Long-term Cross-validation of Everolimus Therapeutic Drug Monitoring Assays: The Zortracker Study

Background: This ongoing academic collaboration was initiated for providing support to set up, validate, and maintain everolimus therapeutic drug monitoring assays and to study long-term interlaboratory performance. Methods: This study was based on EDTA whole blood samples collected from transplant patients treated with everolimus in a prospective clinical trial. Samples were handled under controlled conditions during collection, storage and were shipped on dry ice to minimize freeze–thaw cycles. For more than 1.5 years, participating laboratories received a set of 3 blinded samples on a monthly basis. Among others, these samples included individual patient samples, patient sample pools to assess long-term performance, and patient samples pools enriched with isolated everolimus metabolites. Results: The results between liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and the everolimus Quantitative Microsphere System (QMS, Thermo Fisher) assay were comparable. The monthly interlaboratory variability (coefficient of variation %) for cross-validation samples ranged from 6.5% to 23.2% (average of 14.8%) for LC-MS/MS and 4.2% to 26.4% (average of 11.1%) for laboratories using the QMS assay. A blinded long-term pool sample was sent to the laboratories for 13 months. The result was 5.31 ± 0.86 ng/mL (range, 2.9–7.8 ng/mL) for the LC-MS/MS and 5.20 ± 0.54 ng/mL (range, 4.0–6.8 ng/mL) for QMS laboratories. Enrichment of patient sample pools with 5–25 ng/mL of purified everolimus metabolites (46-hydroxy everolimus and 39-O-desmethyl everolimus) did not affect the results of either LC-MS/MS or QMS assays. Conclusions: Both LC-MS/MS and QMS assays gave similar results and showed similar performance, albeit with a trend toward higher interlaboratory variability among laboratories using LC-MS/MS than the QMS assay.