ASMA DIFFERENTIAL BIOMARKERS (Machine-translation by Google Translate, not legally binding)

专利摘要:
In the present invention, therefore, the usefulness of the study of the expression of 8 proteins, 5 in serum and 3 by western-blot (as described in the examples of the invention), as potential differential biomarkers of several clinical phenotypes: Non-Allergic Asthma (ANA), Allergic Asthma (AA) and Allergy without asthma (A), as well as, differential biomarkers of severity in asthma: ANA Grave vs ANA Moderate/mild (M/I) and AA Severe vs Moderate/mild AA (M/I). Also, the present invention provides a series of biomarkers capable of discriminating between different clinical phenotypes as illustrated throughout the description. (Machine-translation by Google Translate, not legally binding)
公开号:ES2697299A1
申请号:ES201730947
申请日:2017-07-19
公开日:2019-01-22
发明作者:Olombrada Blanca Cárdaba；Muñiz Selene Baos；Ricote David Calzada；Jimeno Lucía Cremades
申请人:Instituto de Investigacion Sanitaria Fundacion Jimenez Diaz；
IPC主号:

专利说明:

[0001]
[0002] Differential biomarkers of asthma
[0003]
[0004] Field of technique
[0005]
[0006] The present invention is framed in the medical field, in particular in the field of medical diagnosis using biomarkers capable of discriminating between individuals with different clinical asthma phenotypes as well as the severity of each of said clinical phenotypes.
[0007]
[0008] BACKGROUND OF THE INVENTION
[0009]
[0010] Chronic inflammatory respiratory diseases, including allergic diseases and asthma, are common, complex and heterogeneous diseases, reasons why their clinical evolution and treatment are not always predictable.
[0011]
[0012] Allergic diseases are adverse reactions of the immune system against theoretically innocuous substances whose prevalence, in addition to being very high worldwide, is increasing, although in a variable way depending on the lifestyle, reflecting its multifactorial character. As highlighted by the European Academy of Allergy and Clinical Immunology (EAACI), today, allergy is a public health problem of pandemic proportions, affecting more than 150 million people in Europe; In fact, it is the most common chronic disease. Taking into account the epidemiological trends, the EAACI estimates that more than half of the European population will suffer some type of allergy in 2025. This high prevalence and its impact on the quality of life make it a serious economic problem (lower productivity of those affected and higher absenteeism) and for public health systems. It is estimated that the most common clinical manifestations of this type of illness, asthma and rhinitis, represent more than 100 million days of work or school absenteeism per year.
[0013]
[0014] Bronchial asthma is an inflammatory disease of the respiratory tract that causes bronchial hyperresponsiveness and / or airflow obstruction characterized by symptoms such as coughing, wheezing or dyspnea. The World Health Organization (WHO) defines it as the most common chronic disease in children and it is estimated that there are more than 300 million affected. Of these, approximately 10% show a severe asthma clinic, with significant morbidity and mortality. In the last Global Initiative for Asthma (GINA) it was defined as "a heterogeneous disease, usually characterized by inflammation Chronic respiratory tract. "It is characterized by the history of respiratory symptoms such as wheezing, shortness of breath, chest tightness and cough that vary in intensity over time and is associated with a variable limitation of expiratory airflow. The main problems to define this pathology is its wide clinical spectrum, from an occasional episode (which can be easily reversed) to a maintained blockade that requires high doses of oral or inhaled corticosteroids .In the medical community, it is generally accepted that The clinical differences observed in the responses to treatment or in the course of the disease over time are related to multiple underlying variations in the genetic, pharmacological, physiological, biological and / or immunological mechanisms that produce subclasses of phenotypes called endotypes. heterogeneity has led to the call for the search for medicine Precision or customized (among others). For this reason, there are more and more groups that talk about endotypes or various forms of the disease, which requires different diagnostic and therapeutic approaches, as recently reviewed by experts in this area.
