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1.  Natural compounds with P2X7 receptor-modulating properties 
Purinergic Signalling  2013;10(2):313-326.
The adenosine 5′-triphosphate (ATP)-gated P2X7 receptor is a membrane-bound, non-selective cation channel, expressed in a variety of cell types. The P2X7 senses high extracellular ATP concentrations and seems to be implicated in a wide range of cellular functions as well as pathophysiological processes, including immune responses and inflammation, release of gliotransmitters and cytokines, cancer cell growth or development of neurodegenerative diseases. In the present study, we identified natural compounds and analogues that can block or sensitize the ATP (1 mM)-induced Ca2+ response using a HEK293 cell line stably expressing human P2X7 and fluorometric imaging plate reader technology. For instance, teniposide potently blocked the human P2X7 at sub-miromolar concentrations, but not human P2X4 or rat P2X2. A marked block of ATP-induced Ca2+ entry and Yo-Pro-1 uptake was also observed in human A375 melanoma cells and mouse microglial cells, both expressing P2X7. On the other hand, agelasine (AGL) and garcinolic acid (GA) facilitated the P2X7 response to ATP in all three cell populations. GA also enhanced the YO-PRO-1 uptake, whereas AGL did not affect the ATP-stimulated intracellular accumulation of this dye. According to the pathophysiological role of P2X7 in various diseases, selective modulators may have potential for further development, e.g. as neuroprotective or antineoplastic drugs.
doi:10.1007/s11302-013-9392-1
PMCID: PMC4040168  PMID: 24163006
Ion channel-coupled purinergic receptors; P2X7; calcium influx; Pore formation; Dye permeability
2.  Correlation of Serum HE4 with Tumor Size and Myometrial Invasion in Endometrial Cancer 
Gynecologic oncology  2011;124(2):270-275.
Objective
To evaluate the utility of serum (HE4) as a marker for high risk disease in patients with endometrial cancer (EC).
Methods
Preoperative serum HE4 levels were measured from a cohort of 75 patients surgically treated for EC. Cases were compared to matched controls without a history of cancer. HE4 levels were analyzed as a function of primary tumor diameter, grade, stage and histological subtype. Wilcoxon rank-sum test, ROC curve, Spearman rank correlation coefficient and contingency tables were used for statistical analyses.
Results
Stage distribution was as follows: 49 stage I, 2 stage II, 20 stage III, 4 stage IV. Type I EC was present in 54 patients, type II in 21. Median HE4 was significantly elevated in both type I and II EC compared to controls (P<0.001 and P=0.019, respectively). There was significant correlation between type I EC, median HE4, deep myometrial invasion (MI) (>50%, P<0.001) and primary tumor diameter (PTD) (>2cm, P=0.002). Low risk patients (type I, MI ≤50% and PTD ≤2cm) had significantly lower median HE4 compared to all other type I EC patients (P<0.01). In comparison to prior investigations, HE4 (cutoff of 8 mfi) was more sensitive than CA125 in detecting advanced stage disease.
Conclusion
Our data suggest that HE4 is elevated in a high proportion of EC patients, is correlated with PTD and MI, and is more sensitive than CA125 in EC. These observations suggest potential utility of HE4 in the preoperative prediction of high risk disease and the necessity for definitive surgical staging.
doi:10.1016/j.ygyno.2011.10.025
PMCID: PMC3913473  PMID: 22037318
Endometrial cancer; human epididymis protein 4 (HE4); tumor marker
3.  Longitudinal Screening Algorithm That Incorporates Change Over Time in CA125 Levels Identifies Ovarian Cancer Earlier Than a Single-Threshold Rule 
Journal of Clinical Oncology  2012;31(3):387-392.
Purpose
Longitudinal algorithms incorporate change over time in biomarker levels to individualize screening decision rules. Compared with a single-threshold (ST) rule, smaller deviations from baseline biomarker levels are required to signal disease. We demonstrated improvement in ovarian cancer early detection by using a longitudinal algorithm to monitor annual CA125 levels.
Patients and Methods
We retrospectively evaluated serial preclinical serum CA125 values measured annually in 44 incident ovarian cancer cases identified from participants in the PLCO (Prostate Lung Colorectal and Ovarian) Cancer Screening Trial to determine how frequently and to what extent the parametric empirical Bayes (PEB) longitudinal screening algorithm identifies ovarian cancer earlier than an ST rule.
