stranded DNA assays in reporting clinical studies: comment ... We would like to call upon investigators to sys- ... New York University Langone Medical Center.
ARTHRITIS & RHEUMATOLOGY Vol. 66, No. 2, February 2014, pp 479–480 © 2014, American College of Rheumatology
LETTERS DOI 10.1002/art.38252
Our arguments are as follows. First, assays assessing the presence and the level of dsDNA antibodies vary significantly (between techniques as well as between manufacturers) despite the use of international units (IU/ml) based on previous attempts to obtain standardization (5). Second, despite the emerging use of enzyme immunoassays (EIAs), it is generally accepted that the Farr assay remains the gold standard for determining SLE-specific DNA antibodies (6). Within this context, details are missing regarding the EIA and manufacturer used in the phase II and phase III belimumab studies, as well as their relationship with the results obtained using a Farr immunoassay (as was used in the phase I belimumab study) (1–4,7). To document this latter relationship as well as the variability between assays, we analyzed anti-dsDNA in a set of 44 patients with SLE, using a Farr assay (Anti-dsDNA Kit; Trinity Biotech) and a selection of commercially available EIAs (Figure 1). All assays included in the study were calibrated against the Wo/80 reference material (5). Using the cutoff values for positivity proposed by the manufacturers, moderate to substantial kappa value agreement between the EIAs was observed ( ⫽ 0.462–0.732). For the comparison between EIAs and the Farr assay, only moderate agreement was observed ( ⫽ 0.573–0.657) (results not shown). Never-
Relevance of different results of different anti–doublestranded DNA assays in reporting clinical studies: comment on the article by Petri et al To the Editor: We read with interest the recent report by Petri and colleagues on baseline predictors of disease flares in patients with serologically active systemic lupus erythematosus (SLE) treated with standard therapy alone—a study that used the combined placebo group of the phase III belimumab trials (1). The definition of serologic activity included the presence of anti–double-stranded DNA (anti-dsDNA) antibodies in concentrations of at least 30 IU/ml. This inclusion criterion was based on a prior phase II placebo-controlled trial showing that belimumab reduced and stabilized disease activity within this subpopulation and was subsequently used in two phase III trials (2–4). In our opinion, however, it should be clearly anticipated that the serologic criterion for study inclusion used in the phase II and phase III studies (anti-dsDNA antibody levels ⱕ30 IU/ml) cannot be extrapolated directly for use in clinical practice to select patients eligible for belimumab therapy. From the same perspective, use of an anti-dsDNA level of ⬎200 IU/ml to predict SLE flares, as reported by Petri et al (1), should be adopted with caution.
Figure 1. Results of anti–double-stranded DNA (anti-dsDNA) antibody assays in 44 patients with systemic lupus erythematosus. For all assays, only results within the measuring range stated by the manufacturer are shown. For the Bio-Rad assay, the scale was aligned with the highest measuring range of the other assays, because no measuring range was specified by the manufacturer. The broken lines represent the cutoff values proposed by the manufacturers. For each comparison, Pearson’s correlation coefficients (r) and kappa agreement values () after dichotomization of the data (using the manufacturers’ cutoff values) are shown. EIA ⫽ enzyme immunoassay; ELISA ⫽ enzyme-linked immunosorbent assay. 479
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theless, it should be stressed that the high variability in test results cannot be reduced solely to an issue of differences in the choice of the cutoff values applied (r ⫽ 0.527–0.956) (Figure 1). For clinical practice, this implies that direct use of criteria for anti-dsDNA antibodies deduced from clinical trials would result in the identification of different patient subsets depending on the assay used (e.g., use of the criterion of an anti-dsDNA antibody level of ⬎30 IU/ml for guidance of belimumab therapy would select 32–100% of the patients in our cohort). We would like to call upon investigators to systematically specify details on the assay and manufacturer when reporting anti-dsDNA results. This has important relevance for interpretation and reproduction of the results reported. Carolien Bonroy, MSc Charlotte Verfaillie, MSc Evy De Witte, MSc Filip De Keyser, MD, PhD Ghent University Hospital Ghent, Belgium 1. Petri MA, van Vollenhoven RF, Buyon J, Levy RA, Navarra SV, Cervera R, et al. Baseline predictors of systemic lupus erythematosus flares: data from the combined placebo groups in the phase III belimumab trials. Arthritis Rheum 2013;65:2143–53. 