[0015]
[0016] Probably due to this great complexity and heterogeneity, so far, there is no cure for asthma. The main objective of asthma treatment is to achieve and maintain control of the disease as soon as possible, in addition to preventing exacerbations and chronic airflow obstruction and to reduce mortality to a minimum. The objectives of the treatment, both in terms of controlling daily symptoms (current control domain), and to prevent exacerbations and an exaggerated loss of lung function (future risk domain), can be achieved in a high percentage of patients with adequate treatment , but there is always a proportion that does not respond adequately to any treatment.
[0017]
[0018] Multiple studies have characterized various phenotypes of the disease in certain groups of patients with recognizable demographic, clinical or pathophysiological characteristics. However, although in patients with severe uncontrolled asthma, such classification may be helpful in guiding specific treatments, for the moment there is no robust evidence to recommend a classification of the disease based on asthma phenotypes in general, and in which controls with the usual treatment, in particular. The asthma phenotypes can be grouped into three main blocks (not mutually exclusive): clinical or physiological, related to triggers and inflammatories (Table 1).
[0019] Table 1. Asthmatic Phenotypes
[0020] Clinical or severe asthma.
[0021] physiological Asthma with severe exacerbations.
[0022] Asthma refractory to treatment, especially in patients without allergy and cortico-dependent asthma.
[0023] Early onset asthma, in children under 12 years old, which is usually allergic. Late-onset asthma, especially women, begins in adulthood and usually goes without allergy.
[0024] Asthma with fixed limitation to airflow, by bronchial remodeling; due to overlapping syndrome, asthma and COPD.
[0025] Asthma and obesity, with severe symptoms.
[0026] Related to allergic asthma, environmental or occupational allergens. Triggers Asthma induced by non-steroidal anti-inflammatory drugs (NSAIDs).
[0027] Asthma induced by menstruation.
[0028] Asthma induced by exercise.
[0029] Inflammatories Eosinophilic asthma is usually allergic and has a good response to inhaled glucocorticoids, in general.
[0030] Neutrophilic asthma, usually occurs in patients with severe disease and severe exacerbations, with a worse response to inhaled glucocorticoids.
[0031] Paucigranulocytic asthma.
[0032] Taken from the Spanish Guide for Asthma Management, GEMA 4.0
[0033]
[0034] Classification of adult asthma. Clinical severity Asthma has usually been classified according to severity, although the definition has evolved over time. The severity of asthma is an intrinsic property of the disease, which reflects the intensity of the pathophysiological abnormalities. Keep in mind that the severity of asthma involves both the intensity of the process and the response to treatment. Gravity is usually assessed when the patient is being treated and classified according to the maintenance treatment needs required to achieve control of symptoms and exacerbations. It is traditionally divided into four categories: intermittent, mild persistent, moderate persistent, and severe persistent (Table 2).
[0035]
[0036] Gravity is not a characteristic of asthma necessarily constant, but can vary over time (in months or years), so it is necessary to periodically reevaluate it. The severity is determined retrospectively in the patient whose asthma it is controlled according to the therapeutic level in which it is found, that is, based on the amount of medication that is necessary to maintain control of the disease, resorting to the reduction of the step if it were necessary to stipulate the minimum amounts of treatment. It can be established in a patient who is not receiving maintenance treatment, but this is infrequent.
[0037]
[0038] Control. The control of asthma is the degree to which the manifestations of asthma are absent or are reduced to the maximum by therapeutic interventions and the treatment objectives are met. The control reflects to a large extent the suitability of the treatment for asthma. However, we must take into account another factor, which differs from one patient to another, and is the response to treatment and the ease and speed with which control is achieved. Although the term control is broad and can encompass all clinical and pathophysiological aspects of asthma, for practical purposes, it includes the clinical characteristics of the disease (symptoms and exacerbations) and pulmonary function tests.
