genetics  2009;18(12):2297-2304.
Because both ovarian and breast cancer are hormone-related and are known to have some predisposition genes in common, we evaluated 11 of the most significant hits (six with confirmed associations with breast cancer) from the breast cancer genome-wide association study for association with invasive ovarian cancer. Eleven SNPs were initially genotyped in 2927 invasive ovarian cancer cases and 4143 controls from six ovarian cancer case–control studies. Genotype frequencies in cases and controls were compared using a likelihood ratio test in a logistic regression model stratified by study. Initially, three SNPs (rs2107425 in MRPL23, rs7313833 in PTHLH, rs3803662 in TNRC9) were weakly associated with ovarian cancer risk and one SNP (rs4954956 in NXPH2) was associated with serous ovarian cancer in non-Hispanic white subjects (P-trend < 0.1). These four SNPs were then genotyped in an additional 4060 cases and 6308 controls from eight independent studies. Only rs4954956 was significantly associated with ovarian cancer risk both in the replication study and in combined analyses. This association was stronger for the serous histological subtype [per minor allele odds ratio (OR) 1.07 95% CI 1.01–1.13, P-trend = 0.02 for all types of ovarian cancer and OR 1.14 95% CI 1.07–1.22, P-trend = 0.00017 for serous ovarian cancer]. In conclusion, we found that rs4954956 was associated with increased ovarian cancer risk, particularly for serous ovarian cancer. However, none of the six confirmed breast cancer susceptibility variants we tested was associated with ovarian cancer risk. Further work will be needed to identify the causal variant associated with rs4954956 or elucidate its function.
doi:10.1093/hmg/ddp138
PMCID: PMC2685754  PMID: 19304784
20.  Senescent Fibroblasts Promote Neoplastic Transformation of Partially Transformed Ovarian Epithelial Cells in a Three-dimensional Model of Early Stage Ovarian Cancer12 
Neoplasia (New York, N.Y.)  2010;12(4):317-325.
Most epithelial ovarian cancers are diagnosed postmenopausally, although the well-established epidemiological risk factors (parity, oral contraceptive use) are premenopausal. We hypothesized that accumulation of senescent fibroblasts, together with concomitant loss of presenescent fibroblasts within the ovarian cortex, promotes initiation and early development of ovarian cancer from ovarian surface epithelial (OSE) cells. To test this, we established immortalized OSE (IOSE) cell lines that mimic early neoplastic transformation by overexpressing the CMYC oncogene (IOSECMYC) and normal ovarian presenescent (PSN) and senescent (SEN) fibroblast cell lines. We then evaluated the ability of PSN and SEN fibroblasts to transform IOSE and IOSECMYC after coculture. SEN fibroblasts significantly enhanced neoplastic development of IOSECMYC cells; there was an up to 15-fold increase in migration of IOSECMYC cells cocultured with SEN fibroblasts compared with PSN fibroblasts. Conditioned medium from SEN fibroblasts promoted anchorage-independent growth of IOSECMYC cells. We studied fibroblast-epithelial cell interactions in heterotypic three-dimensional spheroid models. Dual immunohistochemical staining of spheroids for a proliferation marker (MIB-1) and cytokeratin-18 indicated that SEN fibroblasts induce approximately a five-fold increase in proliferation of IOSECMYC cells relative to cocultures with PSN fibroblasts. SEN, but not PSN fibroblasts, also induced nuclear atypia in epithelial cells in three-dimensional spheroids. These data suggest for the first time that the accumulation of senescent, or loss of presenescent fibroblasts, can promote neoplastic development of partially transformed OSE cells in vitro and illustrates the power of using three-dimensional heterotypic modeling to gain better insights into the etiology underlying the development of epithelial ovarian cancer.
PMCID: PMC2847739  PMID: 20360942
21.  A Genome-Wide Association Study Identifies A New Ovarian Cancer Susceptibility Locus On 9p22.2 
Song, Honglin | Ramus, Susan J. | Tyrer, Jonathan | Bolton, Kelly L. | Gentry-Maharaj, Aleksandra | Wozniak, Eva | Anton-Culver, Hoda | Chang-Claude, Jenny | Cramer, Daniel W. | DiCioccio, Richard | Dörk, Thilo | Goode, Ellen L. | Goodman, Marc T | Schildkraut, Joellen M | Sellers, Thomas | Baglietto, Laura | Beckmann, Matthias W. | Beesley, Jonathan | Blaakaer, Jan | Carney, Michael E | Chanock, Stephen | Chen, Zhihua | Cunningham, Julie M. | Dicks, Ed | Doherty, Jennifer A. | Dürst, Matthias | Ekici, Arif B. | Fenstermacher, David | Fridley, Brooke L. | Giles, Graham | Gore, Martin E. | De Vivo, Immaculata | Hillemanns, Peter | Hogdall, Claus | Hogdall, Estrid | Iversen, Edwin S | Jacobs, Ian J | Jakubowska, Anna | Li, Dong | Lissowska, Jolanta | Lubiński, Jan | Lurie, Galina | McGuire, Valerie | McLaughlin, John | Mędrek, Krzysztof | Moorman, Patricia G. | Moysich, Kirsten | Narod, Steven | Phelan, Catherine | Pye, Carole | Risch, Harvey | Runnebaum, Ingo B | Severi, Gianluca | Southey, Melissa | Stram, Daniel O. | Thiel, Falk C. | Terry, Kathryn L. | Tsai, Ya-Yu | Tworoger, Shelley S. | Van Den Berg, David J. | Vierkant, Robert A. | Wang-Gohrke, Shan | Webb, Penelope M. | Wilkens, Lynne R. | Wu, Anna H | Yang, Hannah | Brewster, Wendy | Ziogas, Argyrios | Houlston, Richard | Tomlinson, Ian | Whittemore, Alice S | Rossing, Mary Anne | Ponder, Bruce A.J. | Pearce, Celeste Leigh | Ness, Roberta B. | Menon, Usha | Kjaer, Susanne Krüger | Gronwald, Jacek | Garcia-Closas, Montserrat | Fasching, Peter A. | Easton, Douglas F | Chenevix-Trench, Georgia | Berchuck, Andrew | Pharoah, Paul D.P. | Gayther, Simon A.
