Serum Autoreactivity Predicts Time to Response to Omalizumab Therapy in Chronic Spontaneous Urticaria.

Omalizumab (anti-IgE) has been shown to be effective in chronic spontaneous urticaria (CSU) in a number of clinical trials.1Zhao Z. Ji C. Yu W. Meng L. Hawro T. Wei J.F. et al.Omalizumab for the treatment of chronic spontaneous urticaria: a meta-analysis of randomized clinical trials.J Allergy Clin Immunol. 2016; 137: 1742-1750.e4Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar However, in clinical practice, some patients with CSU are fast responders to their first injection of omalizumab, often within days, whereas others are slow responders, with therapy taking weeks to be effective. This letter examines the possible underlying mechanisms. Sixty-four patients with CSU (46 women) whose symptoms were not controlled with H1-antihistamines up to 4 times the recommended dose were treated with omalizumab at the Charité Department of Dermatology. Their median age was 47 (range, 23-85 years) years, and the median duration of their CSU was 3 years (range, 4 months to 40 years). Seven patients had received omalizumab in the past but not for at least 6 weeks before enrolment in the study, and their symptoms had returned in full. All analyses were performed according to the Declaration of Helsinki and approval was obtained from the Charité Ethics Committee (EA1/268/13). All patients gave signed informed consent. Omalizumab 300 mg was injected subcutaneously 3 times at 4-week intervals. The first day of response was defined as the first of 7 continuous days when the 7-day urticaria activity score2Zuberbier T. Aberer W. Asero R. Bindslev-Jensen C. Brzoza Z. Canonica G.W. et al.The EAACI/GA(2) LEN/EDF/WAO Guideline for the definition, classification, diagnosis, and management of urticaria: the 2013 revision and update.Allergy. 2014; 69: 868-887Crossref PubMed Scopus (854) Google Scholar was 6 or less. Venous blood was taken before the first omalizumab treatment and centrifuged at 2500 rpm for 10 minutes. By the end of week 12, 56 (88%) had responded to omalizumab and 8 (12%) were nonresponders (Fig 1, A). Of the 56 responders, 39 patients (70%) responded within 8 days (fast responders) and 18 (32%) within the first day. Seventeen patients (30%) responded between 8 days and 3 months (slow responders). The cutoff time of 8 days between fast and slow responders was chosen because it corresponds with the peak plasma level of omalizumab after its initial injection.3Meno-Tetang G.M. Lowe P.J. On the prediction of the human response: a recycled mechanistic pharmacokinetic/pharmacodynamic approach.Basic Clin Pharmacol Toxicol. 2005; 96: 182-192Crossref PubMed Scopus (88) Google Scholar We hypothesized that a slow response to omalizumab occurs in patients with CSU in whom IgG antibodies to unoccupied IgE receptors (FcεRI) activate mast cell mediator release to cause wheal and angioedema formation.4Grattan C.E. Wallington T.B. Warin R.P. Kennedy C.T. Bradfield J.W. A serological mediator in chronic idiopathic urticaria–a clinical, immunological and histological evaluation.Br J Dermatol. 1986; 114: 583-590Crossref PubMed Scopus (261) Google Scholar This hypothesis is based on the knowledge that omalizumab first complexes soluble IgE then sequesters IgE released from mast cells, thus uncovering membrane FcεRI, which subsequently decays slowly over several weeks.5Metz M. Staubach P. Bauer A. Brehler R. Gericke J. Kangas M. et al.Omalizumab normalises levels of high affinity IgE receptor-positive skin cells in patients with chronic spontaneous urticaria: a randomized, double-blind, placebo-controlled study.Allergy. 2014; 69: 87-88Crossref PubMed Scopus (19) Google Scholar To test this, the basophil histamine release assay (BHRA) was used. This assay may be used to detect serum autoantibodies directed against either the cell-bound IgE or unoccupied FcεRI. In this study, basophils were stripped of their IgE to assess FcεRI only. The BHRA was done as previously described (see this article's Online Repository at www.jacionline.org).6Platzer M.H. Grattan C.E. Poulsen L.K. Skov P.S. Validation of basophil histamine release against the autologous serum skin test and outcome of serum-induced basophil histamine release studies in a large population of chronic urticaria patients.Allergy. 