[0039]
[0040] Table 2. Classification of asthma according to severity
[0041] Flashing Persistent Mild Persistent Persistent moderate severe Symptoms No More than 2 times Symptoms a Diurnal symptoms (2 or continuous daily a week less than (several times a week) day) Medication No More than 2 times Every day Several times to relief (agonist (2 times or week a day
[0042] p2-adrenergic less / but not daily
[0043] short-week action)
[0044] Symptoms No more than 2 More than 2 times More than once Often nocturnal times a month a month a week
[0045] Limitation of None Something Quite A lot of activity
[0046] Function> 80% <80%> 60% - <80% <60% pulmonary (FEV1
[0047] or PEF)% theoretical
[0048] Exacerbations None One or none at Two or more at Two or more per year year year
[0049] Taken from the Spanish Guide for Asthma Management, GEMA 4.0. FEV1: Forced expiratory volume in the first second; PEF: Maximum expiratory flow.
[0050] Drugs to treat asthma are classified as control or maintenance, and relief, also called "rescue." Control or maintenance drugs , which must be administered daily for prolonged periods, include inhaled glucocorticoids (GCI) or systemic glucocorticoids. leukotriene receptor antagonists (ARLT), long-acting p2-adrenergic agonists (LABA), tiotropium, and anti-IgE monoclonal antibodies (omalizumab) The chromones and delayed-release theophylline have fallen into disuse because of their lower efficacy. Relief medications are used on demand to treat or prevent bronchoconstriction quickly, and include inhaled short-acting p2-adrenergic agonists (SABAs) (of choice) and inhaled anticholinergics (ipratropium bromide).
[0051]
[0052] Severe asthma is characterized by the need to specify multiple drugs and high doses for treatment. It is associated with a greater consumption of economic resources compared to moderate or mild asthma. Severe uncontrolled asthma (AGNC ) is defined as asthma that persists poorly controlled despite receiving treatment with a combination of GCI / LABA, at high doses in the last year, or oral glucocorticoids for at least six months of the same period .
[0053]
[0054] Technical problems that the present invention solves
[0055]
[0056] As already described above, from a clinical point of view, asthma is a very heterogeneous disease, with a large number of different phenotypes. However, despite this clinical heterogeneity of asthma, allergic mechanisms have been implicated in 50-80% of asthmatic patients and in approximately 50% of severe asthma. This is one of the reasons why asthma has been commonly associated with type 2 (Th2) respiratory inflammation, characterized by elevated levels of IgE, eosinophils and some cytokines such as IL4, IL5, IL13 and IL9, canonically associated with allergic responses and for that reason, a large part of the efforts in the search for new treatments for asthma, have focused on the Th2 cytokine pathway. However, the type 2 immune response is a complex endotype, with several subendotypes, such as the endotypes defined as IL-5-high, IL-13-high or IgE-high that define subgroups of patients that would have therapeutic benefits with different treatment targets. New strategies have been used for the discovery and validation of molecular biomarkers such as omics approaches to reveal mechanisms responsible for asthma endotypes in different tissues. A biomarker is defined as an objective, measurable parameter, which can be the signature of a complex underlying pathway or a key molecule associated with, or that directly plays an essential role in an endotype of a specific disease. Several new experimental treatments, known as biological therapies, are in various stages of clinical development for patients with inflammation driven by a type 2 immune response: anti-IL-4 / IL-13, anti-IL-4, anti-IL antibodies -5 and anti-IgE, as well as CRTH2 (homologous chemokine receptor of a molecule expressed on Th2 lymphocytes). A summary of the state of these biological therapies is currently outlined in Tables 3 and 4. However, at present, the available biomarkers are not specific enough to select the type 2 immune response asthma subendotype that specifically responds to a targeted treatment. This may be due to several factors, such as the genetic (or epigenetic) influence or the ignorance of the predominant inflammatory immune pathway or the contribution in the response of the remodeled tissue itself. In summary, although recent therapeutic advances have unraveled some of the contributions of different phenotypes and endotypes to the pathogenesis of asthma and responses to specific therapies, more information is still needed to optimize the patient's therapeutic responses, trying to avoid the same adverse effects.