Nature genetics  2009;41(9):996-1000.
Epithelial ovarian cancer has a major heritable component, but the known susceptibility genes explain less than half the excess familial risk1. We performed a genome wide association study (GWAS) to identify common ovarian cancer susceptibility alleles. We evaluated 507,094 SNPs genotyped in 1,817 cases and 2,353 controls from the UK and ~2 million imputed SNPs. We genotyped the 22,790 top ranked SNPs in 4,274 cases and 4,809 controls of European ancestry from Europe, USA and Australia. We identified 12 SNPs at 9p22 associated with disease risk (P<10−8). The most significant SNP (rs3814113; P = 2.5 × 10−17) was genotyped in a further 2,670 ovarian cancer cases and 4,668 controls confirming its association (combined data odds ratio = 0.82 95% CI 0.79 – 0.86, P-trend = 5.1 × 10−19). The association differs by histological subtype, being strongest for serous ovarian cancers (OR 0.77 95% CI 0.73 – 0.81, Ptrend = 4.1 × 10−21).
doi:10.1038/ng.424
PMCID: PMC2844110  PMID: 19648919
22.  An Epigenetic Signature in Peripheral Blood Predicts Active Ovarian Cancer 
PLoS ONE  2009;4(12):e8274.
Background
Recent studies have shown that DNA methylation (DNAm) markers in peripheral blood may hold promise as diagnostic or early detection/risk markers for epithelial cancers. However, to date no study has evaluated the diagnostic and predictive potential of such markers in a large case control cohort and on a genome-wide basis.
Principal Findings
By performing genome-wide DNAm profiling of a large ovarian cancer case control cohort, we here demonstrate that active ovarian cancer has a significant impact on the DNAm pattern in peripheral blood. Specifically, by measuring the methylation levels of over 27,000 CpGs in blood cells from 148 healthy individuals and 113 age-matched pre-treatment ovarian cancer cases, we derive a DNAm signature that can predict the presence of active ovarian cancer in blind test sets with an AUC of 0.8 (95% CI (0.74–0.87)). We further validate our findings in another independent set of 122 post-treatment cases (AUC = 0.76 (0.72–0.81)). In addition, we provide evidence for a significant number of candidate risk or early detection markers for ovarian cancer. Furthermore, by comparing the pattern of methylation with gene expression data from major blood cell types, we here demonstrate that age and cancer elicit common changes in the composition of peripheral blood, with a myeloid skewing that increases with age and which is further aggravated in the presence of ovarian cancer. Finally, we show that most cancer and age associated methylation variability is found at CpGs located outside of CpG islands.
Significance
Our results underscore the potential of DNAm profiling in peripheral blood as a tool for detection or risk-prediction of epithelial cancers, and warrants further in-depth and higher CpG coverage studies to further elucidate this role.
doi:10.1371/journal.pone.0008274
PMCID: PMC2793425  PMID: 20019873
23.  Association between invasive ovarian cancer susceptibility and 11 best candidate SNPs from breast cancer genome-wide association study 
Human molecular genetics  2009;18(12):2297-2304.
Because both ovarian and breast cancer are hormone-related and are known to have some predisposition genes in common, we evaluated 11 of the most significant hits (six with confirmed associations with breast cancer) from the breast cancer genome-wide association study for association with invasive ovarian cancer. Eleven SNPs were initially genotyped in 2927 invasive ovarian cancer cases and 4143 controls from six ovarian cancer case-control studies. Genotype frequencies in cases and controls were compared using a likelihood ratio test in a logistic regression model stratified by study. Initially, three SNPs (rs2107425 in MRPL23, rs7313833 in PTHLH, rs3803662 in TNRC9) were weakly associated with ovarian cancer risk and one SNP (rs4954956 in NXPH2) was associated with serous ovarian cancer in non-Hispanic white subjects (P-trend<0.1). These four SNPs were then genotyped in an additional 4060 cases and 6308 controls from eight independent studies. Only rs4954956 was significantly associated with ovarian cancer risk both in the replication study and in combined analyses. This association was stronger for the serous histological sub-type (per minor allele odds ratio (OR) 1.07 95%CI 1.01-1.13, P-trend=0.02 for all types of ovarian cancer and OR 1.14 95%CI 1.07-1.22, P-trend = 0.00017 for serous ovarian cancer). In conclusion, we found that rs4954956 was associated with increased ovarian cancer risk, particularly for serous ovarian cancer. However none of the six confirmed breast cancer susceptibility variants we tested were associated with ovarian cancer risk. Further work will be needed to identify the causal variant associated with rs4954956 or elucidate its function.
