Asthma biomarkers in children. new opportunities, real practice and frontiers

The implementation of the achievements of precision medicine in the field of allergic diseases fundamentally changes the approaches to real clinical practice and the development of new scientific directions [1]. Precision medicine is especially relevant in pediatrics, where it combines the methods of modern classical medicine, the results of epidemiological studies, and the achievements of genetic and molecular diagnostics, including those based on omics technologies, as effectively as possible [2]. This makes it possible to develop fundamentally new approaches to the treatment and prevention of allergic diseases, taking into account endotypes and phenotypes, as well as many individual characteristics (genetic, environmental, lifestyle, etc.). Therefore, biomarkers, which are understood as measurable indicators that can confirm the presence of the disease, its severity, or response to ongoing therapy, play a key role in the development of personalized management of patients with chronic non-infectious inflammatory diseases. Most often, biomarkers include laboratory indicators that can be measured quantitatively and for which a clear boundary between the states of “norm” and “pathology” is defined. A valid biomarker should be measured in an analytical test system with well-established operational characteristics and have scientifically based data on the physiological, toxicological, pharmacological, or clinical significance of the test results.

Both for clinical practice and for scientific research, the choice of the environment in which biomarkers are studied is extremely relevant. Obtaining reliable data depends on this (availability of standardized sample preparation, certified laboratory equipment, the possibility of dynamic observation, etc.), the invasiveness of the procedure for collecting biological material (compliance with ethical aspects). The classic biological material for assessing immunological changes in bronchial asthma (BA) is blood serum. At the same time, some of the mediators produced, for example, by bronchial epithelial cells, may not reach the systemic blood flow or be present in venous blood in concentrations insufficient for determination. Based on the concept of “unified airways” proposed by Jean Busquet, inflammatory molecules of the bronchial wall may also be detected in the nasal epithelium [3]. As less invasive technologies for assessing the molecular landscape of the bronchial epithelium, especially relevant for pediatric patients, the study of exhaled air condensate, brush biopsies of the nasal wall, the assessment of the concentration of proinflammatory metabolites (nitric oxide) or volatile organic substances in exhaled air are proposed as well [4]. As for the exhaled air condensate and for the material obtained by nasal brush biopsy, important limitations are the difficult accurate estimation of the volume of the obtained biomaterial (for calculating the concentration) and the extremely low content of markers, which increases the requirements for the sensitivity and specificity of the test systems used.

Cohort studies and cluster analysis of large data sets revealed four main clinical phenotypes of BA, differing in pathogenetic mechanisms and response to standard therapy [5]:

1) allergic asthma with early onset;

2) allergic asthma with early onset, moderate-severe course;

3) non-allergic eosinophilic asthma with late onset;

4) non-allergic non-eosinophilic asthma with late onset.

The late onset of the disease and non-allergic phenotype are associated with more severe clinical manifestations and less complete response to pharmacotherapy. According to the spectrum of regulatory molecules (interleukins 4, 5, 13 or interleukins 17, 31 and interferons), the T2-endotype of BA (including both allergic phenotypes and eosinophilic asthma) and the non-T2-endotype of BA are distinguished therefore. Most international guidelines suggest dividing the biomarkers of BA into molecules characteristic of the T2-endotype of inflammation and markers of a heterogeneous and less studied group of non-T2-inflammation endotypes [6][7]. Among children with BA, the proportion of patients with non-T2-endotype is extremely small; one of the leading anamnestic markers of BA, along with characteristic complaints and lability of the clinic, is atopy in personal and family history, which unambiguously refers the majority of pediatric patients to the T2-endotype [8].

Atopic dermatitis (AtD) can be confidently attributed to anamnestic markers of the risk of BA formation; at the same time, the nuances of the clinical course of this disease are important. It has been shown that severe uncontrolled forms of AtD, as well as clinically manifest AtD after the age of three years, and especially AtD, whose exacerbations are not associated with food allergies, are more specific anamnestic markers of the risk of BA onset than the diagnosis of AtD as such or a history of AtD [9]. Other important anamnestic markers include clinical signs of bronchial hyperreactivity: reactions with serial cough/shortness of breath or distant wheezing to physical or emotional stress, contact with pungent odors or allergens [10].