Results
The PEB algorithm detected ovarian cancer earlier than an ST rule in a substantial proportion of cases. At 99% specificity, which corresponded to the ST-rule CA125 cutoff ≥ 35 U/mL that was used in the PLCO trial, 20% of cases were identified earlier by using the PEB algorithm. Among these cases, the PEB signaled abnormal CA125 values, on average, 10 months earlier and at a CA125 concentration 42% lower (20 U/mL) than the ST-rule cutoff. The proportion of cases detected earlier by the PEB algorithm and the earliness of detection increased as the specificity of the screening rule was reduced.
Conclusion
The PEB longitudinal algorithm identifies ovarian cancer earlier and at lower biomarker concentrations than an ST screening algorithm adjusted to the same specificity. Longitudinal biomarker assessment by using the PEB algorithm may have application for screening other solid tumors in which biomarkers are available.
doi:10.1200/JCO.2012.43.6691
PMCID: PMC3732015  PMID: 23248253
4.  Interpretation of single and serial measures of HE4 and CA125 in asymptomatic women at high risk for ovarian cancer 
Background
Human epididymis protein 4 (HE4) is approved for clinical use with CA125 to predict epithelial ovarian cancer (EOC) in women with a pelvic mass or in remission after chemotherapy. Previously reported reference ranges for HE4 are inconsistent.
Methods
We report positivity thresholds yielding 90%, 95%, 98% and 99% specificity for age-defined populations of healthy women for HE4, CA125 and Risk of Malignancy Algorithm (ROMA), a weighted average of HE4 and CA125. HE4 and CA125 were measured in 1780 samples from 778 healthy women aged >25 years with a documented deleterious mutation, or aged >35 years with a significant family history. Effects on marker levels of a woman’s age, ethnicity and epidemiologic characteristics were estimated, as were the population-specific means, variances and within- and between-woman variances used to generate longitudinal screening algorithms for these markers.
Results
CA125 levels were lower with Black ethnicity (p=0.008). Smoking was associated with higher HE4 (p=0.007) and ROMA (p<0.019). Continuous oral contraceptive use decreased levels of CA125 (p=0.041), and ROMA (p=0.12). CA125 was lower in women age ≥55, and HE4 increased with age (p<0.01), particularly among women age ≥55.
Conclusions
Due to the strong effect of age on HE4, thresholds for HE4 are best defined for women of specific ages. Age-specific population thresholds for HE4 for 95% specificity ranged from 41.4 pmol/L for women age 30 to 82.1 pmol/L for women age 80.
Impact
Incorporation of serial marker values from screening history reduces personalized thresholds for CA125 and HE4 but is inappropriate for ROMA.
doi:10.1158/1055-9965.EPI-12-0616
PMCID: PMC3493821  PMID: 22962406
ovarian cancer; screening; longitudinal algorithm; HE4; CA125; early detection; high-risk
5.  Prophylactic oophorectomy rates in relation to a guideline update on referral to genetic counseling 
Gynecologic Oncology  2012;126(2):229-235.
Objective
We sought to determine whether prophylactic oophorectomy rates changed after the introduction of a 2007 health plan clinical guideline recommending systematic referral to a genetic counselor for women with a personal or family history suggestive of an inherited susceptibility to breast/ovarian cancer.
Methods
We conducted a retrospective cohort study of female members of Group Health, an integrated delivery system in Washington State. Subjects were women aged ≥35 years during 2004–2009 who reported a personal or family history consistent with an inherited susceptibility to breast/ovarian cancer. Personal and family history information was collected on a questionnaire completed when the women had a mammogram. We ascertained oophorectomies from automated claims data and determined whether surgeries were prophylactic by medical chart review. Rates were age-adjusted and age-adjusted incidence rate ratios (IRR) and 95% confidence intervals (CI) were computed using Poisson regression.
Results
Prophylactic oophorectomy rates were relatively unchanged after compared to before the guideline change, 1.0 versus 0.8/1,000 person-years, (IRR = 1.2; 95% CI: 0.7–2.0), whereas bilateral oophorectomy rates for other indications decreased. Genetic counseling receipt rates doubled after the guideline change (95% CI: 1.7–2.4) from 5.1 to 10.2/1,000 person-years. During the study, bilateral oophorectomy rates were appreciably greater in women who saw a genetic counselor compared to those who did not regardless of whether they received genetic testing as part of their counseling.
