Discussion
Recently, an international panel of MS experts on NAbs to IFNB therapy convened under the auspices of the Neutralizing Antibodies on Interferon beta in Multiple Sclerosis consortium (NAbinMS: a collaborative project funded by the European Union) and issued recommendations for clinical use of data on NAb to IFNB therapy [
21]. In this position paper, the use of the MxA induction assay is suggested to test neutralizing activity to IFNB therapy and a switch to a non-IFNB therapy is recommended in cases of sustained high-titer NAb positivity and/or lack of IFNB bioactivity. However, in clinical practice, other assays are also employed to detect and quantify the level of neutralizing activity to IFNB treatment, such as the CPE and the luciferase assay. These assays differ not only in their sensitivity and specificity but also in their performance in various laboratories resulting in differences with respect to NAb detection and titer quantification [
22]. Furthermore, difficulties in standardizing these assays have considerably hindered inter-laboratory comparison of NAb data [
23].
Here, we assessed the variability and agreement of NAb testing results between different methods and laboratories offering routine NAb testing to detect and quantify neutralizing activity to IFNB treatment in clinical practice. There was a high intra-laboratory agreement for NAb detection and quantification with the MxA induction assay that was used in the BEYOND trial. This finding reveals a robust stability of the neutralizing activity in patient sera stored for several years and a good retest reliability of the MxA induction assay that was used. With respect to the inter-laboratory comparisons, there remained high agreement of NAb detection and quantification between the MxA induction assay and one laboratory using a CPE assay, with just a systematic difference in titers. This contrasted with only moderate agreement between the MxA assay and the CPE assay used by a second laboratory and to the modest agreement between the NAb findings of the two CPE laboratories. Also, the agreement between the MxA assay and both CPE assays with a luciferase assay was only moderate. Of note, when comparing individual titer levels, substantial differences were recorded between laboratories: we frequently observed that high-titer NAb positivity was measured by one lab in a sample that was found to contain low-titer NAb positivity by another lab. Occasionally, even high-titer NAb positivity was reported for samples that were tested NAb negative by another laboratory using a different method.
The focus of this work was to investigate the differences in NAb testing and quantification between laboratories offering NAb services in clinical practice. More research is warranted to better understand the observed discrepancies. This research could constitute the starting point for further standardization of NAb testing. For instance, higher titers have been observed when IFNB-1a vs. IFNB-1b was used as the antigen to test neutralizing activity, a finding that is in line with the somewhat higher titers of our laboratory D using IFNB-1a in a Luciferase assay [
24]. In light of our findings, results of NAb testing obtained in different assays/laboratories must be interpreted or compared with caution. This is of particular importance when titer thresholds are considered for clinical decision-making as there are both systematic shifts and potentially high variability between test methods and among laboratories. Because high-titer NAb positivity may be regarded as a sufficient reason to stop IFNB and switch to a non-IFNB product even if patients are doing well [
21], a false-positive NAb titer might have disadvantageous therapeutic consequences. Therefore, our findings underscore the need for global standardization efforts whenever complex indirect biological assays are to be used. They also support the recommendation of NAb experts to supplement testing for NAbs with more direct measurements of IFNB-induced biological activity in patients undergoing IFNB treatment (such as the measurement of MxA induction following an IFNB injection) [
21]. Yet, the predictive value of measuring IFNB activity needs still to be established in a well-designed prospective trial [
25].
Competing interests
The BEYOND study and NAb testing performed for this manuscript was funded by Bayer Pharma AG.
Dr. Hartung has received personal compensation from Biogen Idec, Teva, sanofi-aventis, Novartis Pharma, Merck Serono and Bayer Schering Pharma AG for speaking and consulting services. The MS Center at the Department of Neurology Heinrich-Heine-University is in part supported by the Walter-and-Ilse-Rose Stiftung.
Dr. Kieseier has received honoraria for lecturing, travel expenses for attending meetings and financial support for research from Baxter, Bayer Schering, Biogen Idec, Medac, Merck Serono, Novartis, Roche, sanofi-aventis, Talecris and Teva Neuroscience, Inc.
Dr. Goodin has participated (or is currently participating) in several industry-sponsored clinical trials in multiple sclerosis. The sponsoring pharmaceutical companies for these trials have included (or do include) Ares-Serono, Merck-Serono, Novartis, Berlex Laboratories, Bayer- Schering Healthcare, Biogen-Idec, Schering AG and Teva Neuroscience, Inc. He has also lectured at both medical conferences and in public on various aspects of the epidemiology, diagnosis and management of multiple sclerosis. In many cases, these talks have been sponsored directly or indirectly by one or another of the above-listed companies. He has also served as a temporary ad hoc consultant to several of these organizations on several occasions.
