Promoting deceased organ and tissue donation registration in family physician waiting rooms (RegisterNow-1 trial): study protocol for a pragmatic, stepped-wedge, cluster randomized controlled registry

Abstract Background There is a worldwide shortage of organs available for transplant, leading to preventable mortality associated with end-stage organ disease. While most citizens in many countries with an intent-to-donate “opt-in” system support organ donation, registration rates remain low. In Canada, most Canadians support organ donation but less than 25% in most provinces have registered their desire to donate their organs when they die. The family physician office is a promising yet underused setting in which to promote organ donor registration and address known barriers and enablers to registering for deceased organ and tissue donation. We developed a protocol to evaluate an intervention to promote registration for organ and tissue donation in family physician waiting rooms. Methods/design This protocol describes a planned, stepped-wedge, cluster randomized registry trial in six family physician offices in Ontario, Canada to evaluate the effectiveness of reception staff providing patients with a pamphlet that addresses barriers and enablers to registration including a description of how to register for organ donation. An Internet-enabled tablet will also be provided in waiting rooms so that interested patients can register while waiting for their appointments. Family physicians and reception staff will be provided with training and/or materials to support any conversations about organ donation with their patients. Following a 2-week control period, the six offices will cross sequentially into the intervention arm in randomized sequence at 2-week intervals until all offices deliver the intervention. The primary outcome will be the proportion of patients visiting the office who are registered organ donors 7 days following their office visit. We will evaluate this outcome using routinely collected registry data from provincial administrative databases. A post-trial qualitative evaluation process will assess the experiences of reception staff and family physicians with the intervention and the stepped-wedge trial design. Discussion Promoting registration for organ donation in family physician offices is a potentially useful strategy for increasing registration for organ donation. Increased registration may ultimately help to increase the number of organs available for transplant. The results of this trial will provide important preliminary data on the effectiveness of using family physician offices to promote registration for organ donation. Trial registration ClinicalTrials.gov, ID: NCT03213171 . Registered on 11 July 2017

AT7867 Is a Potent and Oral Inhibitor of AKT and p70 S6 Kinase That Induces Pharmacodynamic Changes and Inhibits Human Tumor Xenograft Growth

The serine/threonine kinase AKT plays a pivotal role in signal transduction events involved in malignant transformation and chemoresistance and is an attractive target for the development of cancer therapeutics. Fragment-based lead discovery, combined with structure-based drug design, has recently identified AT7867 as a novel and potent inhibitor of both AKT and the downstream kinase p70 S6 kinase (p70S6K) and also of protein kinase A. This ATP-competitive small molecule potently inhibits both AKT and p70S6K activity at the cellular level, as measured by inhibition of GSK3? and S6 ribosomal protein phosphorylation, and also causes growth inhibition in a range of human cancer cell lines as a single agent. Induction of apoptosis was detected by multiple methods in tumor cells following AT7867 treatment. Administration of AT7867 (90 mg/kg p.o. or 20 mg/kg i.p.) to athymic mice implanted with the PTEN-deficient U87MG human glioblastoma xenograft model caused inhibition of phosphorylation of downstream substrates of both AKT and p70S6K and induction of apoptosis, confirming the observations made in vitro. These doses of AT7867 also resulted in inhibition of human tumor growth in PTEN-deficient xenograft models. These data suggest that the novel strategy of AKT and p70S6K blockade may have therapeutic value and supports further evaluation of AT7867 as a single-agent anticancer strategy

Active PI3K Pathway Causes an Invasive Phenotype Which Can Be Reversed or Promoted by Blocking the Pathway at Divergent Nodes

<div><p>The PTEN/PI3K pathway is commonly mutated in cancer and therefore represents an attractive target for therapeutic intervention. To investigate the primary phenotypes mediated by increased pathway signaling in a clean, patient-relevant context, an activating PIK3CA mutation (H1047R) was knocked-in to an endogenous allele of the MCF10A non-tumorigenic human breast epithelial cell line. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036402#s1">Introduction</a> of an endogenously mutated PIK3CA allele resulted in a marked epithelial-mesenchymal transition (EMT) and invasive phenotype, compared to isogenic wild-type cells. The invasive phenotype was linked to enhanced PIP₃ production via a S6K-IRS positive feedback mechanism. Moreover, potent and selective inhibitors of PI3K were highly effective in reversing this phenotype, which is optimally revealed in 3-dimensional cell culture. In contrast, inhibition of Akt or mTOR exacerbated the invasive phenotype. Our results suggest that invasion is a core phenotype mediated by increased PTEN/PI3K pathway activity and that therapeutic agents targeting different nodes of the PI3K pathway may have dramatic differences in their ability to reverse or promote cancer metastasis.</p> </div

MCF10A cells undergo EMT-like changes in the presence of a PI3K mutation.

