Monitoring performance of sites within multicentre randomised trials: a systematic review of performance metrics.

BACKGROUND: Large multicentre trials are complex and expensive projects. A key factor for their successful planning and delivery is how well sites meet their targets in recruiting and retaining participants, and in collecting high-quality, complete data in a timely manner. Collecting and monitoring easily accessible data relevant to performance of sites has the potential to improve trial management efficiency. The aim of this systematic review was to identify metrics that have either been proposed or used for monitoring site performance in multicentre trials. METHODS: We searched the Cochrane Library, five biomedical bibliographic databases (CINAHL, EMBASE, Medline, PsychINFO and SCOPUS) and Google Scholar for studies describing ways of monitoring or measuring individual site performance in multicentre randomised trials. Records identified were screened for eligibility. For included studies, data on study content were extracted independently by two reviewers, and disagreements resolved by discussion. RESULTS: After removing duplicate citations, we identified 3188 records. Of these, 21 were eligible for inclusion and yielded 117 performance metrics. The median number of metrics reported per paper was 8, range 1-16. Metrics broadly fell into six categories: site potential; recruitment; retention; data collection; trial conduct and trial safety. CONCLUSIONS: This review identifies a list of metrics to monitor site performance within multicentre randomised trials. Those that would be easy to collect, and for which monitoring might trigger actions to mitigate problems at site level, merit further evaluation.

Development of a standardised set of metrics for monitoring site performance in multicentre randomised trials: a Delphi study.

BACKGROUND: Site performance is key to the success of large multicentre randomised trials. A standardised set of clear and accessible summaries of site performance could facilitate the timely identification and resolution of potential problems, minimising their impact. The aim of this study was to identify and agree a core set of key performance metrics for managing multicentre randomised trials. METHODS: We used a mixed methods approach to identify potential metrics and to achieve consensus about the final set, adapting methods that are recommended by the COMET Initiative for developing core outcome sets in health care. We used performance metrics identified from our systematic search and focus groups to create an online Delphi survey. We invited respondents to score each metric for inclusion in the final core set, over three survey rounds. Metrics scored as critical by ≥70% and unimportant by <15% of respondents were taken forward to a consensus meeting of representatives from key UK-based stakeholders. Participants in the consensus meeting discussed and voted on each metric, using anonymous electronic voting. Metrics with >50% of participants voting for inclusion were retained. RESULTS: Round 1 of the Delphi survey presented 28 performance metrics, and a further six were added in round 2. Of 294 UK-based stakeholders who registered for the Delphi survey, 211 completed all three rounds. At the consensus meeting, 17 metrics were discussed and voted on: 15 metrics were retained following survey round 3, plus two others that were preferred by consensus meeting participants. Consensus was reached on a final core set of eight performance metrics in three domains: (1) recruitment and retention, (2) data quality and (3) protocol compliance. A simple tool for visual reporting of the metrics is available from the Nottingham Clinical Trials Unit website. CONCLUSIONS: We have established a core set of metrics for measuring the performance of sites in multicentre randomised trials. These metrics could improve trial conduct by enabling researchers to identify and address problems before trials are adversely affected. Future work could evaluate the effectiveness of using the metrics and reporting tool.

Internalisation of uncross-linked rituximab is not essential for the induction of caspase-independent killing in Burkitt lymphoma cell lines.

Characterising the mechanisms underpinning caspase-independent programmed cell death (CI-PCD) induction by uncross-linked rituximab in B-cells may positively impact upon the treatment of disease states in which the classical apoptotic pathway is disabled. The necessity of rituximab internalisation for CI-PCD induction was investigated by flow cytometry and confocal microscopy in human BL cell lines with (e.g. Mutu I) and without (Mutu III) susceptibility to rituximab-induced killing. Flow cytometry demonstrated small, significant and similar amounts of rituximab internalisation by Mutu I cells after 1, 2, 4 and 24 h (p < 0.03, n = 5) and Mutu III cells after 0.5, 2, 4 and 24 h (p < 0.05, n = 4). Confocal microscopy confirmed this. Cytochalasin B and latrunculin A significantly inhibited rituximab-induced CI-PCD (p < or = 0.04, n = 6 and p = 0.01, n = 6, respectively) and internalisation (p = 0.02, n = 5 and p = 0.0002, n = 6, respectively) in Mutu I cells, but confocal microscopy showed no correlation between internalised rituximab and phosphatidylserine exposure. We conclude that rituximab internalisation is not essential for CI-PCD induction in BL cell lines.

A requirement for calcium in the caspase-independent killing of Burkitt lymphoma cell lines by Rituximab.

The therapeutic monoclonal antibody rituximab has previously been shown to kill B cells in a caspase-independent manner. The signalling pathways underpinning this novel death pathway are unknown. The present study showed that rituximab treatment of Burkitt lymphoma cell lines induced a slow rise in intracellular calcium ([Ca(2+)](i)). This rise was only witnessed in cell lines that were killed by antibody, suggesting a critical role for Ca(2+) in mediating rituximab-driven caspase-independent cell death. Inhibition of the two main intracellular store-located Ca(2+) channels, i.e. the ryanodine and inositol-1,4,5-triphosphate receptor channels by dantrolene and xestospongen-c respectively did not prevent the rise in Ca(2+) seen with rituximab or protect cells from subsequent death. In sharp contrast, inhibition of Ca(2+) entry via plasma membrane channels with (2-aminoethoxy) diphenylborate or SKF-96365 or complete chelation of extracellular Ca(2+) with ethyleneglycol bis (aminoethylether) tetra-acetate inhibited the rise in [Ca(2+)](i) and increased cell viability. Together, these data suggest that ligation of the CD20 receptor with rituximab allows a slow sustained influx of Ca(2+) from the external environment that under certain conditions can lead to cell death.

