Variability in total serum IgE over 1 year in severe asthmatics

Background Immunoglobulin E (IgE) is the treatment target of omalizumab, a monoclonal antibody indicated in the treatment of severe allergic asthma. Long-term variability of serum total IgE (sIgEtot) in asthmatics remains poorly documented. Methods In this prospective study, sIgEtot levels were measured over 1 year at 7 time points in 41 severe asthmatics treated with high-dose of inhaled corticosteroids and long-acting β2 agonists. 33 patients were atopic based on at least one positive RAST to common aeroallergens. Patients were divided into three groups according to their baseline sIgEtot level: low (< 76 IU/mL; n = 10), intermediate (76–700 IU/mL; n = 20) or high (> 700 IU/mL; n = 11). Patients also completed the six-item Juniper Asthma Control Questionnaire (ACQ6). The sIgEtot variability and factors predictive for this variability were studied, as well as ACQ6 outcomes. Results The variation in sIgEtot level was mostly the consequence of between patient-variability, which represented 96%, 71% and 96% of the total variability in the low, intermediate and high sIgEtot subgroups, respectively. The residual within-patient variability was therefore limited. In 10/41 patients, sIgEtot levels increased or decreased, for at least one visit, beyond the predefined range of the subgroups to which they were assigned (< 76 IU/mL; 76–700 IU/mL; > 700 IU/mL). There was a significant but weak correlation between sIgEtot and ACQ6 score over all time points (r = 0.15, p = 0.02), but sIgEtot failed to associate with severe exacerbation. sIgEtot decreased by 3% with any additional year of age for the whole group (p = 0.01) and increased by 5% per one unit of allergen exposure score in atopic patients (p = 0.002). Conclusion In severe asthmatics, limited within-patient variability of sIgEtot levels was observed over 1 year as opposed to marked between-subject variability. sIgEtot decreases with age. Variation in sIgEtot weakly associates with asthma control but not with exacerbation.


Introduction
Immunoglobulin E (IgE) is an antibody associated with hypersensitivity and allergic reactions. IgE mainly binds on the high-affinity IgE receptor (FcεRI) on mast cells and basophils. Upon FcεRI cross-linking following allergen exposure, inflammatory mediators including histamine, leukotrienes and pro-inflammatory cytokines are released [1]. However, in murine models, the binding of IgE itself on mast cell surface may already favour cell survival and cytokine release irrespective of the presence of any allergen [2,3]. Although direct evidence for this is still lacking in humans, it has been reported that asthmatics with high sputum IgE levels have also raised sputum levels of TNFα and Interleukin-6, two cytokines released from mast cells [4]. Therefore, IgE might possibly be considered as an inflammatory mediator independent of atopic status and deserves to be monitored.
In a study of a community population with measurements at two time points over an 8-year period, the level of serum total IgE (sIgE tot ) tended to be stable in

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Allergy, Asthma & Clinical Immunology  15:20 subjects above the age of 35 years while levels decreased in children and young adults. Atopic subjects tended to show a decrease with age even after the age of 35 years [5]. Further, a longitudinal study spanning a period of up to 20 years also showed that smoking opposed the natural decline in IgE and synergized with atopy to result in increased levels in subjects over the age of 50 years [6]. However, variation in sIgE tot beyond two or three time points in the same subject has not been studied extensively.
Omalizumab, a monoclonal antibody that binds serum IgE, has become part of asthma treatment for the severe spectrum of the disease in which patients remain uncontrolled despite a combination of high dose inhaled corticosteroids (ICS) with long-acting β 2 agonist (LABA). The drug has been validated in asthmatics whose serum IgE ranges from 30 to 700 IU/mL but most of the effect in terms of reduction of exacerbation was confined to patients with serum IgE ranging from 76 to 700 IU/mL [7]. A retrospective chart review of severe asthmatics with two to four measurements of sIgE tot over an average period of 2 years showed some clinically significant variability in sIgE tot affecting candidacy and dosing of omalizumab [8]. A recent prospective study conducted with 17 moderate-to-severe asthma patients with 6 serial measurements confirmed this variability in sIgE tot over 1 year [9].
To better understand sIgE tot variation and factors influencing this variation, we conducted a 12-month prospective study of sIgE tot levels in severe asthmatics treated with high-dose ICS and LABA. Furthermore, we also examined how fluctuation in IgE may relate to day-to-day environmental exposure, asthma control and exacerbations.

Patient characteristics
Patients enrolled in the study had an established diagnosis of severe asthma as defined by the ERS/ATS consensus statement [10], aged ≥ 18 years, and currently treated with high-dose ICS (daily dose of fluticasone propionate > 500 µg or equivalent) and a LABA. Patients were excluded from the study if: (i) they were being or had been treated with omalizumab (Xolair ® , Novartis, Basel, Switzerland), unless treatment was stopped at least 1 year before inclusion; (ii) they were undergoing immunotherapy; and/or (iii) used an investigational drug at the time of enrollment or within 30 days prior to enrollment in the study.

