Effect of risk-reducing salpingo-oophorectomy on sex steroid hormone serum levels among postmenopausal women: an NRG Oncology/Gynecologic Oncology Group study.

BACKGROUND: Risk-reducing salpingo-oophorectomy is an effective ovarian cancer risk reduction strategy. However, bilateral oophorectomy has also been associated with increased long-term nonneoplastic sequelae, effects suggested to be mediated through reductions in systemic sex steroid hormone levels. Currently, it is unclear whether the postmenopausal ovary contributes to the systemic hormonal milieu or whether postmenopausal ovarian volume or other factors, such as body mass index and age, affect systemic hormone levels. OBJECTIVE: We examined the impact of oophorectomy on sex steroid hormone levels in postmenopausal women. Furthermore, we explored how well ovarian volume measured by transvaginal ultrasound correlated with direct ovarian measures obtained during surgical pathology evaluation and investigated the association between hormone levels and ovarian volumes. STUDY DESIGN: Postmenopausal women who underwent risk-reducing salpingo-oophorectomy (180 cases) or ovarian cancer screening (38 controls) enrolled in an international, prospective study of risk-reducing salpingo-oophorectomy and risk of ovarian cancer algorithm-based screening among women at increased risk of ovarian cancer (Gynecologic Oncology Group-0199) were included in this analysis. Controls were frequency matched to the cases on age at menopause, age at study entry, and time interval between blood draws. Ovarian volume was calculated using measurements obtained from transvaginal ultrasound in both cases and controls and measurements recorded in surgical pathology reports from cases. Serum hormone levels of testosterone, androstenedione, androstenediol, dihydrotestosterone, androsterone, dehydroepiandrosterone, estrone, estradiol, and sex hormone-binding globulin were measured at baseline and follow-up. Spearman correlation coefficients were used to compare ovarian volumes as measured on transvaginal ultrasound and pathology examinations. Correlations between ovarian volumes by transvaginal ultrasound and measured hormone levels were examined using linear regression models. All models were adjusted for age. Paired t tests were performed to evaluate individual differences in hormone levels before and after risk-reducing salpingo-oophorectomy. RESULTS: Ovarian volumes measured by transvaginal ultrasound were only moderately correlated with those reported on pathology reports (Spearman rho [ρ]=0.42). The median time interval between risk-reducing salpingo-oophorectomy and follow-up for the cases was 13.3 months (range, 6.0-19.3), and the median time interval between baseline and follow-up for the controls was 12.7 months (range, 8.7-13.4). Sex steroid levels decreased with age but were not correlated with transvaginal ultrasound ovarian volume, body mass index, or time since menopause. Estradiol levels were significantly lower after risk-reducing salpingo-oophorectomy (percentage change, -61.9 post-risk-reducing salpingo-oophorectomy vs +15.2 in controls; P=.02), but no significant differences were seen for the other hormones. CONCLUSION: Ovarian volumes measured by transvaginal ultrasound were moderately correlated with volumes directly measured on pathology specimens and were not correlated with sex steroid hormone levels in postmenopausal women. Estradiol was the only hormone that declined significantly after risk-reducing salpingo-oophorectomy. Thus, it remains unclear whether the limited post-risk-reducing salpingo-oophorectomy changes in sex steroid hormones among postmenopausal women impact long-term adverse outcomes.

Fibroblast Growth Factor 2 Enhances Zika Virus Infection in Human Fetal Brain.

Zika virus (ZIKV) is an emerging pathogen that can cause microcephaly and other neurological defects in developing fetuses. The cellular response to ZIKV in the fetal brain is not well understood. Here, we show that ZIKV infection of human fetal astrocytes (HFAs), the most abundant cell type in the brain, results in elevated expression and secretion of fibroblast growth factor 2 (FGF2). This cytokine was shown to enhance replication and spread of ZIKV in HFAs and human fetal brain explants. The proviral effect of FGF2 is likely mediated in part by suppression of the interferon response, which would represent a novel mechanism by which viruses antagonize host antiviral defenses. We posit that FGF2-enhanced virus replication in the fetal brain contributes to the neurodevelopmental disorders associated with in utero ZIKV infection. As such, targeting FGF2-dependent signaling should be explored further as a strategy to limit replication of ZIKV.

Zika Virus Hijacks Stress Granule Proteins and Modulates the Host Stress Response.