Conclusion
A doubling in genetic counseling receipt rates lends support to the idea that the guideline issuance contributed to sustained rates of prophylactic oophorectomies in more recent years.
doi:10.1016/j.ygyno.2012.04.046
PMCID: PMC3383401  PMID: 22564716
6.  Impact of screening test performance and cost on mortality reduction and cost-effectiveness of multimodal ovarian cancer screening 
Ongoing ovarian cancer screening trials are investigating the efficacy of a two-step screening strategy using currently available blood and imaging tests (CA125 and transvaginal sonography [TVS]). Concurrently, efforts to develop new biomarkers and imaging tests seek to improve screening performance beyond its current lim its. This study estimates the mortality reduction, years of life saved and cost-effectiveness achievable by annual multimodal screening using rising CA125 to select women for TVS, and predicts improvements achievable by replacing currently available screening tests with hypothetical counter parts with better performance characteristics. An existing stochastic micro-simulation model is refined and used to screen a virtual cohort of 1 million women from age 45 to 85. Each woman is assigned a detailed disease course and screening results timeline. The pre-clinical behavior of CA125 and TVS is simulated using empirical data derived from clinical trials. Simulations in which the disease incidence and performance characteristics of the screening tests are independently varied are performed in order to evaluate the impact of these factors on overall screening performance and costs. Our results demonstrate that when applied to women at average risk, annual screening using rising CA125 to select women for TVS achieves modest mortality reduction (~13%) and falls with in currently accepted cost-effectiveness guidelines. Screening outcomes are relatively insensitive to second-line test performance and costs. Identification of a first line test that perform s substantially better than CA125 and has similar costs is required in order for screening to reduce ovarian mortality by at least 25% and be reasonably cost-effective.
doi:10.1158/1940-6207.CAPR-11-0468
PMCID: PMC3729263  PMID: 22750949
8.  Potential Role of HE4 in Multimodal Screening for Epithelial Ovarian Cancer 
In screening for epithelial ovarian cancer, unnecessary surgery can be reduced by limiting use of transvaginal ultrasound (TVU) to women with increasing CA125 serum levels. Replacing or augmenting TVU with measurement of a serum marker specific for malignancy might further improve screening performance. Serum samples from 112 invasive ovarian cancer patients and 706 matched control subjects from the Prostate, Lung, Colorectal, and Ovarian trial were used to evaluate human epididymis protein 4 (HE4), mesothelin, matrix metalloproteinase 7 (MMP7), SLPI, Spondin2, and insulin-like growth factor binding protein 2 (IGFBP2) for their potential use in screening. TVU results were available for a subset of 84 patients and 516 control subjects used to compare the best marker with TVU. HE4 was found to perform better than TVU as a second-line screen, confirming 27 of 39 cancers with increasing CA125 serum levels compared with 17 cancers confirmed by TVU (P = .03). Serum HE4 levels were found to increase with age and smoking status, suggesting that a longitudinal algorithm might improve its performance.
doi:10.1093/jnci/djr359
PMCID: PMC3206037  PMID: 21917606
9.  Quantitative Comparison of Erythropoietin Receptor Levels in the Epithelial versus Endothelial Fractions of Primary Breast Tumors 
Anticancer Research  2011;31(4):1189-1195.
Background
Erythropoietin (EPO) was shown to reduce tumor survival in recent trials, however, its mechanisms of action are unclear. Efforts to measure tumor EPO receptor (EPOR) are limited by the promiscuity of EPOR antibodies, and concerns as to whether EPOR mRNA measurements are confounded by heterogeneity of tumor vasculature, a known EPOR source.
Materials and Methods
This study compared mRNA levels of EPOR and JAK2 in 11 breast tumor epithelial versus endothelial dissections.
Results
In nine tumors EPOR mRNA was 2.6 (1.2–5.7)-fold lower in the epithelial fraction, however, this reduction was less than the reduction of endothelial markers. In two tumors, EPOR mRNA was 2.9 (1.7–4.0)-fold higher in the epithelial fraction. The inter-tumor variation in EPOR levels exceeded the intra-tumor variation between fractions. Similar results were obtained for JAK2.
Conclusion
Tumor vasculature is not the sole source of EPOR and JAK2, and tumors can be segregated by EPOR and JAK2 levels for correlative analysis with clinical outcomes.
PMCID: PMC3401416  PMID: 21508364
Erythropoietin; breast cancer; erythropoietin receptor; erythropoiesis stimulating agents
10.  Ovarian Cancer Biomarker Performance in Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial Specimens 
Establishing a cancer screening biomarker’s intended performance requires “phase III” specimens obtained in asymptomatic individuals before clinical diagnosis rather than “phase II” specimens obtained from symptomatic individuals at diagnosis. We used specimens from the Prostate, Lung, Colorectal, and Ovarian (PLCO) cancer screening trial to evaluate ovarian cancer biomarkers previously assessed in phase II sets.