P-glycoprotein is implicated in the inhibition of ceramide-induced apoptosis in TF-1 acute myeloid leukemia cells by modulation of the glucosylceramide synthase pathway.

OBJECTIVE: Ceramide, an intermediate of apoptosis induction in response to chemotherapy, can be detoxified by glycosylation at the cytoplasmic surface of the Golgi membrane. P-glycoprotein (p-gp) might augment ceramide glycosylation by translocating glucosylceramide (GC) across the Golgi membrane. We aimed to show that glucosylceramide synthase (GCS) activity is linked to p-gp expression and resistance to ceramide-induced apoptosis in acute myeloid leukemia (AML). METHODS: Apoptosis and cell-cycle analysis were measured using propidium iodide staining and flow cytometry. Fluorescent microscopy assessed p-gp expression in, and rhodamine 123 uptake by, the Golgi. P-gp interaction with GC was assessed by modulation of rhodamine accumulation. The GCS activity assay was based upon the transfer of UDP-(3)H-glucose to C8-ceramide to form radiolabeled GC, by rate-limiting cell-derived GCS. TLC and fluorimetry were used to measure the metabolites of fluorescent ceramide. Cell viability was measured using 7-amino-actinomycin D staining and flow cytometry with an internal standard for cell enumeration. RESULTS: P-gp(+) cell lines (KG1a, TF-1) were resistant to C8-ceramide-induced apoptosis compared to p-gp(-) cell lines (HL-60, U937). P-gp inhibitors GF120918 and cyclosporin A enhanced ceramide-induced apoptosis in the p-gp expressing cells. P-gp expression was identified in the Golgi of these cells. Pgp's efflux function in TF-1 but not KG1a cells was inhibited by glucosylceramide. In the presence of p-gp inhibitors, R123 accumulation in the Golgi of TF-1 cells was lost, and GCS activity and lactosylceramide formation were downregulated. Intact cells were necessary for the involvement of p-gp in the regulation of GCS activity. CONCLUSION: Our data suggests that ceramide induces apoptosis in AML cells and that p-gp confers resistance to ceramide-induced apoptosis, with modulation of the ceramide-glucosylceramide pathway making a marked contribution to this resistance in TF-1 cells.

Resistance to spontaneous apoptosis in acute myeloid leukaemia blasts is associated with p-glycoprotein expression and function, but not with the presence of FLT3 internal tandem duplications.

The ability of acute myeloid leukaemia (AML) blasts to survive in culture has been associated with poor patient response to chemotherapy. Other biological factors predicting an adverse outcome include p-glycoprotein (pgp) expression, which is associated with a reduced remission rate, and the presence of fms-like tyrosine kinase 3 gene (FLT3) internal tandem duplications (ITDs), predictive of a high rate of leukaemic relapse. Our previous work has indicated a drug efflux-independent role for pgp in apoptosis resistance. We measured spontaneous in vitro apoptosis in 58 primary AML samples to establish its relationship with functional and phenotypic pgp and with FLT3 ITDs. Cells were incubated for 48 h in a suspension culture, and the remaining viable cells were counted by flow cytometry. Median survival was 38% of baseline values. Resistance to spontaneous apoptosis was strongly associated with pgp (MRK-16 antibody) expression (P = 0.001) and with pgp functional activity (P < 0.001). FLT3 ITDs, found in 20 cases, were inversely associated with functional pgp activity: thus, the median pgp modulation ratio was 2.0 in FLT3 wild-type cases and 1.38 in ITD cases (P = 0.018). Also, the presence of FLT3 ITDs was not associated with in vitro apoptosis resistance. In conclusion, we have found that the presence of FLT3 ITDs is not related to AML blast survival in vitro, and is inversely associated with pgp activity, whereas pgp expression and activity are associated with resistance to spontaneous apoptosis. These results may help to explain the differing adverse effects of pgp (on remission induction) and FLT3 ITDs (on relapse) in AML.

P-glycoprotein in acute myeloid leukaemia: therapeutic implications of its association with both a multidrug-resistant and an apoptosis-resistant phenotype.

P-glycoprotein (Pgp) expression is an independent prognostic factor for response to remission-induction chemotherapy in acute myeloblastic leukaemia, particularly in the elderly. There are several potential agents for modulating Pgp-mediated multi-drug resistance, such as cyclosporin A and PSC833, which are currently being evaluated in clinical trials. An alternative therapeutic strategy is to increase the use of drugs which are unaffected by Pgp. However, in this review, we explain why this may be more difficult than it appears. Evidence from in vitro studies of primary AML blasts supports the commonly held supposition that chemoresistance may be linked to apoptosis-resistance. We have found that Pgp has a drug-independent role in the inhibition of in vitro apoptosis in AML blasts. Modulation of cytokine efflux, signalling lipids and intracellular pH have all been suggested as ways by which Pgp may affect cellular resistance to apoptosis; these are discussed in this review. For a chemosensitising agent to be successful, it may be more important for it to enhance apoptosis than to increase drug uptake.