Zika virus (ZIKV), a member of the Flaviviridae family, has recently emerged as an important human pathogen with increasing economic and health impact worldwide. Because of its teratogenic nature and association with the serious neurological condition Guillain-Barré syndrome, a tremendous amount of effort has focused on understanding ZIKV pathogenesis. To gain further insights into ZIKV interaction with host cells, we investigated how this pathogen affects stress response pathways. While ZIKV infection induces stress signaling that leads to phosphorylation of eIF2α and cellular translational arrest, stress granule (SG) formation was inhibited. Further analysis revealed that the viral proteins NS3 and NS4A are linked to translational repression, whereas expression of the capsid protein, NS3/NS2B-3, and NS4A interfered with SG formation. Some, but not all, flavivirus capsid proteins also blocked SG assembly, indicating differential interactions between flaviviruses and SG biogenesis pathways. Depletion of the SG components G3BP1, TIAR, and Caprin-1, but not TIA-1, reduced ZIKV replication. Both G3BP1 and Caprin-1 formed complexes with capsid, whereas viral genomic RNA stably interacted with G3BP1 during ZIKV infection. Taken together, these results are consistent with a scenario in which ZIKV uses multiple viral components to hijack key SG proteins to benefit viral replication.IMPORTANCE There is a pressing need to understand ZIKV pathogenesis in order to advance the development of vaccines and therapeutics. The cellular stress response constitutes one of the first lines of defense against viral infection; therefore, understanding how ZIKV evades this antiviral system will provide key insights into ZIKV biology and potentially pathogenesis. Here, we show that ZIKV induces the stress response through activation of the UPR (unfolded protein response) and PKR (protein kinase R), leading to host translational arrest, a process likely mediated by the viral proteins NS3 and NS4A. Despite the activation of translational shutoff, formation of SG is strongly inhibited by the virus. Specifically, ZIKV hijacks the core SG proteins G3BP1, TIAR, and Caprin-1 to facilitate viral replication, resulting in impaired SG assembly. This process is potentially facilitated by the interactions of the viral RNA with G3BP1 as well as the viral capsid protein with G3BP1 and Caprin-1. Interestingly, expression of capsid proteins from several other flaviviruses also inhibited SG formation. Taken together, the present study provides novel insights into how ZIKV modulates cellular stress response pathways during replication.

Threonyl-tRNA synthetase overexpression correlates with angiogenic markers and progression of human ovarian cancer.

BACKGROUND: Ovarian tumors create a dynamic microenvironment that promotes angiogenesis and reduces immune responses. Our research has revealed that threonyl-tRNA synthetase (TARS) has an extracellular angiogenic activity separate from its function in protein synthesis. The objective of this study was to test the hypothesis that TARS expression in clinical samples correlates with angiogenic markers and ovarian cancer progression. METHODS: Protein and mRNA databases were explored to correlate TARS expression with ovarian cancer. Serial sections of paraffin embedded ovarian tissues from 70 patients diagnosed with epithelial ovarian cancer and 12 control patients were assessed for expression of TARS, vascular endothelial growth factor (VEGF) and PECAM using immunohistochemistry. TARS secretion from SK-OV-3 human ovarian cancer cells was measured. Serum samples from 31 tissue-matched patients were analyzed by ELISA for TARS, CA-125, and tumor necrosis factor-α (TNF-α). RESULTS: There was a strong association between the tumor expression of TARS and advancing stage of epithelial ovarian cancer (p < 0.001). TARS expression and localization were also correlated with VEGF (p < 0.001). A significant proportion of samples included heavy TARS staining of infiltrating leukocytes which also correlated with stage (p = 0.017). TARS was secreted by ovarian cancer cells, and patient serum TARS was related to tumor TARS and angiogenic markers, but did not achieve significance with respect to stage. Multivariate Cox proportional hazard models revealed a surprising inverse relationship between TARS expression and mortality risk in late stage disease (p = 0.062). CONCLUSIONS: TARS expression is increased in epithelial ovarian cancer and correlates with markers of angiogenic progression. These findings and the association of TARS with disease survival provide clinical validation that TARS is associated with angiogenesis in ovarian cancer. These results encourage further study of TARS as a regulator of the tumor microenvironment and possible target for diagnosis and/or treatment in ovarian cancer.

Recurrent pregnancy loss in a woman with NLRP7 mutation: not all molar pregnancies can be easily classified as either "partial" or "complete" hydatidiform moles.