Phase II specimens from 180 ovarian cancer cases and 660 benign disease or general population controls were assembled from four Early Detection Research Network (EDRN) or Ovarian Cancer Specialized Program of Research Excellence (SPORE) sites and used to rank 49 biomarkers. Thirty-five markers, including 6 additional markers from a fifth site, were then evaluated in PLCO proximate specimens from 118 women with ovarian cancer and 474 matched controls.
Top markers in phase II specimens included CA125, HE4, transthyretin, CA15.3, and CA72.4 with sensitivity at 95% specificity ranging from 0.73 to 0.40. Except for transthyretin, these markers had similar or better sensitivity when moving to phase III specimens that had been drawn within six months of the clinical diagnosis. Performance of all markers declined in phase III specimens more remote than 6 months from diagnosis.
Despite many promising new markers for ovarian cancer, CA125 remains the single-best biomarker in the phase II and phase III specimens tested in this study.
doi:10.1158/1940-6207.CAPR-10-0195
PMCID: PMC3085251  PMID: 21372036
Ovarian neoplasms; CA125; HE4; Screening tests; CA72.4
11.  A Framework for Evaluating Biomarkers for Early Detection: Validation of Biomarker Panels for Ovarian Cancer 
A panel of biomarkers may improve predictive performance over individual markers. Although many biomarker panels have been described for ovarian cancer, few studies used pre-diagnostic samples to assess the potential of the panels for early detection. We conducted a multi-site systematic evaluation of biomarker panels using pre-diagnostic serum samples from the Prostate, Lung, Colorectal, and Ovarian Cancer (PLCO) screening trial.
Using a nested case-control design, levels of 28 biomarkers were measured laboratory-blinded in 118 serum samples obtained before cancer diagnosis and 951 serum samples from matched controls. Five predictive models, each containing 6–8 biomarkers, were evaluated according to a pre-determined analysis plan. Three sequential analyses were conducted: blinded validation of previously established models (Step 1); simultaneous split-sample discovery and validation of models (Step 2); and exploratory discovery of new models (Step 3). Sensitivity, specificity, sensitivity at 98% specificity, and AUC were computed for the models and CA125 alone among 67 cases diagnosed within one year of blood draw and 476 matched controls. In Step 1, one model showed comparable performance to CA125, with sensitivity, specificity and AUC at 69.2%, 96.6% and 0.892, respectively. Remaining models had poorer performance than CA125 alone. In Step 2, we observed a similar pattern. In Step 3, a model derived from all 28 markers failed to show improvement over CA125.
Thus, biomarker panels discovered in diagnostic samples may not validate in pre-diagnostic samples; utilizing pre-diagnostic samples for discovery may be helpful in developing validated early detection panels.
doi:10.1158/1940-6207.CAPR-10-0193
PMCID: PMC3057372  PMID: 21372037
Early Detection; Screening; Biomarkers; Validation; Study Design
12.  Activation of the MEK – S6 pathway in high-grade ovarian cancers 
The primary objective of this study is to demonstrate the activation and analyze the regulation of the MEK- S6 kinase pathway in high-grade ovarian cancer. Phospho-ERK (pERK), a direct substrate of MEK and two phosphorylation sites on the ribosomal protein, S6, Ser235/236 and Ser240/244, which are both targeted by the MEK and PI3-kinase/AKT pathways, were analyzed in 13 cell lines, 28 primary cancers and 8 cases of cancer cells from ascites. In primary cancers, ERK and S6 phosphorylation was measured by immunohistochemistry (IHC). pERK, pS6, pAKT and p4EBP1 were also measured by Western blotting (WB). The regulation of S6 phosphorylation by the MEK and PI3-kinase pathways was determined in ovarian cancer cell lines. We observed frequent pERK expression in primary ovarian cancers (100 % by WB, 75% by IHC) but not in ovarian cancer cells from ascites (25% of cases by WB). The activation of the AKT pathway, measured by pAKT expression occurred in 7 cases of primary ovarian cancer by WB, but in none of the ascites samples. In ovarian cancer cell lines, the MEK pathway had a greater effect on S6 phosphorylation in cells without hyperactive AKT signaling. Our data suggest that MEK is a potential drug target in high-grade ovarian cancer, however cancer cells with hyperactive AKT and cancer cells in ascites may be less responsive to MEK inhibition. The phosphorylation of S6 as a specific biomarker for either MEK or PI3-kinase pathway activation should be used with caution.
doi:10.1097/PAI.0b013e3181e53e1c
PMCID: PMC2989426  PMID: 20661131
ovarian cancer; pathway activation; MEK; AKT; S6
13.  An integrative genomic approach identifies p73 and p63 as activators of miR-200 microRNA family transcription 
Nucleic Acids Research  2011;40(2):499-510.