2. Wallace DJ, Stohl W, Furie RA, Lisse JR, McKay JD, Merrill JT, et al. A phase II, randomized, double-blind, placebo-controlled, dose-ranging study of belimumab in patients with active systemic lupus erythematosus. Arthritis Rheum 2009;61:1168–78. 3. Navarra SV, Guzman RM, Gallacher AE, Hall S, Levy RA, Jimenez RE, et al. Efficacy and safety of belimumab in patients with active systemic lupus erythematosus: a randomised, placebo-controlled, phase 3 trial. Lancet 2011;377:721–31. 4. Manzi S, Sanchez-Guerrero J, Merrill JT, Furie R, Gladman D, Navarra SV, et al. Effects of belimumab, a B lymphocyte stimulatorspecific inhibitor, on disease activity across multiple organ domains in patients with systemic lupus erythematosus: combined results from two phase III trials. Ann Rheum Dis 2012;71:1833–8. 5. Feltkamp TE, Kirkwood TB, Maini RN, Aarden LA. The first international standard for antibodies to double stranded DNA. Ann Rheum Dis 1988;47:740–6. 6. Launay D, Schmidt J, Lepers S, Mirault T, Lambert M, Kyndt X, et al. Comparison of the Farr radioimmunoassay, 3 commercial enzyme immunoassays and Crithidia luciliae immunofluorescence test for diagnosis and activity assessment of systemic lupus erythematosus. Clin Chim Acta 2010;411:959–64. 7. Furie R, Stohl W, Ginzler EM, Becker M, Mishra N, Chatham W, et al. Biologic activity and safety of belimumab, a neutralizing anti-B-lymphocyte stimulator (BLyS) monoclonal antibody: a phase I trial in patients with systemic lupus erythematosus. Arthritis Res Ther 2008;10:R109.
DOI 10.1002/art.38251
Reply To the Editor: We appreciate the comments of Dr. Bonroy et al regarding the varied assays available for the detection of
anti-dsDNA antibodies. However, the goal in conducting post hoc analyses of data from the phase II (1) and phase III (2,3), belimumab trials was not to establish a cutoff measure for anti-dsDNA antibody levels but rather to identify real-world measures that clinicians could use to identify SLE patients at increased risk of moderate to severe disease flares. The BLISS (Study of Belimumab in Subjects with SLE) trials used a validated Quest enzyme-linked immunosorbent assay to measure anti-dsDNA. To our knowledge, the Farr assay is no longer available in clinical settings. It is likely that other validated anti-dsDNA assays, which provide titers to identify high values, can likewise be used to predict patients at risk of disease flares. Of importance to clinicians following up their SLE patients who are receiving standard therapy, our data show that those with renal, neurologic, or vasculitic involvement and those who have elevated anti-dsDNA, high B lymphocyte stimulator, or low C3 levels may be at increased risk of clinically meaningful flares. Michelle A. Petri, MD Johns Hopkins University School of Medicine Baltimore, MD Ronald F. van Vollenhoven, MD, PhD Karolinska Institute Stockholm, Sweden Jill Buyon, MD New York University Langone Medical Center New York, NY Roger A. Levy, MD, PhD Hospital Universita ´rio Pedro Ernesto-UERJ Rio de Janeiro, Brazil Sandra V. Navarra, MD University of Santo Tomas Hospital Manila, The Philippines Ricard Cervera, MD, PhD, FRCP Hospital Clı´nic Barcelona Catalonia, Spain Z. John Zhong, PhD William W. Freimuth, MD, PhD Human Genome Sciences, Inc. Rockville, MD 1. Wallace DJ, Stohl W, Furie RA, Lisse JR, McKay JD, Merrill JT, et al. A phase II, randomized, double-blind, placebo-controlled, dose-ranging study of belimumab in patients with active systemic lupus erythematosus. Arthritis Rheum 2009;61:1168–78. 2. Navarra SV, Guzman RM, Gallacher AE, Hall S, Levy RA, Jimenez RE, et al. Efficacy and safety of belimumab in patients with active systemic lupus erythematosus: a randomised, placebo-controlled, phase 3 trial. Lancet 2011;377:721–31. 3. Furie R, Petri M, Zamani O, Cervera R, Wallace DJ, Tegzova D, et al. A phase III, randomized, placebo-controlled study of belimumab, a monoclonal antibody that inhibits B lymphocyte stimulator, in patients with systemic lupus erythematosus. Arthritis Rheum 2011;63:3918–30.