Recurrent hydatidiform moles is an uncommon occurrence. Over the past decade, genetic studies of women with multiple recurrent molar pregnancies have revealed that maternal mutations in two different genes, NLRP7 and C6orf221, result in recurrent moles. We report a 23 year old woman, born of unrelated parents, who has experienced three molar pregnancies in succession. Whilst the first pregnancy was classified as a complete hydatidiform mole, the second and third moles defied classification as complete or partial mole using conventional histology, p57 nuclear staining pattern and ploidy studies. Molecular and cytogenetic studies proved that all three molar pregnancies were diploid and biparental in origin. Gene sequencing analysis showed that the patient is homozygous for a previously described mutation in NLRP7. A SNP microarray ruled out the presence of deletion of the NLRP7 locus. This case draws attention to the fact that recurrent molar pregnancies may be the result of specific, identifiable gene mutations, even in patients from non-consanguineous backgrounds. When pathologists encounter patients with molar pregnancies that are diploid and p57 negative and yet have fetal elements such as nucleated red blood cells or immature fetal tissues, it should heighten their suspicion of a possible genetic basis and appropriate molecular genetic workup performed with counseling offered.

Negative regulation of HIF-1α by an FBW7-mediated degradation pathway during hypoxia.

Hypoxia inducible factor-1α (HIF-1α) stimulates expression of genes associated with angiogenesis and is associated with poor outcomes in ovarian and other cancers. In normoxia, HIF-1α is ubiquitinated and degraded through the E3 ubiquitin ligase, von Hippel-Lindau; however, little is known about the regulation of HIF-1α in hypoxic conditions. FBW7 is an E3 ubiquitin ligase that recognizes proteins phosphorylated by glycogen synthase kinase 3ß (GSK3ß) and targets them for destruction. This study used an ovarian cancer cell model to test the hypothesis that HIF-1α phosphorylation by GSK3ß in hypoxia leads to interaction with FBW7 and ubiquitin-dependent degradation. Expression of constitutively active GSK3ß reduced HIF-1α protein and transcriptional activity and increased ubiquitination of HIF-1α in hypoxia, whereas pharmacologic inhibition of GSK3 or expression of siGSK3ß promoted HIF-1α stabilization and activity. A mechanism through FBW7 was supported by the observed decrease in HIF-1α stabilization when FBW7 was overexpressed and both the elevation of HIF-1α levels and decrease in ubiquitinated HIF-1α when FBW7 was suppressed. Furthermore, HIF-1α associated with FBW7γ by co-immunoprecipitation, and the interaction was weakened by inhibition of GSK3 or mutation of GSK3ß phosphorylation sites. The relevance of this pathway to angiogenic signaling was supported by the finding that endothelial cell tube maturation was increased by conditioned media from hypoxic SK-OV-3 cell lines expressing suppressed GSK3ß or FBW7. These data introduce a new mechanism for regulation of HIF-1α during hypoxia that utilizes phosphorylation to target HIF-1α for ubiquitin-dependent degradation through FBW7 and may identify new targets in the regulation of angiogenesis.

Aspiration cytology of ovarian cystic masses: histologic correlation and review of the literature.

OBJECTIVE: To determine the diagnostic accuracy of cytologic evaluation of ovarian cystic masses. STUDY DESIGN: Sixty-seven ovarian cystic masses with fine needle aspiration cytology and concurrent or subsequent cystectomy/oophorectomy with histology were examined. Correlations with malignancy were made with 4 parameters: serum CA-125, radiographic size and architecture, and cytology. RESULTS: Histologic examination of the 67 cases revealed 10 malignancies including 3 primary ovarian carcinomas, 2 metastatic neoplasms, and 5 borderline tumors. In the 10 malignant cases, the cytologic diagnoses were that of benign (n=2), benign but non-diagnostic/paucicellular (n=3), and atypical/malignant (n=5), giving an overall sensitivity for cytology of 50%. However, there were no false positives (specificity of 100%). Reasons for the low sensitivity of cytology were the paucicellular nature of aspirate (n=3), focality of ovarian borderline tumors (n=5), and surface involvement by metastatic cancer (n=2). The 4 parameters were independent of one another and none proved to have significant correlation with malignancy (p>0.05). Thirty-nine percent of the aspirates had low cellularity (6% non-diagnostic/33% paucicellular). CONCLUSIONS: Cytology was the only parameter with 100% specificity and 100% positive predictive value. However, paucicellular specimens are a common problem in aspiration from this site.

Use of Primary Human Fetal Astrocytes and Tissue Explants as Ex Vivo Models to Study Zika Virus Infection of the Developing Brain.