[0057] Table 3. Asthma treatments with approaches guided by biomarkers
[0058] Biomarker Treatment with the Associations Comments (points to keep in mind, produce a variability / fluctuation) response
[0059] Blood
[0060] Eosinophils Anti-IL-5 Exacerbations Available easily
[0061] Anti-IgE FP reduction Significant fluctuation Anti-IL-4 / IL-13 Fixed obstruction of
[0062] Corticosteroids the ways
[0063] Respiratory CRTH2 antagonists
[0064] Specific IgE Anti-IgE Exacerbations
[0065] AIT AHR (AIT)
[0066] Periostina Anti-IL-13 Decrease in Research
[0067] DPP-4 FP Dependent test Exacerbations
[0068] Induced sputum
[0069] Eosinophils Anti-IL-5 Exacerbations In Investigation
[0070] ICS Significant fluctuation
[0071] IL-13 Anti-IL-13 Unknown In Investigation
[0072] Exhaled air
[0073] FENO Anti-IL-5 Exacerbations, Easily available
[0074] Anti-IgE decrease of significant Fluctuation Anti-IL-13 FP
[0075] ICS
[0076] Metabolomics ICS Unknown In research
[0077] (VOC)
[0078] There is a significant overlap between the biomarkers used to predict the response to different strategies directed by the endotype. In addition, few biomarkers are readily available, most fluctuate significantly, and none are validated or guaranteed their quality. TIA, immunotherapy with allergen; DPP-4, dipeptidyl peptidase 4; ICS, inhaled corticosteroids; FP, pulmonary function; VOC, volatile organic components.
[0079] Table 4. Treatments directed by endotypes in asthma of type Th2
[0080] Biomarker Medicament Target Effects State predictive regulation Eosinophils in Omalizumab IgE Reduces exacerbations Approved by FDA blood Improves symptoms and EMA and Periostine quality of life
[0081] FENO
[0082] Eosinophils in Mepolizumab IL-5 Reduces the number of FDA approved blood / sputum eosinophils, exacerbations and in evaluation by FENO OCS EMA
[0083] Enhanced FEV1 Tested for CRSwNP Eosinophils in Reslizumab IL-5 Reduces the number of In evaluation by eosinophilic blood , FDA exacerbations
[0084] Improvement FEV1
[0085] Eosinophils in Benralizumab IL- Reduces the number of Phase III blood 5Ra eosinophils and basophils,
[0086] exacerbations
[0087] Improvement FEV1
[0088] Eosinophils in Dupilumab IL- Reduces exacerbations Phase III blood 4Ra Improved FEV1 Tested for Improved symptoms and CRSwNP, AD and quality of life EoE Periostine Tralokinumab IL-13 Reduces Phase II number
[0089] DPP-4 eosinophils and exacerbations
[0090] Improvement FEV1
[0091] Periostin Lebrikizumab IL-13 Reduces Exacerbations Phase III Best FEV1
[0092] The IgE, IL-5, and IL-4 / IL-13 pathways can be approached with monoclonal antibodies (mAbs). There is a remarkable overlap between the so-called predictive biomarkers and significant heterogeneity in the clinical response. CRSwNP, Chronic rhinosinusitis with nasal polyps; DPP-4, dipeptidyl peptidase 4; EMA, European Medicines Agency; EoE, eosinophilic esophagitis; FDA, Food and Drug Administration of the United States; IL-4Ra, receptor a of IL-4; IL-5Ra, Receiver a of IL-5; OCS, oral corticosteroids.
