Analytical validation of an interferon-inducible gene expression kit as a potential diagnostic test for anifrolumab

Background: Anifrolumab, a fully human monoclonal antibody that binds to the type I interferon receptor, is in Phase III development for moderate to severe systemic lupus erythematosus (SLE). Patients with greater interferon-inducible gene signatures (IFNGS) have enhanced response to anifrolumab treatment. AstraZeneca and QIAGEN developed an in-vitro diagnostic test for interferon-inducible gene expression (IFIGx). Efficacy of anifrolumab will be evaluated in patients with high and low IFNGS. Objectives: We aimed to analytically validate the IFIGx kit for use in a pivotal clinical study and potentially to support future regulatory submission. Methods: The IFIGx kit measures expression of four interferon-inducible genes (IF127, IF114, IF114L and RSAD2) compared with three housekeeping genes. Measurements were performed on mRNA extracted from whole blood from adults with SLE. A score was generated that identified patients as “IFNGS test–high” or “IFNGS test–low.” Analytical validation involved six studies measuring lot interchangeability, linearity, repeatability, reproducibility, cross contamination, and system verification. Results: Repeatability was 100%. Reproducibility was >96%. No cross contamination was observed. Results of all studies validated the IFIGx kit (table 1). Abstract AB1212 – Table 1 Summary of Analytical Validation of Anifrolumab IFIGx Test Study Description Results Lot interchangeability Verification that scores and assay Ct values were robust when different lots of kit components were used Acceptance criterion was<0.58 Ct Lot interchangeability verified Largest observed change=0.16 Ct Linearity Verification, using linear and quadratic regression analyses, that mRNA input concentration (10 ng/µL) is in assay’s linear range Linearity verified Change in score over the concentration range on either side of 10 ng/µL=0.0043 Ct Repeatability Verification of Dx result repeatability when the same operator tested 60 random samples using the same kit lot and instrument Repeatability verified Observed repeatability=100% Reproducibility Verification of Dx result reproducibility when multiple operators tested 48 random samples using multiple kit lots and instruments at different sites For verification, overall rate of correct calls must be≥95% Reproducibility verified Overall rate=99.7% After six samples with values close to the cut-off were added for further confirmation, overall rate=96.5% Cross contamination Investigation of inter- and intra-run cross contamination No cross contamination No cross contamination found in reverse transcription or PCR steps System verification Verification of functionality and utility of the IFIGx software and IFIGx assay package System verification confirmed Software flags produced as expected Ct, cycle threshold (PCR cycle at which fluorescence rises above background level); Dx, diagnostic; IFIGx, interferon-inducible gene expression; PCR, polymerase chain reaction; mRNA, messenger ribonucleic acid. Conclusions: The IFIGx kit was shown to be a robust, reproducible diagnostic test for IFNGS. The IFIGx kit has demonstrated value in prior anifrolumab studies, and will be used both for patient stratification in Phase III studies and to support anifrolumab regulatory filings. Reference: Furie, et al. Arthritis Rheumatol. 2017;69:376–86. Disclosure of Interest: P. Brohawn Employee of: MedImmune LLC, B. Higgs Employee of: MedImmune LLC, S. Patel Employee of: AstraZeneca, A. Moody Employee of: QIAGEN Manchester Ltd, P. Cooper Employee of: QIAGEN Manchester Ltd, K. Ranade Employee of: MedImmune LLC DOI: 10.1136/annrheumdis-2018-eular.2547 Citation: Ann Rheum Dis, volume 77, supplement Suppl, year 2018, page A1704Session: Diagnostics and imaging procedures