Zika virus (ZIKV) infection during pregnancy can result in congenital Zika syndrome which is characterized by microcephaly and other neurodevelopmental disorders. In this chapter, we describe methods to model ex vivo ZIKV infection in astrocytes and tissue explants from human fetal brain. These cell- and tissue-based platforms have been useful to elucidate mechanisms of ZIKV persistence and might lead to important clues about virus-induced neuropathogenesis. In addition, these ex vivo model systems allow researchers to conduct drug discovery and development experiments in more representative settings of the developing human brain.

In vitro study of pharmacobezoar formation in simulated acetaminophen overdose.

Objectives: There have been few studies of pharmacobezoar formation, but they can be an important contributor to overdose toxicity. Pharmacobezoars may explain the delayed peak or "double hump" pharmacokinetics, which were noted in previous case reports with delayed toxicity of acetaminophen (APAP). We validated the presence of APAP bezoar formation in a controlled modified in vitro environment simulating acute APAP overdose.Methods: This study involved the APAP and control groups (ferrous sulfate and chlorpheniramine). The APAP study group contained three subgroups of APAP with different dosage, i.e., 25 g (50 tabs)/37.5 g (75 tabs)/50 g (100 tabs). The positive control group containing ferrous sulfate, i.e., 15 g (50 tabs), has been reported previously to form pharmacobezoars in overdose. The negative control group containing chlorpheniramine, i.e., 200 mg (50 tabs), has not been reported to form pharmacobezoars in previous case studies. Tablets from each study group were placed into a separate pig stomach. Each stomach contained 28 ml USP standard simulated gastric acid. The stomach was placed in a plastic box filled with water maintaining at 37 °C. Each test group was examined for 4 h in the stomach. The primary outcome was the presence of clump formation. Positive clump formation was defined as tablets sticking together and the ability to maintain shape upon dissecting the pig stomach and lifting with fingers. Tablet clumps would then undergo dissolution testing with subsequent analysis of dissolution profiles.Results: Formation of tablets clumps was confirmed in APAP overdose in the in vitro environment. Clumps were noted to be present in the 37.5 g and 50 g APAP groups, while 25 g APAP was unlikely to form clumps. The dissolution profile of clump demonstrated slower release without reaching plateau at 60 min, as compared to corresponding individual tabs of APAP. f1 and f2 analyses showed the dissolution profile of clump was different compared to that of referenced individual tab.Conclusions: APAP clump formation was confirmed in acute overdose of 37.5 g or more. Dissolution tests revealed delayed and steady release of tablet residue from the clumps, which could explain prolonged or delayed toxicity in large APAP overdose.

Interplay between Zika Virus and Peroxisomes during Infection.

Zika virus (ZIKV) has emerged as an important human pathogen that can cause congenital defects in the fetus and neurological conditions in adults. The interferon (IFN) system has proven crucial in restricting ZIKV replication and pathogenesis. The canonical IFN response is triggered by the detection of viral RNA through RIG-I like receptors followed by activation of the adaptor protein MAVS on mitochondrial membranes. Recent studies have shown that a second organelle, peroxisomes, also function as a signaling platforms for the IFN response. Here, we investigated how ZIKV infection affects peroxisome biogenesis and antiviral signaling. We show that ZIKV infection depletes peroxisomes in human fetal astrocytes, a brain cell type that can support persistent infection. The peroxisome biogenesis factor PEX11B was shown to inhibit ZIKV replication, likely by increasing peroxisome numbers and enhancing downstream IFN-dependent antiviral signaling. Given that peroxisomes play critical roles in brain development and nerve function, our studies provide important insights into the roles of peroxisomes in regulating ZIKV infection and potentially neuropathogenesis.

Targeted Elimination of Peroxisomes During Viral Infection: Lessons from HIV and Other Viruses.

Peroxisomes are membrane-bound organelles that are best known for their roles in lipid metabolism. Mounting evidence indicates that they are also important nodes for antiviral signaling. While research over the past few decades has revealed effective viral strategies to block antiviral signalling pathways from the plasma membrane, mitochondria and/or the nucleus, until recently, very little was known about how viruses interfere with peroxisome-based antiviral signaling. In this essay, we review how viruses use a variety of strategies to interfere with peroxisome biogenesis, a phenomenon that has implications for evasion of the host immune system as well as pathogenesis.

Spleno-parametrial ectopic varices: an unusual porto-systemic shunt in portal hypertension.