[0093] On the other hand, although allergic asthma (AA) affects a significant proportion of patients, 10% to 33% of subjects with asthma are non-allergic (NA) subjects, defined as subjects with asthma but without any associated allergic sensitization. In these subjects, the mechanisms that contribute to the type 2 immune response are less clear. In many cases, instead of eosinophilic inflammation, there is a prevalence of neutrophils. The endotype of immune response asthma not mediated by Th2 is much less known than the Th2 type and until now effective therapies targeting this endotype have not been demonstrated. Therefore, efforts directed towards this type of asthma are clearly an unmet need and biologically targeted therapies are a field to be developed.
[0094]
[0095] Brief description of the figures
[0096]
[0097] Fig. 1. Preliminary study of the expression data by PCA ( Principal Component Assay ) as explained in example 1, in particular in the section on the study of gene expression.
[0098]
[0099] Fig. 2 Quantification of MSR1. Relative quantification of the 2 bands detected by the MSR1 antibody of all the subjects studied in the control group (C) and the group with non-allergic asthma (ANA). * Statistically significant comparison (p <0.001) between the group with non-allergic asthma and the control group
[0100]
[0101] Fig. 3. Quantification of SERPINB2. Relative quantification of the detected band around 43kDa by the SERPINB2 antibody of all the subjects studied for this control group protein (C), nonallergic asthma (ANA), allergic asthma (AA) and allergy without asthma (A). * Statistically significant comparison (p <0.0001) between the control and the indicated group. Statistically significant comparison (p <0.05) between the group with allergy without asthma and the one indicated.
[0102]
[0103] Fig. 4 Quantification PHLDA1. Relative quantification of the band detected around 43kDa by the PHLDA1 antibody of all the subjects studied for this control group protein (C), nonallergic asthma (ANA), allergic asthma (AA) and allergy without asthma (A).
[0104] Fig. 5 Levels of protein in serum . A) Average levels of IL8 in the serum of the control groups (C), non-allergic asthma (ANA), allergic asthma (AA) and allergy without asthma (A). B) Average levels of IL10 in the serum of groups C, ANA, AA and A. C) Mean levels of CHI3L1 in the serum of groups C, ANA, AA and A. D) Mean levels of PI3 in the serum of groups C, ANA, AA and A. D) Mean levels of POSTN in the serum of groups C, ANA, AA and A. * Statistically significant comparison (p <0.001) between group A and the indicated group. Statistically significant comparison (p <0.0001) between group C and the indicated group. ** Statistically significant comparison (p <0.05) between the indicated groups. ## Statistically significant comparison (p <0.001) between the indicated groups.
[0105]
[0106] Description of the invention
[0107]
[0108] Definitions
[0109]
[0110] For the purposes of the present invention, the following definitions are included below:
[0111]
[0112] - In the present invention, "Asthma" is understood to be a chronic inflammatory disease of the respiratory tract, in whose pathogenesis various cells and mediators of inflammation intervene, conditioned in part by genetic factors and which is associated with bronchial hyperresponsiveness (BHR) and a variable airflow obstruction, totally or partially reversible, either by the action of medications or spontaneously - In the present invention, "Non-allergic asthma" is understood as the clinical diagnosis of asthma carried out by a specialist Allergist or Pulmonologist, according to the criteria of the Spanish Guide to Asthma Management (GEMA) and without any associated allergic symptoms (clinical history not suspicious of any allergy and negative skin tests against battery of common allergens).
[0113] - In the present invention, "Allergic asthma" means the clinical diagnosis of asthma performed by a specialist Allergist or Pulmonologist, according to the criteria of the Spanish Guide to Asthma Management (GEMA), associated with characterized allergic symptoms (history of suspicion of allergic diseases, positive skin tests against an allergen of a battery of common allergens, high total IgE).
[0114] - the term "selection" is understood as the examination or testing of a group of individuals belonging to the general population, at risk of suffering from asthma, in order to distinguish healthy or allergic individuals from those suffering from asthma, more particularly with the objective of distinguishing the severity of asthma among those individuals suffering from asthma.