2005; 60: 1152-1156Crossref PubMed Scopus (46) Google Scholar Analysis of the omalizumab responders showed that most BHRA-positive patients responded only after the second injection (Fig 1, B), with a median time to response of 29 days, whereas BHRA-negative patients had a median time to response of only 2 days (Fig 1, C). Furthermore, only 1 of the 39 fast responders was BHRA positive, whereas 8 of the 17 slow responders were BHRA positive (P = .0001; Fisher exact test; Fig 1, D). The hypothesis was also tested using the autologous serum skin test (ASST)7Konstantinou G.N. Asero R. Maurer M. Sabroe R.A. SchmidGrendelmeier P. Grattan C.E. EAACI/GA(2)LEN task force consensus report: the autologous serum skin test in urticaria.Allergy. 2009; 64: 1256-1268Crossref PubMed Scopus (148) Google Scholar in which 50 μL of fresh undiluted autologous serum was injected intradermally into the volar forearm. Similar volumes of 0.9% NaCl saline and 100 μg/mL histamine were used as negative and positive controls. The ASST was taken to be positive when the serum-induced wheal had a diameter at least 1.5 mm greater than the saline-induced wheal at 30 minutes. For clinical reasons, the ASST was performed on 51 patients only. Twelve of the 33 fast responders were ASST positive, whereas 10 of the 13 slow responders showed a positive ASST result (P = .012; Fisher exact test; Fig 1, E and F). There were no statistical differences between fast and slow omalizumab responders with respect to age, body mass index, or disease activity or duration. Similarly, pretreatment IgE levels, neutralization rates of IgE, and free IgE and omalizumab levels posttreatment were not significantly different between slow and fast responders to omalizumab (Table I).Table ICSU responders to omalizumab: Demographic, clinical, and laboratory characteristicsCharacteristicAll complete responders (n = 56)Complete response within 8 dComplete response after 8 dP valueAge (y)48 (33-60)49 (37-58)42 (31-63).544Sex Female40 (71.4%)28 (71.8%)12 (70.6%).583 Male16 (28.6%)11 (28.2%)5 (29.4%)BMI27.3 ± 4.827.9 ± 4.926.1 ± 4.6.192UAS7∗Before start of omalizumab treatment.24.0 ± 9.723.0 ± 9.326.5 ± 4.6.215Disease duration∗Before start of omalizumab treatment.36 (16-102)40 (18-103)24 (14-85).240ASST+∗Before start of omalizumab treatment.23 of 46 (50.0%)12 of 33 (36.4%)11 of 13 (84.6%)<.01BHRA+∗Before start of omalizumab treatment.9 of 55 (16.4%)1 of 38 (2.6%)8 of 17 (47.1%)<.001Anti-FcεRI+∗Before start of omalizumab treatment.5 of 44 (11.4%)4 of 30 (13.3%)1 of 14 (7.1%).485Anti-IgE+∗Before start of omalizumab treatment.1 of 44 (2.3%)1 of 30 (3.3%)0 of 14 (0.0%).682Total IgE∗Before start of omalizumab treatment.205.4 ± 229.6239.2 ± 250.6130.0 ± 155.0.104Free IgE after omalizumab†Four weeks after start of omalizumab treatment. Note that not all the patients were tested for ASST and/or BHRA, because of various reasons including time constraints, availability of personnel to do the serum preparations, and injections and patient consent.31.8 ± 47.731.2 ± 39.633.2 ± 64.7.893% IgE neutralization†Four weeks after start of omalizumab treatment. Note that not all the patients were tested for ASST and/or BHRA, because of various reasons including time constraints, availability of personnel to do the serum preparations, and injections and patient consent.94.7 ± 13.496.7 ± 2.290.0 ± 24.5.106Omalizumab (μg/mL)†Four weeks after start of omalizumab treatment. Note that not all the patients were tested for ASST and/or BHRA, because of various reasons including time constraints, availability of personnel to do the serum preparations, and injections and patient consent.16.2 ± 7.816.6 ± 7.215.2 ± 9.2.557Data are given as n (%) for sex, ASST, and BHRA; median (IQR) for age and duration of disease (in months); or mean ± SD for BMI, UAS7, total IgE, free IgE after omalizumab, % neutralization, omalizumab. Statistical differences between responder groups in age, ASST+, BHRA+, anti-FcεRI+, and anti-IgE+ were analyzed using χ2 or Fisher exact test when appropriate. Statistical differences between responder groups in BMI, UAS7, and total IgE were analyzed using unpaired t test. P values in boldface indicate statistical significance.BMI, Body mass index; IQR, interquartile range; UAS7, 7-day urticaria activity score.