Study design
This was a prospective, multicenter study to assess variability of sIgE tot levels in severe asthmatics conducted in 5 Belgian centers. The 12-month variability in sIgE tot levels in the total population was assessed and the proportion of the total variability explained by withinand between-patient variability was calculated (primary objective). In accordance with reimbursement criteria in Belgium, patients were stratified post hoc into three groups on the basis of baseline sIgE tot : < 76 IU/mL (low IgE), 76-700 IU/mL (intermediate IgE), and > 700 IU/ mL (high IgE). These three groups were selected based on the study of Bousquet et al., which demonstrated that omalizumab reduces the annualized rate of asthma exacerbations more in patients with sIgE tot ≥ 76 IU/mL [7]; and based on the upper limit of serum IgE inclusion criteria of the INNOVATE registration study (< 700 IU/ mL) [11]. These two studies are currently used as the basis for the reimbursement of omalizumab in Belgium. An additional objective of the study was to evaluate demographic, environmental, and treatment factors that may influence sIgE tot variation and whether sIgE tot relates to asthma control and asthma exacerbation.
At baseline patients completed the six-item Juniper Asthma Control Questionnaire (ACQ 6 ) [12]; blood was sampled to measure sIgE tot levels; and atopic status of the patient was evaluated with a radio allergosorbent test (RAST) directed towards common allergens: grass mix, birch, mould mix, house dust mite (Dermatophagoides pteronyssinus), and cat and dog dander. Patients were considered atopic if RAST levels to any allergen exceeded 0.35 kU/L. Additionally, an environmental questionnaire was completed which enquired about the frequency of patients' exposure (1: "once, more than 1 month ago"; 2: "once, more than 1 week ago"; 3: "once, within the last week"; 4: "occasionally"; 5: "frequently" or 6: "constantly") to house dusts, pollens, molds, cats and dogs, smoke, humidifier/vaporizer, and carpeting.
Subsequently, 6 visits were planned every 2 months for a follow-up period of 12 months. At each visit, sIgE tot was measured, the ACQ 6 and environmental questionnaires were completed, and the treatment prescribed by the physician was registered. Treatment prescription, continuation, or discontinuation was per the physician's best clinical judgment and in accordance with the Belgian reimbursement criteria. Also, to allow for the monitoring of exacerbations during the study, patients had to report if and how many times they had an exacerbation since the last visit. The presence of an exacerbation in the week preceding the visit was recorded specifically. A severe exacerbation was defined as an exacerbation requiring a treatment containing systemic corticosteroids for at least three consecutive days and/or an emergency room visit and/or a hospitalization.
This study was approved by the ethical committee of the CHU Liege under approval number B70720096731. All patients had to provide an informed consent before entering the study.

Statistical analysis
Descriptive statistics were calculated using frequencies, percentages, means, standard deviations, medians and ranges as appropriate. Comparisons in baseline characteristics between the three subgroups (low, intermediate, high sIgE tot ) were tested using ANOVA with Tukey post hoc test comparison for age and a general linear model with contrasts for other (categorical) variables. Random-intercepts regression analysis was used to model log-transformed sIgE tot , with 'patient' as the random variable. The model allowed us to calculate intra-cluster correlations to determine how much of the variability observed in sIgE tot levels over time could be attributed to variability between individual patients, compared to variability within each patient. It also allowed us to test predictors of sIgE tot (exposure to allergens, active/passive exposure to tobacco smoke, change in asthma treatment, treatment with maintenance systemic corticosteroids, and ACQ 6 score). Modeling of exacerbations (absent/present) was performed by logistic regression analysis, using generalized estimating equations to account for repeated measurements over time. This analysis was used to assess influence of treatment changes and time of year. The correlation between sIgE tot and ACQ 6 score at baseline was evaluated with a Spearman correlation. As this was a real-life study, data were collected from routine clinical practice and there may be missing data. Missing values were not imputed in the descriptive and statistical analyses. p values < 0.05 were considered statistically significant.