In order to identify a risk group for the development of BA or to confirm the diagnosis of BA among children with recurrent obstructive complications of acute respiratory infections, an assessment of the concentration of molecules predictors of the development of BA may be used [7][9][11]. Quite conditionally, two groups of prognostic markers can be distinguished. The first category includes less labile markers reflecting a child's genetic predisposition to a violation of the barrier properties of epithelial tissues (thymic stromal lymphopoietin (TSLP), the presence of filaggrin mutations, etc.) or an altered immune response to environmental stimuli (specific IgE to inhaled allergens). In children, reliable determination of specific IgE (sIgE) is necessary for adequate administration of elimination measures and allergen-specific immunotherapy. Rational use of the achievements of molecular allergology makes it possible to fundamentally change the course of the disease.

The second group of prognostic markers includes substances whose concentration changes quite rapidly and is directly related to inflammation of the bronchial wall (periostin, exhaled nitrogen oxide (FeNO), eosinophilic cationic protein (ECP), leukotrienes, etc.) [7][11]. Monitoring of the concentration of markers of inflammatory activity after confirmation of the diagnosis of BA can also be used as a tool for assessing the response to therapy, which is currently symptomatic/pathogenetic in nature, but not curative. Basic therapy of BA, modified on the basis of biomarkers, could probably reduce the drug load and the risk of future exacerbations. Currently, the correction of the volume of anti-inflammatory treatment based on the assessment of markers is unjustified from the standpoint of pharmacoeconomics and does not demonstrate significant advantages in the general population. This approach is most promising for patients with severe, difficult-to-control BA, who are considered candidates for monoclonal antibody therapy [7].

Taking into account the continuing increase in the prevalence of BA, including the detection of this disease in patients who did not have a hereditary predisposition in the family history, protective biomarkers that reduce the risk of BA formation (for example, protein CC16, or uteroglobin) are also interesting for the study [12]. The concentration of uteroglobin in the blood serum and in the bronchoalveolar lavage fluid is lower in patients with BA compared with healthy volunteers [12][13]. It was shown that the level of protein CC16 was initially lower in children who were subsequently diagnosed with BA; further, it constantly decreases with an increase in the length of the disease [14]. A pronounced inverse correlation of the level of secretory protein with serum CC16 and allergic sensitization to inhaled allergens was found [14].

Within the framework of this publication, it is impossible to cover the characteristics of all used and promising biomarkers of BA, so only the advantages and disadvantages of only the most relevant of them will be presented (Table 1).

Eosinophils are among the main inflammatory effector cells in the T2 endotype of BA, which is why determining their number in peripheral blood is often recommended as an available biomarker. Most experts consider the excess of 300–500 cells/ml to be absolute peripheral blood eosinophilia, and more than 5% of leukocytes are relative [15]. Eosinophilia in clinical blood analysis is regarded as a surrogate marker of respiratory tract eosinophilia; however, there are patients who do not have this relationship [16]

The significance of eosinophilia for the primary diagnosis of BA is low, since the content of these cells can also increase in other allergic diseases (rhinitis, dermatitis, etc.), parasitic invasions, and some autoimmune diseases [17]. At the same time, the level of eosinophils in the blood ≥4% belongs to the small criteria of both the original and modified predictive asthma index (API) [18]. Various thresholds of peripheral blood eosinophilia are associated with a threefold increase in the risk of BA developing by the age of 6 (more than 300 cells/µl in the first year of life) [19], and with an established BA diagnosis — with a higher frequency of exacerbations and poorer disease control (>400 cells/µl) [20]. In the pediatric cohort, peripheral blood eosinophilia (≥ 300 cells/µl) is associated with a greater severity of BA, a greater number of exacerbations, a decrease in FEV₁/FVC, bronchial hyperreactivity, and thickening of the bronchial wall [21].

