Cancer patients acceptance, understanding, and willingness-to-pay for pharmacogenomic testing.

BACKGROUND: Pharmacogenomics is gaining increasing importance in the therapeutics of cancer; yet, there is little knowledge of cancer patients' attitudes toward the use of pharmacogenomic testing in clinical practice. We carried out this study to explore cancer patients' acceptance, understanding, and willingness-to-pay for pharmacogenomic testing. MATERIALS AND METHODS: A broad cross-section of gastrointestinal, lung, breast, and other cancer patients were interviewed in terms of their acceptance of pharmacogenomic testing using hypothetical time, efficacy, and toxicity trade-off and willingness-to-pay scenarios. RESULTS: Among the 96% of 123 adjuvant patients accepting chemotherapy under optimal conditions, 99% wanted pharmacogenomic testing that could identify a subset of patients benefiting from chemotherapy, accepting median incurred costs of $2000 (range $0-25,000) and turnaround time for test results of 16 days (range 0-90 days). Among the 97% of 121 metastatic patients accepting chemotherapy, 97.4% wanted pharmacogenomic testing that could detect the risk of severe toxicity, accepting median incurred costs of $1000 (range $0-10,000) and turnaround time for results of 14 days (range 1-90 days). The majority of patients wanted to be involved in decision-making on pharmacogenomic testing; however, one in five patients lacked a basic understanding of pharmacogenomic testing. CONCLUSION: Among cancer patients willing to undergo chemotherapy, almost all wanted pharmacogenomic testing and were willing-to-pay for it, waiting several weeks for results. Although patients had a strong desire to be involved in decision-making on pharmacogenomic testing, a considerable proportion lacked the necessary knowledge to make informed choices.

Tracheostomies in term and preterm infants: A single-center Canadian retrospective cohort.

OBJECTIVE: To examine patient characteristics, hospital course, and medical outcomes of neonatal tracheostomy at a single center. DESIGN: Retrospective cohort study. SETTING: Level III neonatal intensive care units (NICUs) in Edmonton, Canada. PATIENTS: Infants admitted to NICU who underwent tracheostomy between January 2013 and December 2017 inclusive. MAIN OUTCOME MEASURES: Hospital course, discharge, and 3-year post-tracheostomy outcomes were compared between preterm infants <29 weeks gestation and infants with congenital anomalies. RESULTS: Forty-three infants were identified; seven were lost to follow-up and excluded. Of the 36 analyzed, 86% survived to discharge. At discharge, 13% were decannulated, 36% required no mechanical ventilation, and 52% required mechanical ventilation. Median hospitalization was 295 days. At 3 years post-tracheostomy, 97% were alive. Proportions of infants with tracheostomy in situ was 80%, 73%, and 60% at 1, 2, and 3 years post tracheostomy. Tracheostomy incidence was 2.7% for preterm infants <29 weeks gestational age with 55% for subglottic stenosis. All preterm infants received postnatal steroids. Preterm infants underwent tracheostomy at later chronological age (123 vs. 81 days, p < 0.001), but similar corrected gestational age (42 + 5 vs. 51 + 2 weeks, p = 0.095). Preterm infants had more intubation attempts (17 vs. 4, p < 0.001), total extubations (8 vs. 2, p < 0.001), and days on ventilation before tracheostomy (100 vs. 78, p < 0.001). CONCLUSIONS: Infants who underwent tracheostomy in a Canadian public healthcare setting demonstrated decreasing tracheostomy dependence and high survival post tracheostomy, despite prolonged hospitalization. Preterm infants had more intubation and extubation events which may have contributed to airway injury.

Versican mediates mesenchymal-epithelial transition.

Versican is a large extracellular chondroitin sulfate proteoglycan that belongs to the family of lecticans. Alternative splicing of versican generates at least four isoforms named V0, V1, V2, and V3. We show here that ectopic expression of versican V1 isoform induced mesenchymal-epithelial transition (MET) in NIH3T3 fibroblasts, and inhibition of endogenous versican expression abolished the MET in metanephric mesenchyme. MET in NIH3T3 cells was demonstrated by morphological changes and dramatic alterations in both membrane and cytoskeleton architecture. Molecular analysis showed that V1 promoted a "switch" in cadherin expression from N- to E-cadherin, resulting in epithelial specific adhesion junctions. V1 expression reduced vimentin levels and induced expression of occludin, an epithelial-specific marker, resulting in polarization of V1-transfected cells. Furthermore, an MSP (methylation-specific PCR) assay showed that N-cadherin expression was suppressed through methylation of its DNA promoter. Exogenous expression of N-cadherin in V1-transfected cells reversed V1's effect on cell aggregation. Reduction of E-cadherin expression by Snail transfection and siRNA targeting E-cadherin abolished V1-induced morphological alteration. Transfection of an siRNA construct targeting versican also reversed the changed morphology induced by V1 expression. Silencing of endogenous versican prevented MET of metanephric mesenchyme. Taken together, our results demonstrate the involvement of versican in MET: expression of versican is sufficient to induce MET in NIH3T3 fibroblasts and reduction of versican expression decreased MET in metanephric mesenchyme.