BACKGROUND: Cirrhotic liver disease commonly is complicated by portal hypertension, and the resultant porto-systemic shunts are an important cause of morbidity and mortality in cirrhotic patients. Identification of these shunts and their management is an important part of the medical care provided to this population. CASE: We present the case of a patient with nonalcoholic steatohepatitis or nonalcoholic fatty liver disease and resultant portal hypertension who developed an unusual porto-systemic shunt, which at first was thought to represent a highly vascular gynecologic mass. The splanchnic blood was shunted through a recanalized vein interconnecting the splenic vein with the parametrial venous plexus. CONCLUSION: Unrecognized portal hypertension and resultant porto-systemic shunts may mask themselves as vascular masses and result in catastrophic surgical outcomes if not fully characterized preoperatively.

Erythropoietin disrupts hypoxia-inducible factor signaling in ovarian cancer cells.

OBJECTIVE: The objective of this study was to evaluate the effects of recombinant erythropoietin (EPO) on HIF-1alpha induced angiogenic pathways in ovarian cancer cells. METHODS: Using Western blots and both quantitative and non-quantitative RT-PCR, HIF-1alpha protein and VEGF transcription levels were assessed. Cell growth was measured using flow cytometry. RESULTS: EPO treatment decreased hypoxia-induced HIF-1alpha protein levels and VEGF transcription, with no effect on cell growth. Inhibition of HIF-1alpha signaling by EPO was also observed in MCF-7 breast cancer cells. CONCLUSION: These novel findings suggest that EPO may exhibit anti-angiogenic properties, thus encouraging further exploration of signaling pathways between EPO and HIF-1alpha.

Factor V Leiden, prothrombin 20210A and the risk of venous thrombosis among cancer patients.

Cancer patients have an increased risk of venous thrombosis (VT). The association of factor V Leiden (FVL) and the prothrombin 20210A variant with VT in cancer patients is not established. We genotyped 101 cancer patients with VT and 101 cancer patients without VT for these polymorphisms. Five cases and three controls were heterozygous for FVL, yielding an odds ratio of 1.7 (95% confidence interval (CI) 0.3-10.7). Five cases and no controls were heterozygous for prothrombin 20210A, for an odds ratio of 6.7 (95% CI 0.9-infinity). Prothrombin 20210A may be associated with VT risk among cancer patients.

Synchronous renal cell carcinoma and gynecologic malignancies.

BACKGROUND: Synchronicity of renal cell carcinoma and gynecologic malignancies is a rare condition and standardized treatment does not exist. CASES: Three cases of synchronous renal cell carcinoma and gynecologic malignancies are described. All three cases underwent optimal cytoreductive surgery for the gynecologic malignancy and a radical nephrectomy for the renal cell carcinoma. Adjuvant treatment, after surgery, was individualized in each case. Estrogen and progesterone receptors were positive in all the gynecologic tumors but negative in the renal cell tumors. CONCLUSIONS: This is apparently the largest report of synchronous renal cell carcinoma and gynecologic malignancies. Despite this rare condition, surgery should still be considered as primary treatment for these patients.

VEGF and HIF-1alpha expression are increased in advanced stages of epithelial ovarian cancer.

OBJECTIVE: Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that is elevated in epithelial ovarian cancer. Hypoxia inducible factor-1alpha (HIF-1alpha) is a transcription factor that plays a regulatory role in the expression of VEGF. Currently, there is limited information regarding VEGF and HIF-1alpha expression in epithelial ovarian cancer specimens. The objective of this study was to measure VEGF and HIF-1alpha expression in epithelial ovarian cancer samples and to compare VEGF and HIF-1alpha expression between ovarian cancer and normal ovarian tissue METHODS: . Serial sections of paraffin-embedded ovarian tissues from a study group of 16 control patients and 37 patients diagnosed with epithelial ovarian cancer were analyzed for expression of VEGF and HIF-1alpha using immunohistochemistry. RESULTS: There was a strong correlation between the average expression scores of VEGF and HIF-1alpha expression (r(2) = 0.991). There was a significant increase in expression of VEGF in stage III and IV tumors over that in controls (P < 0.005). There was also a significant increase in HIF-1alpha expression in stage III and IV tumors over that in controls (P < 0.005 and P < 0.05, respectively). CONCLUSION: VEGF and HIF-1alpha are both expressed in epithelial ovarian cancer. VEGF expression correlated with HIF-1alpha expression, suggesting that HIF-1alpha may contribute to the overexpression of VEGF observed in epithelial ovarian cancer.