The content of eosinophils in peripheral blood less than 470 cells/ml is a more sensitive predictor of the absence of recurrence of bronchial obstruction than negative results of specific IgE or prick tests [22]. Children with atopic BA and eosinophilia ≥300 cells/µl more significantly clinically respond to iGCS [23]. A decrease in the number of peripheral blood eosinophils is observed with an increase in the dose of iGCS [24].

Proteins produced by eosinophils are also used as biomarkers of BA: the main protein, eosinophilic peroxidase, eosinophilic neurotoxin, and eosinophil cationic protein (ECP). The latter is the most accessible for use in routine pediatric practice, since a standardized commercial test system has been developed for it. This protein is located in eosinophil granules and is released upon activation of these cells (for example, through IL-5) by the release of granules; the ECP level is strongly directly correlated with the intensity of allergic inflammation of the respiratory tract [25]. The concentration of ECP in the blood serum increases with exacerbation of BA and normalizes, correlating with a decrease in airway resistance, after eight weeks of treatment with montelukast [26]. At the same time, in children with a higher baseline serum ECP level, a more significant improvement in lung function was observed as a result of treatment with iGCS [27], which makes it possible to recommend this marker for the selection of young children for treatment with iGCS and titration of the dose [28].

The nitrogen oxide of exhaled air is formed in the respiratory tract by the enzyme inducible nitric oxide synthase, the activity of which depends on the pro-inflammatory cytokines of the T2 response, namely IL-4 and IL-13. It has been shown that the level of FeNO is maximal in the group of patients with allergic asthma, rather high in the group of patients with non-allergic asthma and minimal in the group of healthy volunteers [29]. The content of FeNO directly correlates with the severity of reversible bronchial obstruction, the number of peripheral blood eosinophils, and the degree of bronchial hyperreactivity [30]. Various threshold values of this biomarker have been proposed, but it remains generally accepted that an indicator of less than 25 particles per billion (ppb) is not associated with eosinophilic inflammation, and a result of more than 50 ppb confidently indicates eosinophilic inflammation [30]. The level of FeNO is affected by gender, smoking, atopy, overweight, as well as nasal polyps, even in the absence of BA. In a three-year observational study of patients with severe BA course, it was shown that the subgroup with the highest level of FeNO (≥50 ppb) was characterized by a greater number of exacerbations and a shorter duration of the period without exacerbations of BA compared with the subgroup of patients with consistently low levels of FeNO (<25 ppb). A decrease in FeNO levels is inversely correlated with the patient's adherence to the use of inhaled glucocorticosteroids (iGCS) [31].

The concentration of total IgE in the blood serum may be increased in patients of different ages with atopic diseases; however, parasitic invasions, invasive mycoses, some infections, as well as autoimmune diseases and primary immunodeficiency are also alternative reasons for increasing this indicator [32]. In children with an established diagnosis of atopic BA, the level of total serum IgE moderately correlates with the severity of BA, hyperreactivity of the respiratory tract, and thickening of the bronchial wall [21]. The use of total serum IgE as a diagnostic biomarker of BA in real practice is impractical due to its low specificity and sensitivity. High levels of total IgE in the blood serum may indicate the presence of sensitization; this parameter is taken into account when determining the indications and calculating the dose of one of the monoclonal antibody preparations (omalizumab). During treatment with this medicine, repeated determination of total serum IgE is uninformative and is not recommended for monitoring the effectiveness of therapy [33].