doi:10.1093/hmg/ddp138
PMCID: PMC2685754  PMID: 19304784
24.  COMBINING MULTIPLE SERUM TUMOR MARKERS IMPROVES DETECTION OF STAGE I EPITHELIAL OVARIAN CANCER 
Gynecologic oncology  2007;107(3):526-531.
Objective
Currently available tumor markers for ovarian cancer are still inadequate in both sensitivity and specificity to be used for population-based screening. Artificial neural network (ANN) as a modeling tool has demonstrated its ability to assimilate information from multiple sources and to detect subtle and complex patterns. In this paper, an ANN model was evaluated for its performance in detecting early stage epithelial ovarian cancer using multiple serum markers.
Methods
Serum specimens collected at four institutions in the US, the Netherlands, and the United Kingdom were analyzed for CA125II, CA72-4, CA15-3, and macrophage colony stimulating factor (M-CSF). The four tumor marker values were then used as inputs to an ANN derived using a training set from 100 apparently healthy women, 45 women with benign conditions arising from the ovary, and 55 invasive epithelial ovarian cancer patients (including 27 stage I/II cases). A separate validation set from 27 apparently healthy women, 56 women with benign conditions, and 35 women with various types of malignant pelvic masses was used to monitor the ANN’s performance during training. An independent test dataset from 98 apparently healthy women and 52 early stage epithelial ovarian cancer patients (38 stage I and 4 stage II invasive cases and 10 stage I borderline ovarian tumor cases) was used to evaluate the ANN.
Results
ROC analysis confirmed the overall superiority of the ANN-derived composite index over CA125II alone (p=0.0333). At a fixed specificity of 98%, the sensitivities for ANN and CA125II alone were 71% (37/52) and 46% (24/52) (p=0.047), respectively, for detecting early stage epithelial ovarian cancer, and 71% (30/42) and 43% (18/42) (p=0.040), respectively, for detecting invasive early stage epithelial ovarian cancer.
Conclusions
The combined use of multiple tumor markers through an ANN improves the overall accuracy to discern healthy women from patients with early stage ovarian cancer. Analysis of multiple markers with an ANN may be a better choice than the use of CA125II alone in a two-step approach for population screening in which a secondary test such as ultrasound is used to keep the overall specificity at an acceptable level.
doi:10.1016/j.ygyno.2007.08.009
PMCID: PMC2171045  PMID: 17920110
Ovarian cancer; tumor marker; early detection; multivariate model; artificial neural network
25.  Epigenotyping in Peripheral Blood Cell DNA and Breast Cancer Risk: A Proof of Principle Study 
PLoS ONE  2008;3(7):e2656.
Background
Epigenetic changes are emerging as one of the most important events in carcinogenesis. Two alterations in the pattern of DNA methylation in breast cancer (BC) have been previously reported; active estrogen receptor-α (ER-α) is associated with decreased methylation of ER-α target (ERT) genes, and polycomb group target (PCGT) genes are more likely than other genes to have promoter DNA hypermethylation in cancer. However, whether DNA methylation in normal unrelated cells is associated with BC risk and whether these imprints can be related to factors which can be modified by the environment, is unclear.
Methodology/Principal Findings
Using quantitative methylation analysis in a case-control study (n = 1,083) we found that DNA methylation of peripheral blood cell DNA provides good prediction of BC risk. We also report that invasive ductal and invasive lobular BC is characterized by two different sets of genes, the latter particular by genes involved in the differentiation of the mesenchyme (PITX2, TITF1, GDNF and MYOD1). Finally we demonstrate that only ERT genes predict ER positive BC; lack of peripheral blood cell DNA methylation of ZNF217 predicted BC independent of age and family history (odds ratio 1.49; 95% confidence interval 1.12–1.97; P = 0.006) and was associated with ER-α bioactivity in the corresponding serum.
Conclusion/Significance
This first large-scale epigenotyping study demonstrates that DNA methylation may serve as a link between the environment and the genome. Factors that can be modulated by the environment (like estrogens) leave an imprint in the DNA of cells that are unrelated to the target organ and indicate the predisposition to develop a cancer. Further research will need to demonstrate whether DNA methylation profiles will be able to serve as a new tool to predict the risk of developing chronic diseases with sufficient accuracy to guide preventive measures.
doi:10.1371/journal.pone.0002656
PMCID: PMC2442168  PMID: 18628976

Results 1-25 (29)