Prognostic gene expression signature for high-grade serous ovarian cancer.

BACKGROUND: Median overall survival (OS) for women with high-grade serous ovarian cancer (HGSOC) is ∼4 years, yet survival varies widely between patients. There are no well-established, gene expression signatures associated with prognosis. The aim of this study was to develop a robust prognostic signature for OS in patients with HGSOC. PATIENTS AND METHODS: Expression of 513 genes, selected from a meta-analysis of 1455 tumours and other candidates, was measured using NanoString technology from formalin-fixed paraffin-embedded tumour tissue collected from 3769 women with HGSOC from multiple studies. Elastic net regularization for survival analysis was applied to develop a prognostic model for 5-year OS, trained on 2702 tumours from 15 studies and evaluated on an independent set of 1067 tumours from six studies. RESULTS: Expression levels of 276 genes were associated with OS (false discovery rate < 0.05) in covariate-adjusted single-gene analyses. The top five genes were TAP1, ZFHX4, CXCL9, FBN1 and PTGER3 (P < 0.001). The best performing prognostic signature included 101 genes enriched in pathways with treatment implications. Each gain of one standard deviation in the gene expression score conferred a greater than twofold increase in risk of death [hazard ratio (HR) 2.35, 95% confidence interval (CI) 2.02-2.71; P < 0.001]. Median survival [HR (95% CI)] by gene expression score quintile was 9.5 (8.3 to -), 5.4 (4.6-7.0), 3.8 (3.3-4.6), 3.2 (2.9-3.7) and 2.3 (2.1-2.6) years. CONCLUSION: The OTTA-SPOT (Ovarian Tumor Tissue Analysis consortium - Stratified Prognosis of Ovarian Tumours) gene expression signature may improve risk stratification in clinical trials by identifying patients who are least likely to achieve 5-year survival. The identified novel genes associated with the outcome may also yield opportunities for the development of targeted therapeutic approaches.

SNX9 promotes metastasis by enhancing cancer cell invasion via differential regulation of RhoGTPases.

Despite current advances in cancer research, metastasis remains the leading factor in cancer-related deaths. Here, we identify sorting nexin 9 (SNX9) as a new regulator of breast cancer metastasis. We detected an increase in SNX9 expression in human breast cancer metastases compared with primary tumors and demonstrated that SNX9 expression in MDA-MB-231 breast cancer cells is necessary to maintain their ability to metastasize in a chick embryo model. Reciprocally, SNX9 knockdown impairs the process. In vitro studies using several cancer cell lines derived from a variety of human tumors revealed a role for SNX9 in cell invasion and identified mechanisms responsible for this novel function. We showed that SNX9 controls the activation of RhoA and Cdc42 GTPases and also regulates cell motility via the modulation of well-known molecules involved in metastasis, namely RhoA-ROCK and N-WASP. In addition, we have discovered that SNX9 is required for RhoGTPase-dependent, clathrin-independent endocytosis, and in this capacity, can functionally substitute to the bona fide Rho GAP, GRAF1 (GTPase Regulator Associated with Focal Adhesion Kinase). Together, our data establish novel roles for SNX9 as a multifunctional protein scaffold that regulates, and potentially coordinates, several cellular processes that together can enhance cancer cell metastasis.

Extracellular vesicles such as prostate cancer cell fragments as a fluid biopsy for prostate cancer.

Extracellular vesicles (EVs) are cell-derived vesicles generated through a process of cell membrane shedding or storage vesicle release, as occurs during apoptosis, necrosis or exocytosis. Initially perceived as cellular by-products or 'dust' of insignificant biological importance, recent research has shed light on the role of EVs as mediators of intercellular communication, blood coagulation and disease progression. The prostate is a source of EVs and their abundance in complex biological fluids such as plasma, serum and urine make them compelling entities for a 'fluid biopsy'. As such, prostate cancer cell fragments (PCCF) are EVs generated by the tumor resident within the prostate and are also present in blood, expressing a portion of biomarkers representative of the primary tumor. High-throughput analytical techniques to determine biomarker expression on EVs is the last hurdle towards translating the full potential of prostate EVs for clinical use. We describe current state-of-the-art methods for the analysis of prostate-derived EVs in patient fluids such as plasma and the challenges that lie ahead in this emerging field of translational research.

The interaction between caveolin-1 and Rho-GTPases promotes metastasis by controlling the expression of alpha5-integrin and the activation of Src, Ras and Erk.