Specific IgE blood sera to various allergens currently appear to be one of the key prognostic biomarkers of the development and course of BA. According to prospective cohort studies conducted in various countries, early sensitization (concentration of specific IgE in blood serum from 6 to 24 months of life) to inhaled allergens directly indicates a high risk of BA formation in children by school age [34]. As for high-quality clinical interpretation, it is fundamentally important to evaluate sensitization using modern precision technologies, for example, ImmunoCAP or the ISAC allergy chip. The use of the ISAC allergy chip in children aged 3–4 years allows identifying sensitization to the most significant inhaled allergens and assessing the risk of the development and/or persistence of BA. Risk molecules, depending on the cohort studied, include Betv1 (birch pollen), Feld1 (cat), Phlp1 and Phlp5 (timothy pollen), Derp1/2 (Dermatophagoides pteronyssinus), Derf1/2 (Dermatophagoides farinae) [35]. The presence of slgE to major allergens of milk, eggs, peanuts, and fish also has a prognostic significance of increasing the risk of BA, although less pronounced than inhalation sensitization. There is no doubt that in young children with AtD, polysensitization to food allergens acts as a biomarker of the development of BA [34][36].

Current biomarkers of BA also include products of the 5-lipoxygenase pathway of arachidonic acid oxidation: leukotrienes (LT): LTC4 and LTE4, LTD4 (cysteinyl-leukotrienes, CysLT) and LTV4. Sources of CysLT include many types of upper and lower respiratory tract inflammatory cells, such as eosinophils, mast cells, basophils, macrophages, platelets, and neutrophils. The release of CysLT from peripheral blood leukocytes of atopic patients occurs both with specific (allergens) and non-specific (platelet activation factor) stimulation [37]. Cysteinyl leukotrienes stimulate the production, mobilization, and activation of inflammatory cells, including eosinophils, as well as Th2 cells, ILC 2, monocytes/macrophages and dendritic cells. LTE4 is a stable metabolite of LTC4/LTD4, and it is available for measurement in urine [38]. Levels of LTE4 in urine increase during the exacerbations of BA and correlate with the degree of obstructive disorders. An increase in the concentration of LTE4 in urine was shown under the influence of significant inhalation allergens in children with allergic asthma, as well as when provoked by aspirin in patients with hypersensitivity to the latter [39][40].

Periostin is a protein of the extracellular matrix of the human epithelium, which is actively secreted by fibroblasts, epithelial cells, and some other cells. The physiological role of periostin is to ensure the adhesion and migration of epithelial cells; in addition, periostin is actively secreted during their damage and the development of inflammatory processes [41]. In various biomaterials (blood, sputum, bronchial biopsies, and bronchoalveolar lavage fluid), periostin concentrations were significantly higher in patients with asthma than in those without asthma [42]. Periostin participates in the acute phase of asthmatic inflammation, providing synthesis and secretion by eosinophils IL-6, IL-8, transforming growth factors beta-1 and -2, cystenyl leukotrienes and prostaglandin E2 [43]. In the chronic phase of inflammation, periostin forms accumulations in the basal membrane of the bronchi, providing its thickening and remodeling [44]. It has been shown that the concentration of periostin in patients with BA is stable regardless of the time of year, the period of the disease, or the time of day [44]. It is assumed that patients with a higher concentration of serum periostin are characterized by persistent inflammation of the bronchial wall, a higher risk of future exacerbations, as well as a greater likelihood of remodeling of the bronchial wall (connective tissue rearrangement of the submucosal epithelium layer) [45][46]. According to the author’s data, the concentration of serum periostin was significantly higher in patients who did not reach a controlled course of BA during one year of follow-up, regardless of age group [47]. In addition, in the same observation, a moderate inverse correlation of nasal periostin concentration and forced expiratory volume in the first second was shown in the group of adolescents with BA.