∗ Before start of omalizumab treatment.† Four weeks after start of omalizumab treatment. Note that not all the patients were tested for ASST and/or BHRA, because of various reasons including time constraints, availability of personnel to do the serum preparations, and injections and patient consent. Open table in a new tab Data are given as n (%) for sex, ASST, and BHRA; median (IQR) for age and duration of disease (in months); or mean ± SD for BMI, UAS7, total IgE, free IgE after omalizumab, % neutralization, omalizumab. Statistical differences between responder groups in age, ASST+, BHRA+, anti-FcεRI+, and anti-IgE+ were analyzed using χ2 or Fisher exact test when appropriate. Statistical differences between responder groups in BMI, UAS7, and total IgE were analyzed using unpaired t test. P values in boldface indicate statistical significance. BMI, Body mass index; IQR, interquartile range; UAS7, 7-day urticaria activity score. When comparing BHRA with ASST, it must be realized that BHRA is more specific in that it was designed to identify serum IgG antibodies to FcεRI. However, it is time consuming and may not be available in all clinics. In contrast, the ASST is easy to perform clinically but is less specific, because it will also detect IgG antibodies against mast cell–bound IgE, and may contain other histamine-releasing factors that can lead to a positive ASST response.7Konstantinou G.N. Asero R. Maurer M. Sabroe R.A. SchmidGrendelmeier P. Grattan C.E. EAACI/GA(2)LEN task force consensus report: the autologous serum skin test in urticaria.Allergy. 2009; 64: 1256-1268Crossref PubMed Scopus (148) Google Scholar Even so, there was a significant relationship between BHRA and ASST results (Spearman ρ = 0.448, P < .01). All BHRA-positive patients were ASST positive. BHRA-positive and ASST-positive omalizumab responders are 4.5 and 5.5 times more likely to have a slow response to treatment compared with BHRA-negative and ASST-negative responders; the relative risks with 95% confidence limits were 4.54 for BHRA (2.42-8.53, z test P < .001) and for ASST 5.50 (1.37-22.11, z test P < .05). The strength of this study is the highly significant correlation between the length of time to the onset of activity of omalizumab and BHRA, strongly suggesting that a positive BHRA may predict a slow response to omalizumab. Limitations of the study are that all the data are derived from an open observational study without controls. A further weakness is that the story is not entirely black and white. For example, 1 patient with positive BHRA and ASST became symptom free within 1 day of the first omalizumab injection. Also, there were 9 BHRA-negative patients in the slow responder group. However, 5 of these did have a positive ASST result. These apparent anomalies suggest that in some patients, CSU may have more than 1 mechanism.8Chang T.W. Chen C. Lin C.J. Metz M. Church M.K. Maurer M. The potential pharmacologic mechanisms of omalizumab in patients with chronic spontaneous urticaria.J Allergy Clin Immunol. 2015; 135: 337-342Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar In conclusion, there are significant correlations between a positive BHRA and ASST and the time to symptom relief with omalizumab. The fact that a positive BHRA is predictive of a slow response to omalizumab suggests that omalizumab works via reducing FcεRI expression in these patients. Larger prospective studies are needed to confirm these preliminary observations. The ASST was performed as previously described.E1Konstantinou G.N. Asero R. Maurer M. Sabroe R.A. Schmid-Grendelmeier P. Grattan C.E. EAACI/GA(2)LEN task force consensus report: the autologous serum skin test in urticaria.Allergy. 