Although microRNAs (miRNAs) are important regulators of gene expression, the transcriptional regulation of miRNAs themselves is not well understood. We employed an integrative computational pipeline to dissect the transcription factors (TFs) responsible for altered miRNA expression in ovarian carcinoma. Using experimental data and computational predictions to define miRNA promoters across the human genome, we identified TFs with binding sites significantly overrepresented among miRNA genes overexpressed in ovarian carcinoma. This pipeline nominated TFs of the p53/p63/p73 family as candidate drivers of miRNA overexpression. Analysis of data from an independent set of 253 ovarian carcinomas in The Cancer Genome Atlas showed that p73 and p63 expression is significantly correlated with expression of miRNAs whose promoters contain p53/p63/p73 family binding sites. In experimental validation of specific miRNAs predicted by the analysis to be regulated by p73 and p63, we found that p53/p63/p73 family binding sites modulate promoter activity of miRNAs of the miR-200 family, which are known regulators of cancer stem cells and epithelial–mesenchymal transitions. Furthermore, in chromatin immunoprecipitation studies both p73 and p63 directly associated with the miR-200b/a/429 promoter. This study delineates an integrative approach that can be applied to discover transcriptional regulatory mechanisms in other biological settings where analogous genomic data are available.
doi:10.1093/nar/gkr731
PMCID: PMC3258134  PMID: 21917857
14.  Desmoglein 2 is a receptor for adenovirus serotypes 3, 7, 11, and 14 
Nature medicine  2010;17(1):96-104.
We have identified desmoglein 2 (DSG2) as the primary high-affinity receptor used by adenovirus (Ad) serotypes Ad3, Ad7, Ad11, and Ad14. These serotypes represent important human pathogens causing respiratory tract infections. In epithelial cells, adenovirus binding to DSG2 triggers events reminiscent of epithelial-to-mesenchymal transition, leading to transient opening of intercellular junctions. This improves access to receptors, e.g. CD46 and Her2/neu, that are trapped in intercellular junctions. In addition to complete virions, dodecahedral particles (PtDd), formed by viral penton and fiber in excess during viral replication, can trigger DSG2-mediated opening of intercellular junctions as shown by studies with recombinant Ad3 PtDd. Our findings shed light on adenovirus biology and pathogenesis and have implications for cancer therapy.
doi:10.1038/nm.2270
PMCID: PMC3074512  PMID: 21151137
15.  Use of a Symptom Index, CA125 and HE4 to predict ovarian cancer 
Gynecologic oncology  2009;116(3):378.
BACKGROUND
Prior studies suggest that combining the Symptom Index (SI) with a serum HE4 test or a CA125 test may improve prediction of ovarian cancer. However, these three tests have not been evaluated in combination.
METHODS
A prospective case-control study design including 74 women with ovarian cancer and 137 healthy women was used with logistic regression analysis to evaluate the independent contributions of HE4, CA125, and the SI to predict ovarian cancer status in a multivariate model. The diagnostic performance of various decision-rules for combinations of these tests was assessed to evaluate potential use in predicting ovarian cancer.
RESULTS
The SI, HE4, and CA125 all made significant independent contributions to ovarian cancer prediction. A decision-rule based on any one of the three tests being positive had a sensitivity of 95% with specificity of 80%. A rule based on any two of the three tests being positive had a sensitivity of 84% with a specificity of 98.5%. The SI alone had sensitivity of 64% with specificity of 88%. If the SI index is used to select women for CA125 and HE4 testing, specificity is 98.5% and sensitivity is 58% using the 2-of-3-positive decision rule.
CONCLUSIONS
A 2-of-3-positive decision rule yields acceptable specificity, and higher sensitivity when all 3 tests are performed than when the SI is used to select women for screening by CA125 and HE4. If positive predictive value is a high priority, testing by CA125 and HE4 prior to imaging may be warranted for women with ovarian cancer symptoms.
doi:10.1016/j.ygyno.2009.10.087
PMCID: PMC2822097  PMID: 19945742
ovarian cancer; CA125; HE4; Symptom Index; sensitivity; specificity
16.  Correction: Analysis of Epithelial and Mesenchymal Markers in Ovarian Cancer Reveals Phenotypic Heterogeneity and Plasticity 
PLoS ONE  2011;6(2):10.1371/annotation/8c637352-3614-406c-89dc-e78d10fa069c.
doi:10.1371/annotation/8c637352-3614-406c-89dc-e78d10fa069c
PMCID: PMC3039027
17.  Analysis of Epithelial and Mesenchymal Markers in Ovarian Cancer Reveals Phenotypic Heterogeneity and Plasticity 
PLoS ONE  2011;6(1):e16186.