Proteins containing a caveolin-binding domain (CBD), such as the Rho-GTPases, can interact with caveolin-1 (Cav1) through its caveolin scaffold domain. Rho-GTPases are important regulators of p130(Cas), which is crucial for both normal cell migration and Src kinase-mediated metastasis of cancer cells. However, although Rho-GTPases (particularly RhoC) and Cav1 have been linked to cancer progression and metastasis, the underlying molecular mechanisms are largely unknown. To investigate the function of Cav1-Rho-GTPase interaction in metastasis, we disrupted Cav1-Rho-GTPase binding in melanoma and mammary epithelial tumor cells by overexpressing CBD, and examined the loss-of-function of RhoC in metastatic cancer cells. Cancer cells overexpressing CBD or lacking RhoC had reduced p130(Cas) phosphorylation and Rac1 activation, resulting in an inhibition of migration and invasion in vitro. The activity of Src and the activation of its downstream targets FAK, Pyk2, Ras and extracellular signal-regulated kinase (Erk)1/2 were also impaired. A reduction in α5-integrin expression, which is required for binding to fibronectin and thus cell migration and survival, was observed in CBD-expressing cells and cells lacking RhoC. As a result of these defects, CBD-expressing melanoma cells had a reduced ability to metastasize in recipient mice, and impaired extravasation and survival in secondary sites in chicken embryos. Our data indicate that interaction between Cav1 and Rho-GTPases (most likely RhoC but not RhoA) promotes metastasis by stimulating α5-integrin expression and regulating the Src-dependent activation of p130(Cas)/Rac1, FAK/Pyk2 and Ras/Erk1/2 signaling cascades.

Validation of flow cytometric detection of platelet microparticles and liposomes by atomic force microscopy.

BACKGROUND: Platelet microparticles (PMPs) are a promising prognostic marker for thrombotic disorders because of their release during platelet activation. The use of flow cytometry for the enumeration of PMPs in plasma has generated controversy due to their size, which is below the stated detection limits of conventional flow cytometry instruments. The potential impact of this is an underestimation of PMP counts. OBJECTIVES/METHODS: To address this possibility, we used a combination of fluorescence-activated cell sorting (FACS) and atomic force microscopy (AFM) to determine the size distribution of PMPs present in plasma from acute myocardial infarction (AMI) patients and normal volunteers, and PMPs generated by expired platelet concentrates and washed platelets treated with agonists such as thrombin and calcium ionophore (A23187). RESULTS: According to AFM image analysis, there was no statistically significant difference in height or volume distributions in PMPs from thrombin-activated, calcium ionophore-activated, expired platelet concentrates and plasma from healthy volunteers and AMI patients. Based on volume, expired platelets released the greatest proportion of exosomes (< 1.0 × 10(-22) L(3) in volume) in relation to the entire PMP population (29.7%) and the smallest proportion of exosomes was observed in AMI patient plasma (1.8%). Moreover, AFM imaging revealed that PMPs from expired platelets exhibited smooth surfaces compared with other PMP types but this was not statistically significant. CONCLUSIONS: We confirm that flow cytometry is capable of analyzing PMPs from plasma by using AFM to perform nanoscale measurements of individual PMP events isolated by FACS. This method also provided the first quantitative nanoscale images of PMP ultrastructure.

Exon-array profiling unlocks clinically and biologically relevant gene signatures from formalin-fixed paraffin-embedded tumour samples.

BACKGROUND: Degradation and chemical modification of RNA in formalin-fixed paraffin-embedded (FFPE) samples hamper their use in expression profiling studies. This study aimed to show that useful information can be obtained by Exon-array profiling archival FFPE tumour samples. METHODS: Nineteen cervical squamous cell carcinoma (SCC) and 9 adenocarcinoma (AC) FFPE samples (10-16-year-old) were profiled using Affymetrix Exon arrays. The gene signature derived was tested on a fresh-frozen non-small cell lung cancer (NSCLC) series. Exploration of biological networks involved gene set enrichment analysis (GSEA). Differential gene expression was confirmed using Quantigene, a multiplex bead-based alternative to qRT-PCR. RESULTS: In all, 1062 genes were higher in SCC vs AC, and 155 genes higher in AC. The 1217-gene signature correctly separated 58 NSCLC into SCC and AC. A gene network centered on hepatic nuclear factor and GATA6 was identified in AC, suggesting a role in glandular cell differentiation of the cervix. Quantigene analysis of the top 26 differentially expressed genes correctly partitioned cervix samples as SCC or AC. CONCLUSION: FFPE samples can be profiled using Exon arrays to derive gene expression signatures that are sufficiently robust to be applied to independent data sets, identify novel biology and design assays for independent platform validation.

Kruppel-like factor 5 modulates p53-independent apoptosis through Pim1 survival kinase in cancer cells.

Although Kruppel-like factor 5 (KLF5) is a transcription factor that has been implicated in pathways critical to carcinogenesis, controversy persists as to whether it functions as a tumor suppressor or as an oncogene. Here, we describe a novel role for KLF5 in a p53-independent apoptotic pathway. Using RNA-interference technology, we show that cells deficient in KLF5 have increased sensitivity to DNA damage, regardless of p53 status. Both p53 and p53-dependent factors are unaffected by KLF5 depletion. Instead, the apoptotic phenotype consequent to damage is associated with reduced bad phosphorylation, and downregulation of Pim1. Consistently, transfection of wild-type Pim1 is sufficient to rescue this phenotype. Previous data have shown a number of putative Sp1-binding consensus sequences on the Pim1 promoter. Remarkably, chromatin immunoprecipitation studies show that KLF5 binds to the Pim1 promoter, and that binding increases soon after damage. These results identify a novel, p53-independent apoptotic pathway through which KLF5 functions in response to DNA damage. Therapeutic deregulation of this pathway could be used to modulate chemosensitivity.