The effect of central loops in miRNA:MRE duplexes on the efficiency of miRNA-mediated gene regulation.

MicroRNAs (miRNAs) guide posttranscriptional repression of mRNAs. Hundreds of miRNAs have been identified but the target identification of mammalian mRNAs is still a difficult task due to a poor understanding of the interaction between miRNAs and the miRNA recognizing element (MRE). In recent research, the importance of the 5' end of the miRNA:MRE duplex has been emphasized and the effect of the tail region addressed, but the role of the central loop has largely remained unexplored. Here we examined the effect of the loop region in miRNA:MRE duplexes and found that the location of the central loop is one of the important factors affecting the efficiency of gene regulation mediated by miRNAs. It was further determined that the addition of a loop score combining both location and size as a new criterion for predicting MREs and their cognate miRNAs significantly decreased the false positive rates and increased the specificity of MRE prediction.

MiRNA-directed regulation of VEGF and other angiogenic factors under hypoxia.

MicroRNAs (miRNAs) are a class of 20-24 nt non-coding RNAs that regulate gene expression primarily through post-transcriptional repression or mRNA degradation in a sequence-specific manner. The roles of miRNAs are just beginning to be understood, but the study of miRNA function has been limited by poor understanding of the general principles of gene regulation by miRNAs. Here we used CNE cells from a human nasopharyngeal carcinoma cell line as a cellular system to investigate miRNA-directed regulation of VEGF and other angiogenic factors under hypoxia, and to explore the principles of gene regulation by miRNAs. Through computational analysis, 96 miRNAs were predicted as putative regulators of VEGF. But when we analyzed the miRNA expression profile of CNE and four other VEGF-expressing cell lines, we found that only some of these miRNAs could be involved in VEGF regulation, and that VEGF may be regulated by different miRNAs that were differentially chosen from 96 putative regulatory miRNAs of VEGF in different cells. Some of these miRNAs also co-regulate other angiogenic factors (differential regulation and co-regulation principle). We also found that VEGF was regulated by multiple miRNAs using different combinations, including both coordinate and competitive interactions. The coordinate principle states that miRNAs with independent binding sites in a gene can produce coordinate action to increase the repressive effect of miRNAs on this gene. By contrast, the competitive principle states when multiple miRNAs compete with each other for a common binding site, or when a functional miRNA competes with a false positive miRNA for the same binding site, the repressive effects of miRNAs may be decreased. Through the competitive principle, false positive miRNAs, which cannot directly repress gene expression, can sometimes play a role in miRNA-mediated gene regulation. The competitive principle, differential regulation, multi-miRNA binding sites, and false positive miRNAs might be useful strategies in the avoidance of unwanted cross-action among genes targeted by miRNAs with multiple targets.

Versican G3 domain promotes blood coagulation through suppressing the activity of tissue factor pathway inhibitor-1.

We have detected versican, a member of the large chondroitin sulfate proteoglycans, and its degraded C-terminal G3 fragments in human plasma and observed that the versican G3 domain promoted blood coagulation. Silencing G3 expression with small interfering RNA reduced the effect of G3 on coagulation. Plasma coagulation assays suggest that G3 enhances coagulation irrespective of its actions on platelets and white blood cells. To examine how versican affected blood coagulation, we used normal human plasma and different types of coagulation factor-deficient plasmas. The experiments indicated that versican enhanced coagulation through the extrinsic pathway, and that Factor VII was the target molecule. FVII activity assays showed that G3 activated FVII in the presence of plasma but not with purified FVII directly. Yeast two-hybrid, immunoprecipitation, and gel co-migration assays showed that G3 interacted with the tissue factor pathway inhibitor-1 (TFPI-1). TFPI-1 activity assays suggested that G3 inhibited TFPI-1 activity, allowing FVIIa and FXa to facilitate the coagulation process. G3-induced blood coagulation was further confirmed with a mouse model in a real-time manner. Taken together, these results indicate that versican may represent a new target for the development of therapies against atherosclerosis.