[0115] - The expression "positively regulated", "increased concentration level" or "over expressed" referred to any of the proteins or combinations thereof described in the present invention, refers to an increase in their level of protein concentration with with respect to a given "threshold value" or "limit value" of at least 5%, of at least 10%, of at least 15%, of at least 20%, of at least 25%, of at least less 30%, of at least 35%, of at least 40%, of at least 45%, of at least 50%, of at least 55%, of at least more than 60%, of at least more than 65%, of at least 70%, of at least 75%, of at least 80%, of at least 85%, of at least 90%, of at least 95%, of at least 100%, of at least 110%, of at least 120%, of at least 130%, of at least 140%, of at least 150%, or more.
[0116] - The expression "negatively regulated", "reduced concentration level" or "underexpressed" referred to any of the proteins or combinations thereof described in the present invention, refers to a reduction in their level of protein concentration with respect to a given "threshold value" or "limit value" of at least 5%, of at least 10%, of at least 15%, of at least 20%, of at least 25%, of at least 30%, of at least 35%, of at least 40%, of at least 45%, of at least 50%, of at least 55%, of at least more than 60%, of less than 65%, of at least 70%, of at least 75%, of at least 80%, of at least 85%, of at least 90%, of at least 95%, of at least 100%, of at least 110%, of at least 120%, of at least 130%, of at least 140%, of at least 150%, or more.
[0117] - In the present memory in order to separate the integer from the decimals, the Anglo-Saxon mode is used, therefore using a point instead of a comma.
[0118] - The term "threshold value" or "limit value", when referring to the concentration levels of the proteins described in the present invention, refers to a level of the reference concentration indicative that a subject is likely to suffer from asthma or a type of asthma determined as intermittent asthma or mild, moderate or severe persistent asthma with a sensitivity and specificity given if the Patient concentration levels are above or below said threshold, limit or reference levels.
[0119] - The term "comprising" is intended to include, but is not limited to, what follows the expression "comprising". Therefore, the use of the term "comprising" indicates that the elements cited are necessary or mandatory, but that other elements are optional and there is a possibility that they are present or not.
[0120] - By "consisting of" is meant to include, and is limited to, what follows the expression "consisting of". Therefore, the expression "consisting of" indicates that the elements cited are necessary or mandatory and that no other element can be present.
[0121] - It should also be borne in mind that the term "kit", as used herein, is not limited to any specific device and includes any device suitable for practicing the invention.
[0122] - SERPINB2 (member of the group of inhibitors of the family of serine proteases, enzymes that inhibit the neutrophil cathepsin G protease and mast cell chymase SERPINB2 has been detected in different types of cells, playing a role in inflammation and remodeling ( Swartz, JM, Bystrom, J., Dyer, KD, Nitto, T., Wynn, TA, Rosenberg, HF, 2004. Plasminogen activator inhibitor-2 (PAI-2) in eosinophilic leukocytes, J. Leukoc.Biol. 812-819).
[0123] - MSR1 ("Junk" receptor of class A macrophages, also called SR-A or CD204.) These receptors are integral trimeric membrane glycoproteins, initially described as specific for macrophages (Naito, M., Kodama, T., Matsumoto, A ., Doi, T., Takahashi, K., 1991. Tissue distribution, intracellular localization, and in vitro expression of bovine macrophage scavenger receptors, Am. J. Pathol., 139, 1411-1423), which have been implicated in many processes Physiological and pathological factors associated with this cell type were subsequently found in other types of cells (usually tissue localization), such as vascular smooth muscle cells, endothelial cells (EC), human lung epithelial cells, etc. (Tomokiyo, R. , Jinnouchi, K., Honda, M., Wada, Y., Hanada, N., Hiraoka, T., et al., 2002. Production, characterization, and interspecies reactivities of monoclonal antibodies against human class A macrophage scavenger receptors. Atherosc lerosis 161, 123-132), a fact that increases its physiopathological potential, indicated as a central pivot of health and disease (Kelley, JL, Ozment, TR, Li, C., Schweitzer, JB, Williams, DL, 2014. Scavenger Receptor-A (CD204): a twoedged sword in health and disease. Crit. Rev. Immunol. 34, 241-261).