Variability in sIgE tot over 12 months
The between-patient variability represented 93.4% of the total variability for the entire sample, and 96%, 71% and 96% in the low, intermediate, and high sIgE tot subgroups, respectively. Therefore, within-patient variability represented 4%, 29% and 4% of the total variability in the low, intermediate and high sIgE tot subgroups, respectively. The within-patient variability was significantly higher in the intermediate sIgE tot group than in the other groups (p < 0.0001) ( Table 2). Detailed individual trajectories over time by baseline IgE strata are shown in Fig. 1, panels a, c, and d. Ten of the 41 patients (24%) reached values beyond the limit of their own baseline sIgE tot subgroup during at least one of the follow-up visits. Of these 10 patients, 4 were in the low, 4 in the intermediate, and 2 in the high sIgE tot subgroup, therefore representing 40% of the patients in the low sIgE tot subgroup, 20% of the patients in the intermediate sIgE tot subgroup and 18% of the patients in the high sIgE tot , respectively.
Two patients had sIgE tot levels in the three categories over the 1 year period of observation. One patient initially in the low sIgE group had a rise in IgE above 700 IU/mL for at least 2 consecutive measurements before returning to intermediate levels at the two last visits (Fig. 1b). This patient was subsequently found to have developed a parasitic infection.
The relevant clinical data for this patient are presented in Table 3. The patient did not have any positive RAST results at baseline and was therefore considered non-atopic.

Relationship between variation in sIgE tot and demographics, environmental exposure and treatment features
Age was a significant predictor of sIgE tot levels over 12 months. sIgE tot decreased by 3% with each additional year of age (p = 0.01). Allergen exposure for patients with a history of a positive RAST test was associated with change in sIgE tot . Exposure scores ranged from 0 to 12 and reflected the sum of the frequencies of exposure for the different allergens for which the patient had shown a positive RAST. A unit increase in the exposure score was associated with a 5% rise sIgE tot (p = 0.002). No relationship was found between sIgE tot and gender, smoke exposure, and asthma treatment.

Relationship between sIgE tot and lung function, asthma control, and exacerbation
There was no relationship between lung function (% predicted FEV1) and sIgE tot variation at baseline or throughout the study period. The odds of severe asthma   Over the entire study period, there was no correlation between sIgE tot and severe exacerbations since the previous visit nor with exacerbation that occurred the week before the visits. At baseline there was no relationship between sIgE and ACQ 6 in the whole group (Spearman's ρ = 0.01, p = 0.96) nor in any of the three groups. There was a significant but weak correlation between sIgE tot and ACQ 6 score across all time points (r = 0.15, p = 0.02). Figure 2 shows the variation in sIgE tot (panel a) and asthma control (panel b) according to the seasons. In the total study group, there was no association between seasons (March-May, June-August; September-November; December-February) and sIgE tot (p = 0.47) or ACQ 6 score (p = 0.43).