<p>(A) Comparative gene expression analysis of the parental and knock-in clone. Expression profile differences of commonly associated epithelial and mesenchymal genes by microarray are shown by heat map for a ratio of gene expression levels (H1047R A to parental). (B) Effects of GDC-0941 and erlotinib combination treatment in parental and knock-in cell lines. Percent growth inhibition data from a viability assay is shown at EC₅₀ doses of single agent GDC-0941 and 2.5 µM erlotinib after drug incubation. Each bar indicates mean % inhibition ±SEM from quadruplicate wells.</p

Compounds used for viability and 3-D cell culture assays.

<p>Specificities and CellTiter-Glo viability assay EC₅₀s for parental and H1047R clones.</p

PI3K signaling responses to PI3K pathway inhibition.

<p>(A) Knock-in cells (H1047R A) were cultured with inhibitors at concentrations used in the 3-D culture assay and analyzed by Western blotting for indicated treatment times. (B) PIP₃ levels assessed at 48 hours post treatment with 3-D culture concentrations of inhibitors in the knock-in clone (H1047R A). (C) PI3K and Akt inhibitor effects can be distinguished in 3-D culture through variations in cellular morphology. Phase contrast images of the knock-in clone (H1047R A) show representative phenotypes of treatment with 0.5 µM GDC-0941, 5 µM AKT1/2i, or the same compound doses in combination after 2 days in culture.</p

MCF10A knock-in cells show a more invasive phenotype in 3-D cell culture.

<p>(A) Parental and H1047R A cells were cultured for 2 days in the presence or absence of GDC-0941 (0.5 µM), PI103 (0.5 µM), PI3Ki-A/D (2 µM), AKT1/2i (5 µM) or mTOR1/2i (5 µM). (B) A mathematical distribution of acinar size (area) and shape (shape factor) was used to assess morphology changes with drug treatments on day 2. Data are plotted as the mean (horizontal line), middle 50% of data (box), and 95% confidence interval (lines). Pair-wise comparisons to the DMSO control were done by Student's t test. GDC-0941 and PI103 treatments resulted in significant morphology changes in both the parental and H1047R A clone (p<0.02, area or shape factor). PI3Ki-A/D treatment resulted in significant morphological changes in parental (p<0.006, area) or the H1047R clone (p<0.003, area or shape factor). Statistical significance was also achieved in the H1047R clone with the AKT1/2i (p<0.0002, area or shape factor) or mTOR1/2i (p = 0.03, shape factor).</p

MCF10A knock-in cells show enhanced migration through matrigel.

<p>(A) Cells were plated under the conditions indicated and allowed to migrate for 24 hours. Representative images for each condition are shown. (B) Quantification of migrated cells in triplicate wells.</p

Effects of the H1047R mutation on PI3K pathway signaling, cell viability and proliferation.

<p>(A) Analysis of PIP₃ levels in parental and knock-in clones. (B) Parental and knock-in clones were cultured with dose-titrated small molecule inhibitors and cell viability was assessed after four days. (C) Parental and knock-in clones were cultured in the presence or absence of GDC-0941 dosed at an EC₅₀ concentration for the time points indicated and analyzed by Western blotting. (D) The parental and knock-in clones were cultured in the presence or absence of GDC-0941 for 48 hours and proliferation was assessed in triplicate samples by BrdU labeling.</p

siRNA knockdown of p110α, Akt or mTOR phenocopies small molecule inhibitor treatments.

<p>(A) Knock-in cells (H1047R A) were transfected with the indicated siRNAs and plated in the 3-D culture assay for 5 days. (B) Assessment of siRNA knockdown by Western blot in the knock-in clone (H1047R A) 48 hours after transfection (NT = Non-targeting control siRNA).</p