In our studies of ovarian cancer cells we have identified subpopulations of cells that are in a transitory E/M hybrid stage, i.e. cells that simultaneously express epithelial and mesenchymal markers. E/M cells are not homogenous but, in vitro and in vivo, contain subsets that can be distinguished based on a number of phenotypic features, including the subcellular localization of E-cadherin, and the expression levels of Tie2, CD133, and CD44. A cellular subset (E/M-MP) (membrane E-cadherinlow/cytoplasmic E-cadherinhigh/CD133high, CD44high, Tie2low) is highly enriched for tumor-forming cells and displays features which are generally associated with cancer stem cells. Our data suggest that E/M-MP cells are able to differentiate into different lineages under certain conditions, and have the capacity for self-renewal, i.e. to maintain a subset of undifferentiated E/M-MP cells during differentiation. Trans-differentiation of E/M-MP cells into mesenchymal or epithelial cells is associated with a loss of stem cell markers and tumorigenicity. In vivo xenograft tumor growth is driven by E/M-MP cells, which give rise to epithelial ovarian cancer cells. In contrast, in vitro, we found that E/M-MP cells differentiate into mesenchymal cells, in a process that involves pathways associated with an epithelial-to-mesenchymal transition. We also detected phenotypic plasticity that was dependent on external factors such as stress created by starvation or contact with either epithelial or mesenchymal cells in co-cultures. Our study provides a better understanding of the phenotypic complexity of ovarian cancer and has implications for ovarian cancer therapy.
doi:10.1371/journal.pone.0016186
PMCID: PMC3021543  PMID: 21264259
19.  Evaluating Erythropoietin-Associated Tumor Progression Using Archival Tissues from a Phase III Clinical Trial 
Stem cells (Dayton, Ohio)  2009;27(9):2353-2361.
Despite the prevalence of anemia in cancer, recombinant erythropoietin (Epo) has declined in use because of recent Phase III trials showing more rapid cancer progression and reduced survival in subjects randomized to Epo. Since Epo receptor (EpoR), Jak2, and Hsp70 are well-characterized mediators of Epo signaling in erythroid cells, we hypothesized that Epo might be especially harmful in patients whose tumors express high levels of these effectors. Because of the insensitivity of immunohistochemistry for detecting low level EpoR protein, we developed assays to measure levels of EpoR, Jak2 and Hsp70 mRNA in formalin-fixed paraffin-embedded (FFPE) tumors. We tested 23 archival breast tumors as well as 136 archival head and neck cancers from ENHANCE, a Phase III trial of 351 patients randomized to Epo versus placebo concomitant with radio-therapy following complete resection, partial resection, or no resection of tumor. EpoR, Jak2, and Hsp70 mRNA levels varied >30-fold, >12-fold, and >13-fold across the breast cancers, and >30-fold, >40-fold, and >30-fold across the head and neck cancers, respectively. Locoregional progression-free survival (LPFS) did not differ among patients whose head and neck cancers expressed above- versus below-median levels of EpoR, Jak2 or Hsp70, except in the subgroup of patients with unresected tumors (n = 28), where above-median EpoR, above-median Jak2, and below-median Hsp70 mRNA levels were all associated with significantly poorer LPFS. Our results provide a framework for exploring the relationship between Epo, cancer progression, and survival using archival tumors from other Phase III clinical trials.
doi:10.1002/stem.156
PMCID: PMC2911228  PMID: 19544471
Erythropoietin; Erythropoietin receptor; Erythropoiesis stimulating agents; Growth factors; Tumor progression
20.  The Temporal Stability of the Symptom Index Among Women at High-Risk for Ovarian Cancer 
Gynecologic oncology  2009;114(2):225-230.
Objective
To evaluate the temporal stability of self-reported symptoms known to be associated with ovarian cancer.
Methods
This report is a longitudinal analysis of symptom reporting from 123 women who participated in the Seattle-based Ovarian Cancer Early Detection Study (OCEDS). The OCEDS population includes women at increased risk of ovarian cancer based on a family history of cancer or a BRCA I/II mutation. Data on symptoms were collected at two time points using a Symptoms Index that included abdominal pain, pelvic pain, feeling full quickly, inability to eat normally, abdominal bloating, and increased abdominal size.