2009; 64: 1256-1268Crossref PubMed Scopus (238) Google Scholar Briefly, venous blood was withdrawn before the first administration of omalizumab. Samples were centrifuged at 2500 rpm for 10 minutes and the serum separated. Fresh serum was used for the ASST or stored at −80°C for the serum-induced BHRA and other analyses. A volume of 50 μL of fresh undiluted autologous serum was injected intracutaneously into the volar surface of the forearm. A similar volume of 0.9% NaCl saline was injected as negative control, and histamine (50 μL of 100 μg/mL, intracutaneous or skin prick test) was used simultaneously as positive control. Wheal and flare responses were measured at 30 minutes. The ASST response was taken to be positive when the serum-induced wheal had a diameter at least 1.5 mm greater than the saline-induced wheal. The serum-induced BHRA may be used to detect autoantibodies in patient sera directed against either the cell-bound IgE or against free IgE receptors (FcεRI). In this study, basophils were stripped of their IgE to assess serum antibodies to FcεRI. Basophils were prepared and histamine release assessed as previously described.E2Platzer M.H. Grattan C.E. Poulsen L.K. Skov P.S. Validation of basophil histamine release against the autologous serum skin test and outcome of serum-induced basophil histamine release studies in a large population of chronic urticaria patients.Allergy. 2005; 60: 1152-1156Crossref PubMed Scopus (54) Google Scholar Briefly, blood bank buffy coats containing 1% to 2% basophils were obtained from fresh healthy donor blood. Because cell responses vary between donor cells, 4 blood bank buffy coats were combined. The buffy coats were mixed with equal volumes of RPMI containing IL-3 in a final concentration of 1 ng/mL, and the samples were stored overnight at 2°C to 8°C. The next day, the buffy coats were centrifuged to remove plasma and RPMI. Thereafter, the samples were washed with physiological saline, exposed to low pH using a stripping buffer (pH 3.6 from RefLab, Copenhagen, Denmark) to remove IgE from the basophils, and resuspended in Pipes buffer (RefLab, Copenhagen, Denmark) before incubation with patient sera. Each serum was tested at dilutions of 20% and 10%; data are shown using 20% dilutions. Total histamine content was determined after cell lysis with 100 μL of 7% HClO4 for 60 minutes at 37°C. After centrifugation, 25 μL supernatants were transferred to glass fiber–coated microtiter plates and histamine was measured according to RefLab (Copenhagen, Denmark) instructions. Triplicate histamine determinations of each sample were performed. Assay variation was less than 7%. Assay sensitivity was 5 ng histamine/mL. The release of histamine in cell supernatants was expressed as the percentage of total histamine content from lysed donor cells. BHRA was considered positive if histamine levels were more than 16.5% spontaneous. As negative control, the buffy coat cells responded to a mixture of healthy sera with less than 5%. As a positive control, we used anti-IgE, resulting in 45% release. The mean ± SEM histamine release of the BHRA-negative patients was 2.8% ± 0.4% and that of the BHRA-positive patients 63% ± 8%. Serum levels of total IgE, IgG-anti-IgE, and IgG-anti-FcεRI were determined as previously described.E3Staubach P. Onnen K. Vonend A. Metz M. Siebenhaar F. Tschentscher I. et al.Autologous whole blood injections to patients with chronic urticaria and a positive autologous serum skin test: a placebo-controlled trial.Dermatology. 2006; 212: 150-159Crossref PubMed Scopus (107) Google Scholar Briefly, recombinant FcεRI was fractionated by 14% reducing SDS-PAGE and transferred to nitrocellulose. Blots were blocked for 1 hour in 1% (v/v) Tween-20 in PBS buffer (154 mM NaCl, 20 mM KH2PO4, pH 7.6). For detection of autoantibodies, sera were diluted 100-fold in PBS. mAb directed to the N-terminal tag RGSH4 (Qiagen, Hilden, Germany) was diluted 2000-fold in PBS. After a 12-hour incubation at room temperature with diluted sera, the blots were washed and incubated with rabbit antihuman IgG or rabbit antimouse (Jackson Immunoresearch Laboratories, West Grove, Pa) conjugated with alkaline phosphatase. The NBT/BCIP staining system was used as chromogenic substrate. A commercially available recoveryELISA kit was used for the quantification of free IgE, IgE neutralization rates, and omalizumab levels in patient serum (BioTeZ Berlin Buch GmbH, Berlin, Germany) as described previously.E4Strohner P. Staatz A. Sarrach D. Steiss J.O. Becher G. The recoveryELISA–a newly developed immunoassay for measurement of therapeutic antibodies and the target antigen during antibody therapy.Clin Chem Lab Med. 2012; 50: 1263-1269Crossref PubMed Scopus (13) Google Scholar