Vimentin autoantibodies induce platelet activation and formation of platelet-leukocyte conjugates via platelet-activating factor.

Anti-vimentin antibodies (AVA) are associated with autoimmunity and solid organ transplantation, conditions associated with vascular disease, but their contribution to disease pathogenesis is unknown. Here, we have examined interactions between AVA (mAb and serum from patients) and various leukocyte populations using whole blood and flow cytometry. Normal blood treated with patient sera containing high AVA-IgM titers or with a vimentin-specific monoclonal IgM led to activation of platelets and other leukocytes, as demonstrated by induced expression of P-selectin, fibrinogen, tissue factor, and formation of platelet:leukocyte (P:L) conjugates and a reduction in platelet counts. This activity was antigen (vimentin)-specific and was not mediated by irrelevant IgM antibodies. Flow cytometry demonstrated that AVA do not bind directly to resting platelets in whole blood, but they bind to approximately 10% of leukocytes. Supernatant, derived from AVA-treated leukocytes, induced platelet activation, as measured by the generation of platelet microparticles, when added to platelet-rich plasma. When AVA were added to whole blood in the presence of CV-6209, a platelet-activating factor (PAF) receptor inhibitor, platelet depletion was inhibited. This suggests that PAF is one of the mediators released from AVA-activated leukocytes that leads to P:L conjugation formation and platelet activation. In summary, AVA bind to leukocytes, resulting in release of a PAF and prothrombotic factor that exert a paracrine-activating effect on platelets. Overall, this proposed mechanism may explain the pathogenesis of thrombotic events in autoimmune diseases associated with AVA.

Survey on mammographic screening among women aged 40 to 65 years old at polyclinics.

INTRODUCTION: Breast cancer is the commonest female cancer in Singapore. It is steadily rising with an incidence of 53.1 cases per 100,000 persons per year among women. Screening for detection of early lesions which are highly curable helps to reduce mortality. METHODS: Over three afternoon sessions in December 2003, 224 female patients aged 40-65 years, participated in interviews conducted by the National Healthcare Group Polyclinics, Singapore. The survey sought information on mammographic screening history, the time interval since the previous mammographic screening, and the reasons for not going for the screening. RESULTS: The survey found that only 26.4 percent (28 out of 106) among those aged 40 to 49 years had mammographic screening done within the past one year, and 43.2 percent (51 out of 118) among those aged 50 to 65 years had screening done within the last two years. Chinese women were twice more likely than Malay women to have a mammogram done. The commonest reasons for not wanting to have mammographic screening among women who did not have a mammogram done or had mammogram done more than two years ago, were lack of time (42.5 percent), fear of pain during the procedure (26.9 percent), and the belief that cancer would not happen to them (24.6 percent). CONCLUSION: Despite publicity on breast cancer being the commonest cancer among women in Singapore and cure being possible if the malignancy was detected early, close to half of the women aged 40-65 years old who attended the National Healthcare Group Polyclinics did not have mammographic screening done. One-quarter of the women who did not have mammogram screening did not do so as they did not think cancer would happen to them.

Evaluation of ketamine abuse using hair analysis: concentration trends in a Singapore population.

This paper presents a method for the detection of ketamine in hair. Hair samples (25 mg) were washed, pulverized, and digested in hydrochloric acid (0.5 M) overnight at 45 degrees C. The samples were extracted by an automated solid-phase extraction procedure, and the extracts were subsequently analyzed using gas chromatography-mass spectrometry in selected ion monitoring mode. Linearity up to 120 ng/mg was obtained for both ketamine and norketamine with correlation coefficients of 0.9987 and 0.9985, respectively. Limit of detection was found to be at 0.4 ng/mg for both drugs while the limit of quantitation was found to be 0.6 and 0.8 ng/mg for ketamine and norketamine, respectively. The validated method was used in the analysis of 91 hair segments obtained from 54 ketamine abusers. Based upon the voluntary confession of the ketamine abusers, a correlation between the amount of ketamine detected and the frequency of abuse was observed.

Glycolysis and pyruvate oxidation in cardiac hypertrophy--why so unbalanced?

Cardiac hypertrophy, induced by chronic pressure or volume overload, is associated with abnormalities in energy metabolism as well as characteristic increases in muscle mass and alterations in the structure of the heart. Hypertrophied hearts display increased rates of glycolysis and overall glucose utilization, but rates of pyruvate oxidation do not rise in step with rates of pyruvate generation. Glycolysis and glucose oxidation, therefore, become markedly less 'coupled' in hypertrophied hearts than in non-hypertrophied hearts. Because the pyruvate dehydrogenase complex (PDC) contributes so powerfully to the control of glucose oxidation, we set out to test the hypothesis that the function of PDC is impaired in cardiac hypertrophy. In this review we describe evidence indicating that the alterations in glucose metabolism in hypertrophied hearts cannot be explained simply by changes in PDC expression or control. Additional mechanisms that may lead to an altered balance of pyruvate metabolism in cardiac hypertrophy are discussed, with commentaries on possible changes in pyruvate transport, NADH shuttles, lactate dehydrogenase, and amino acid metabolism.