In response to a mechanical violation of integrity or microbial attack, cells of various epithelial barriers (skin, bronchial and intestinal walls) secrete “anxiety mediators” — alarmins (short-lived interleukins-25 and -33 and TSLP) [48][49]. Under physiological conditions, TSLP stimulates the protective reaction of innate lymphoid and phagocytic cells with the development of inflammation. A pathological increase in the concentration of this chemoattractant leads to the chronization of the inflammatory process and the involvement of the adaptive link of the immune response. Under the influence of TSLP, dendritic cells and T-helper cells acquire T2 polarization, that is, they begin to actively synthesize interleukins 4, 5 and 13, form chronic inflammation and a pathological reaction to allergens [50]. TSLP affects the maturation and differentiation of dendritic cells and lymphocytes in the red bone marrow, and also supports T2-polarization of the cytokine profile of eosinophils and mast cells [51]. An increase in the concentration of TSLP in blood serum has been described, in addition to BA, in AtD, allergic rhinitis, BA, and eosinophilic esophagitis [7][52][53].

When performing a bronchial wall biopsy, the material obtained from patients with BA was characterized by a higher concentration of TSLP in comparison with healthy volunteers [48]. In patients with BA, a direct pronounced correlation of the concentration of interleukin-5 and other mediators characteristic of T2 inflammation with the level of TSLP in blood serum was described [51]. At the same time, it was shown that in patients with a more severe, uncontrolled course of BA, the content of TSLP in blood serum was higher [54]. According to the authors’ own data, a strong direct correlation was found between the concentration of TSLP in blood serum and the duration of periods of loss of BA control during the year of follow-up (r=0.74). The concentration of TSLP in blood serum was maximal in patients with atopic asthma with tick-borne sensitization (792.6±114.1 pg/ml). Patients whose FEV₁ index against the background of optimal BA therapy did not reach 80% of the required level during the year had a higher serum TSLP content [55].

The main pharmacological target in BA is inflammation, so the markers of response to therapy can be molecules and indicators that reflect the activity of the inflammatory process in the bronchial wall. It is possible to identify indicators that act as predictors of a good response to a particular group of medicines before their use, as well as markers that are used as indicators of effectiveness at the end of the course of therapy. It is known that patients with initially more pronounced T2 polarization of the immune inflammatory response respond more quickly and more fully to therapy with iGCS [56]. Therefore, predictors of a good response to iGCS will be the hereditary burden of allergic diseases, comorbid asthma, allergic rhinitis, and AtD, high rates of absolute blood eosinophilia, total and specific serum IgE, high FeNO results [6][7][11]. It has been shown that patients with high levels of LTE4 in urine, on the contrary, respond more fully, faster, and more persistently to therapy with antileukotriene drugs than to treatment with iGCS [38]. As for the selection of modern targeted treatment methods (monoclonal antibodies), the level of eosinophils in peripheral blood, total IgE, FeNO and, indirectly, specific IgE as an indicator of the atopic phenotype of BA are actively used as biomarkers [11][57]. In particular, peripheral blood eosinophilia is used as a biomarker for selecting therapy and monitoring the effectiveness of treatment with anti-IL-5 (mepolizumab and reslizumab), anti-IL-5Ra (benralizumab) antibodies [58]. At the same time, data on the independence of the clinical response from the baseline level of T2-inflammation biomarkers were published for omalizumab and dupilumab [59].

Thus, the development and implementation of biomarkers into real pediatric clinical practice today make it possible to identify a risk group for the development of BA, offer rational prevention, in some cases justify the diagnosis and assess the severity of the disease. For children with an already established diagnosis of BA, the use of biomarkers for controlling inflammation and predicting the response to therapy makes it possible to assess the likelihood of future exacerbations and adherence to treatment, optimize anti-inflammatory therapy, and identify children with a severe BA course who need to initiate and conduct biological therapy in a differentiated manner. Scientific studies offer a whole palette of biomarkers in the management of children at risk of developing or suffering from BA; each molecule has certain indications for use and limitations in use (Table 2). Timely, reasonable, and individual selection of biomarkers for measurement in a particular patient can contribute to the prevention of irreversible changes in the bronchial wall (remodeling), reduce the drug load, and improve the prognosis of the course of asthma in children.