Discussion
There are limited data in the literature on the stability of IgE measurements over time in asthmatics. In the current study, we investigated variations in sIgE tot in severe asthmatics over a 1 year period with repeated measurements every 2 months. The principal finding is  that most of the variability sIgE tot was due to betweenpatient variability whereas within-patient variability of sIgE tot levels was rather limited. sIgE tot was found to decrease with age. The relative stability in sIgE tot suggests that the mechanisms regulating IgE levels are sustained over time in most patients. Nonetheless, 24% of patients showed sIgE tot level scores beyond the range of their own group for at least one visit. This was particularly the case in patients with baseline sIgE tot < 76 IU/mL as 4 out of 10 patients had a measurement above 76 IU/mL in at least one subsequent blood sample.
Omalizumab is a biologic with proven efficacy to improve asthma quality of life and reduce asthma exacerbations in severe allergic asthmatics. The findings from our study suggest that regular monitoring of sIgE tot in routine clinical practice may assist in optimizing treatment by aligning it with observed variations in sIgE tot and thus avoid under-treatment with omalizumab. As the 40% of patients with sIgE tot < 76 IU/mL at baseline but subsequent serum levels in the 76-700 IU/mL range underscore, without repeated blood sampling these patients might not have been initiated on omalizumab, which may have translated into a subsequent preventable need for corticosteroid therapy or into preventable exacerbations and hospitalizations. Likewise, the 20% of patients with sIgE tot between 700 and 1000 IU/ mL who subsequently showed serum levels below the 700 IU/mL threshold might not have been initiated on omalizumab without repeated testing. However, patients with very high serum IgE levels (> 2000 IU/mL) remain fairly stable in this "high" zone. Although betweenpatient variability was the major trend identified in our study, limited within-patient variability was still present. Therefore, regular monitoring of sIgE over 1 year results in an increase from 50 to 66% of patients falling into the range between 76 and 700 IU/mL. The limited patient variability resulted in the fact that two-third (27/41) of our patients treated with high doses ICS/LABA had a serum IgE values between 76 and 700 IU/mL after a 1 year period of observation (including 7 measurements for most of them), a figure clearly higher than 50% of patients as observed at baseline.
Despite the limited within-patient variability, one patient with baseline sIgE tot < 76 IU/mL showed significant changes in serum levels over the observation period. This patient who was non-atopic, maintained low IgE levels (< 76 IU/mL) at the first two visits, after which there was a marked increase to 818 IU/mL at the third and 1159 IU/mL at the fourth visit, before declining at subsequent visits without, however, returning to low serum levels. This patient was a severe corticosteroiddependent asthmatic in whom a parasitic infection was detected and treated accordingly. Remarkably, when the sIgE tot increased sharply at visit 3, the patient's ACQ 6 scores rose from 2.3 to 4.2 without any change in FEV 1 but with an augmentation of the OCS dose as treatment adjustment. This case points at the importance of considering possible etiologies when a sharp rise in IgE is detected. IgE levels are known to increase in the case of parasitic infection, namely helminth infections [13]; yet other causes of elevated IgE outside an atopic status should also be considered including infections by mycobacterium tuberculosis, Epstein-Barr virus, cytomegalovirus, malignancies, or chronic inflammatory/ dysimmune disorders [14]. Table 3 Clinical data for the outliner patient belonging to the low serum total IgE (sIgEtot) subgroup (see Fig. 1b Though not powered for that purpose, our study also showed an inverse relationship between age and serum IgE, a finding we have recently reported in a large cross-sectional study in asthmatics [15] and which was previously established in longitudinal population studies [5,6]. A decrease in sIgE tot by 3% with every year Fig. 2 Variation in sIgE tot and asthma control in severe asthmatic patients (n = 41) according to the seasons. Data from the 6 follow-up visits are shown according to the month of the visit (the baseline data are not included). In these graphics, the geometric mean sIgE tot with standard deviation (SD) (a) and the geometric mean six-item Juniper Asthma Control Questionnaire (ACQ 6 ) score with SD (b) are given increase in age was observed in the present study. Not unexpectedly, allergen exposure score was associated with a rise of sIgE tot in sensitized subjects, supporting a role for allergen exposure in IgE synthesis in addition to its role in mast cell activation thereby confirming a recent study [9].
In contrast to what is known for eosinophils [16,17], there are limited data on the relationship between sIgE tot and asthma control and severity. A higher sIgE tot level in severe asthmatics as compared to that in mild to moderate asthmatics has recently been reported in a large real-life cross-sectional study even if there was a considerable overlap between the groups [15]. In the same study, there was however no correlation between ACQ 7 and serum IgE. In the study reported here, though not present at baseline, a weak but significant correlation was observed between sIgE tot and ACQ 6 score throughout the study period, which suggests that fluctuation in IgE over time in a subject may relate to day-to-day asthma symptoms. By contrast, no relationship was found between sIgE tot and severe exacerbation rates. Recent data from SARP (Severe Asthma Research Program) have shown an inverse and surprising relationship between serum IgE and propensity to severe exacerbation in asthmatics [18].
Omalizumab convincingly reduces asthma exacerbation in clinical trial and routine practice in patients with serum IgE ranging from 30 to 700 IU/ mL. The efficacy of omalizumab was shown when free circulating IgE was reduced by more than 95% and maintained at lower than 30 IU/mL [19]; however, sIgE tot itself prior to treatment initiation does not predict the ability of the drug to prevent exacerbation [20]. Rather than considering the baseline IgE level, the IgE level at which omalizumab is able to maintain free circulating IgE may be more relevant in terms of reduction in exacerbations. It is also likely that airway (and not serum) IgE, which can be assessed in sputum [4,21] and bronchial biopsies [22,23], plays a more important role in the disease than circulating IgE though a convincing correlation was reported between serum and sputum IgE levels [4]. We might have found a stronger relationship between IgE and exacerbation if we had measured airway IgE rather than serum IgE in the present study.
The main limitation of our study is the limited number of patients included and followed up throughout the protocol. This may have resulted in a lack of association between sIgE and seasons in contrast to what was reported by others [9]. Moreover, we did not found a relationship between smoke exposure and serum IgE. Our negative finding does not preclude, however, any real effect of smoke exposure on serum IgE because of the limited number of current smoking asthmatics in our cohort. Future studies with larger sample sizes are needed to confirm our findings and explore associations with other possible determinants of IgE variation. Asthmatics in this study were selected on the basis of disease severity defined by the requirement of high-dose ICS combined with a LABA. Therefore, the IgE variability depicted here may not be applicable to a population of mild untreated asthmatics. As we did not have detailed history data regarding atopic dermatitis we could not assess the influence of the disease evolution on sIgE tot over the study period; specifically, whether change in atopic dermatitis severity may have influenced change in serum IgE in our study. In our study the majority of patients with atopic dermatitis were in the intermediate IgE group.
We conclude that in severe asthmatics receiving highdose ICS combined with a LABA, there was limited within-patient variability of sIgE tot but significant between-patient variability. Yet 30% of the patients not initially eligible for receiving omalizumab based on a single measurement may actually benefit from repeated measurements to qualify for treatment.