Dr. Arnason has served as a consultant within the past year to Bayer Schering Healthare, sanofi-aventis, Questcor, Inc. and Acorda, Inc. He has participated in a clinical trial sponsored by Acorda and has received research grant support from Questcor.
Dr. Comi has received personal compensation for speaking and consultancy activities from Bayer Schering, Serono Symposia International Foundation, Merck Serono International, sanofi-aventis, Novartis, Biogen Dompè and Teva Pharmaceutical Industries, Ltd. in the past two years.
Dr. Cook has received personal compensation for consultations from advisory boards or lectures from Bayer Healthcare, Merck Serono, Teva Pharmaceutical Industries Ltd, Biogen Idec, sanofi-aventis and Actinobacter.
Dr. Filippi serves on scientific advisory boards for Teva Pharmaceutical Industries, Ltd. and Genmab A/S; has received funding for travel from Bayer Schering Pharma, Biogen-Dompè, Genmab A/S, Merck Serono and Teva Pharmaceutical Industries, Ltd.; serves on editorial boards of the American Journal of Neuroradiology, BMC Musculoskeletal Disorders, Clinical Neurology and Neurosurgery, Erciyes Medical Journal, Journal of Alzheimer’s Disease, Journal of Neuroimaging, Journal of Neurovirology, Magnetic Resonance Imaging, Multiple Sclerosis and Neurological Sciences; serves as a consultant to Bayer Schering Pharma, Biogen-Dompè, Genmab A/S, Merck Serono and Teva Pharmaceutical Industries Ltd.; serves on speakers’ bureaus for Bayer Schering Pharma, Biogen-Dompè, Genmab A/S, Merck Serono and Teva Pharmaceutical Industries Ltd.; and receives research support from Bayer Schering Pharma, Biogen-Dompè, Genmab A/S, Merck Serono, Teva Pharmaceutical Industries Ltd. and Fondazione Italiana Sclerosi Multipla.
Dr. Jeffery has received honoraria and consulting fees from Berlex, Serono, Teva Pharmaceutical Industries Ltd, Glaxo and Pfizer.
Dr. Kappos has received financial support for research activities from Abbott, Bayer, Bayer HealthCare Pharmaceuticals, Bayer Schering Pharma, Bayhill, Biogen Idec, Centocor, Eisai, Elan, Genzyme, Merck Serono, Neurocrine, Novartis, sanofi-aventis, Roche, Teva, UCB and Wyeth.
Dr. Bogumil is a salaried employee of Bayer HealthCare Pharmaceuticals, Montville, NJ.
Dr. Stemper, Dr. Sandbrink, Dr. Schwenke, and Heubach are salaried employees of Bayer Pharma AG, Berlin, Germany.
Dr. Lehr was a salaried employee of Bayer Pharma AG, Berlin, Germany.
Yukiko Nakada and Haruhiko Nakajima are salaried employees of Mitsubishi Chemical Medience Corporation.
Dr. Pohl and Dr. Reischl are salaried employees of Bayer Pharma AG, Berlin, Germany.
Authors’ contributions
HPH has been actively involved in the conception, design and drafting the protocol of the BEYOND study and the reported biomarker analysis. He reviewed the statistical analysis, contributed to interpretation of the results, drafted the first version of this manuscript, actively contributed to further development of the manuscript and approved its final version. DSG, BGWA, GC, SC, MF, DRJ and LK were actively involved in the conception, design and drafting the protocol of the BEYOND study. They reviewed the statistical analysis of the reported biomarker data, contributed to data interpretation, gave critical revision for important intellectual content of the submitted manuscript, and approved its final version. SL and SS were responsible for the conception, design and biometrical analyses reported in this manuscript, have created the statistical analysis plan, interpreted the results, have actively contributed to writing and reviewing of the submitted manuscript, and have approved its final version. CP has been actively involved in supervising the BEYOND study and in setting up the protocol of the reported biomarker analysis, reviewed the statistical analysis, interpreted the results and has actively contributed to the writing and reviewing of the submitted manuscript. He approved the final version of the manuscript. TB and RS were actively involved in the conception, design and drafting the protocol and in supervising the BEYOND study. They reviewed the statistical analysis of the reported biomarker data, contributed to data interpretation, gave critical revision for important intellectual content of the submitted manuscript and approved its final version. BK, JFH and BS actively contributed to the conception and design of the reported biomarker analysis, reviewed the statistical analysis, interpreted the results, actively contributed to the writing of the submitted manuscript and approved its final version. YN and HN were responsible for the CPE assay in the Mitsubishi Chemical Medience Corporation and approved the final version of the submitted manuscript. JR actively contributed to the conception and design of the reported biomarker analysis, reviewed the statistical analysis, interpreted the results, actively contributed to the writing of the submitted manuscript and approved its final version. He coordinated and supervised the NAb laboratory comparison study. All authors read and approved the final manuscript.