Results
There was a median of 101 days between the two time points, with a range of 72–332 days. The median age of the women was 51, with a range of 32–79 years. Abdominal bloating was the most commonly reported symptom at both time points. The symptom least commonly reported at the two time points was inability to eat normally. The Symptoms Index was negative at both time points for 86% of all women and positive at both time points for 2% of all women. There were no statistically significant patterns of change for symptom reporting between time points.
Conclusions
The Symptoms Index and women’s report of abdominal pain, pelvic pain, feeling full quickly, unable to eat normally, abdominal bloating, increased abdominal size were stable between two time points in this sample. These findings provide evidence that longitudinal measurements of symptoms reporting by women in a screening study are likely to be reliable.
doi:10.1016/j.ygyno.2009.03.015
PMCID: PMC2736546  PMID: 19427026
21.  Antifouling Surface Layers for Improved Signal-to-Noise of Particle-Based Immunoassays 
A ten fold improvement in the signal-to-noise (S/N) ratio of an optically encoded silica particle-based immunoassay was achieved through incorporating a protein resistant poly(ethylene glycol) (PEG) surface layer and optimizing antibody immobilization conditions. PEG was activated using 2,2,2-trifluoroethanesulfonyl chloride (tresyl) and required a minimum reaction time of 1.5 hrs. The activated PEG had a reactive half life of approximately 5 hrs when stored in acidified dimethyl sulfoxide (DMSO). By increasing the protein incubation time and concentration, a maximum antibody loading on the particle surface of 1.6×10−2 molecules per nm2 was achieved. The assay S/N ratio was assessed using a multiplexed multicomponent optically encoded species-specific immunoassay. Encoded particles were covalently grafted or nonspecifically coated with either bovine or mouse IgG for the simultaneous detection of complimentary anti-IgG `target' or uncomplimentary anti-IgG `noise'. The versatility and potential as a serum-based assay platform was demonstrated by immobilizing either a polyclonal antibody or an engineered single-chain variable fragment (scFv) capture probe on particles for the detection of the ovarian cancer biomarker, mesothelin (MSLN). The MLSN antigen was spiked into PBS buffer or 50% human serum. Both capture probe orientations and media conditions showed similar low level detection limits of 5 ng/mL; however, a 40% decrease in maximum signal intensity was observed for assays run in 50% serum.
doi:10.1021/la903148n
PMCID: PMC2891774  PMID: 19928944
Immunoassay; molecular diagnostic; signal-to-noise; cancer biomarker; scFv; bead-based assay
22.  The epithelial phenotype confers resistance of ovarian cancer cells to oncolytic adenoviruses 
Cancer research  2009;69(12):5115-5125.
We studied the propensity of primary ovarian cancer cells to oncolytic adenoviruses. Using gene expression profiling of cancer cells either resistant or susceptible to viral oncolysis, we discovered that the epithelial phenotype of ovarian cancer represents a barrier to infection by commonly used oncolytic adenoviruses targeted to CAR or CD46. Specifically, we found that these adenovirus receptors were trapped in tight junctions and not accessible for virus binding. Accessibility to viral receptors was critically linked to depolarization and the loss of tight and adherens junctions, both hallmarks of epithelial-to-mesenchymal transition (EMT). We showed that specific, so far little explored adenovirus serotypes (Ad3, 7, 11, and 14) that use receptor(s) other than CAR and CD46 were able to trigger EMT in epithelial ovarian cancer cells and cause efficient oncolysis. Our studies on ovarian cancer cultures and xenografts also revealed a number of interesting cancer cell biology features. Tumors in situ as well as tumor xenografts in mice mostly contained epithelial cells and cells that were in a hybrid stage where they expressed both epithelial and mesenchymal markers (E/M cells). These E/M cells are the only xenograft-derived cells that can be cultured, and with passaging undergo EMT and differentiate into mesenchymal cells. Our study provides a venue for improved virotherapy of cancer as well as new insights into cancer cell biology.
doi:10.1158/0008-5472.CAN-09-0645
PMCID: PMC2738419  PMID: 19491256
23.  CA125 in Ovarian Cancer 
Biomarkers in medicine  2007;1(4):513-523.
Summary
Twenty five years after its discovery, circulating CA125 antigen is recommended for clinical use in the US for ovarian cancer (OC) screening of high risk women with ovaries despite its limited sensitivity and specificity. Recent findings suggest that CA125 might also serve as a predictive marker for pre-invasive OC. Methods to quantify circulating CA125 evolved towards sensitive and reliable double determinant ELISA assays. The CA125 gene, MUC16, was cloned 20 years after the protein discovery and revealed a very complex and unusual glycoprotein structure suggesting an immunological role. Recent evidence points toward CA125 function in the induction of materno-fetal tolerance through the alteration of NK phenotype. Two receptors for CA125 have been described: mesothelin and galectin-1. The specific location and functional proprieties of CA125 make it a therapeutic target of choice; clinical trials have demonstrated that anti-CA125 injections are well tolerated and suggest a potential survival benefit.
doi:10.2217/17520363.1.4.513
PMCID: PMC2872496  PMID: 20477371
CA 125; biomarker; detection; disease monitoring; ovarian cancer; pregnancy; Glycoprotein; NK; mesothelin; galectin-1
24.  No Effect of Aspirin on Mammographic Density in a Randomized Controlled Clinical Trial 
Background:
Epidemiologic studies suggest a reduced risk of breast cancer among women who regularly use aspirin; a plausible mechanism is through aspirin effect on mammographic breast density, a breast cancer risk factor, possibly mediated through aspirin interference with estrogen synthesis.
Methods:
In a 2-arm randomized placebo-controlled clinical trial, we evaluated the effects of 6-months administration of 325 mg/day aspirin on total mammographic breast dense area and percent of the mammographic breast image occupied by dense areas (% density) in 143 postmenopausal women. Eligible women, recruited 2005-7, were healthy, not taking hormone therapy, with elevated mammographic breast density (American College of Radiology Breast Imaging Reporting and Data System (BI-RADS®) density category 2, 3 or 4) within 6 months prior to enrollment.
Results:
Women were a mean (s.d.) 59.5 (5.5) years. Geometric mean baseline percent density was 17.6% (95% CI 14.8, 20.9) in women randomized to aspirin and 19.2% (95% CI 16.3, 22.7) in women randomized to placebo. Percent density decreased in women randomized to aspirin by an absolute 0.8% vs. an absolute decrease of 1.2% in controls (p = 0.84). Total breast area and dense area decreased to a similar degree in women assigned to aspirin and in those assigned to placebo, with no differences statistically significantly different between trial arms.
Conclusions:
A single daily administration of adult-dose aspirin for 6 months had no effect on mammographic density in postmenopausal women. If aspirin affects breast cancer risk in postmenopausal women, it may do so through alternative pathways than mammographic breast density.
doi:10.1158/1055-9965.EPI-08-1088
PMCID: PMC2689709  PMID: 19423529
aspirin; NSAIDs; mammographic density; breast cancer
25.  INFLUENCE OF OVARIAN CANCER RISK STATUS ON THE DIAGNOSTIC PERFORMANCE OF THE SERUM BIOMARKERS MESOTHELIN, HE4 AND CA125 
Objective
To evaluate the impact of ovarian cancer risk on the performance of the serum biomarkers mesothelin, HE4 and CA125.
Methods
We measured mesothelin, HE4 and CA125 levels from women with invasive ovarian cancer (n=143), benign gynecological conditions (n=124) and healthy women (n=344). Demographic, epidemiologic, reproductive, medical and family history data were collected using a standardized questionnaire. Pedigree and BRCA 1/2 test results were used to stratify women into average and high risk groups. The diagnostic accuracy of each biomarker was characterized using receiver operating characteristic (ROC) curve methods.
Results
Baseline characteristics did not vary by risk or case status. The distribution of stage and histology was similar in average and high risk women. All three markers discriminated ovarian cancer cases from risk-matched healthy and benign controls. Marker performance did not vary by risk status. The sensitivity at 95% specificity for discriminating cases from risk-matched healthy control women in the average and high risk groups respectively was 53.9% and 39.0% for mesothelin, 80.4% and 87.8% for HE4, and 79.4% and 82.9% for CA125. The performance of the markers was not as robust when cases were compared to benign controls. AUC values for cases vs. healthy and benign controls did not vary by risk status.
Conclusions
The ability of serum mesothelin, HE4 and CA 125 levels to discriminate ovarian cancer cases from healthy and benign controls is not influenced by risk status. Our findings support the pursuit of additional studies evaluating the early detection potential of these markers in high-risk populations.
doi:10.1158/1055-9965.EPI-08-1034
PMCID: PMC2714056  PMID: 19423517
ovarian cancer screening; biomarkers; high-risk; molecular diagnosis and prognosis; molecular markers in prevention research; gynecological cancers: ovarian

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