[0124] - Lower MSR1 (lower molecular weight band recognized by the specific monoclonal antibody of human MSR1, in the protein analysis by western).
[0125] - MSR1 superior (band of higher molecular weight recognized, by the monoclonal antibody specific to human MSR1, in the protein analysis by western).
[0126] - PHLDA1 (pleckstrin homology-like domain, family A, member 1, a nuclear protein rich in proline-histidine that has been described in humans as a putative marker of epithelial stem cells in the intestine (Sakthianandeswaren, A., Christie, M., D'Andreti, C., Tsui, C., Jorissen, RN, Li, S., et al., 2011. PHLDA1 expression marks the putative epithelial stem cells and contributes to intestinal tumorigenesis Cancer Res. 71, 3709-3719) .
[0127] - CHI3L1 ( chitinase 3-like 1, a glycoprotein, member of the 18 family of glycosyl transferases or YKL-40) It lacks activity chitinase and is secreted by activated macrophages, chondrocytes, neutrophils and synovial fluid cells. role inflammation and tissue remodeling and its role as a possible biomarker has been reviewed in signaling mechanisms regulated by YKL-40.
[0128] - IL10 (Cytokine with pleiotropic effects, produced mainly by monocytes and in smaller quantity by lymphocytes, which has been described as a potential driver of the immune response.
[0129] - IL8 (Interleukin 8, or member of the CXC family of chemokines, considered one of the main mediators of the inflammatory response and very important for the survival and chemotaxis of neutrophils.) It is secreted by different cell types and is considered to be related to the pathogenesis of bronchiolitis, respiratory disease caused by viral infection, as well as being involved in acute lung damage, affecting, among other things, the survival of neutrophils - PI3 (Peptidase 3 inhibitor derived from the skin or specific inhibitor of elastase (Trappin-2 or Elafin) that functions as an antimicrobial peptide, is a potent inhibitor of serine proteases, preventing excessive damage at inflammatory sites, modulating a wide range of parameters that are critical for inflammation, although with pleiotropic effects and / or Periostina (secreted protein composed of 4 domains of fascillin 1 aligned in tandem, of which we suspect has that it is an adhesion molecule and that it may be involved in subepithelial fibrosis.
[0130] In the prior art a variety of statistical and mathematical methods are known to establish the threshold level or concentration limit. A threshold or limit concentration level for a particular biomarker can be selected, for example, based on data from the receiver operating characteristic (ROC) graphs, as described in the Examples and Figures of the present invention. One skilled in the art will appreciate that these threshold or limit concentration levels can be varied, for example, by moving along the ROC plot for any of the biomarkers described in the present invention, or for any combination thereof, to obtain different values. of sensitivity or specificity, thus affecting the overall performance of the assay. For example, if the goal is to have a clinically sound diagnostic method, one should try to have a high sensitivity. However, if the objective is to have an economic method, one should try to achieve a high specificity. The best limit refers to the value obtained from the ROC plot for a particular biomarker, or a particular combination, that produces the best sensitivity and specificity. Sensitivity and specificity values are calculated over the range of thresholds (limits). In this way, the threshold or limit values can be selected in such a way that the sensitivity and / or specificity are at least about 70%, and can be, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% at least 60% of the patient population tested, or at least 65%, 70%, 75% or 80% of the patient population tested.
[0131]
[0132] Accordingly, each of the embodiments cited throughout the present invention is preferably performed by determining the concentration levels of at least one of the biomarkers described in the present invention in an isolated sample from a subject to be diagnosed or screened, and comparing the concentration levels of said biomarker with predetermined threshold or limit values.
[0133]
[0134] Detailed description of the invention
[0135]
[0136] Considering the above knowledge, the present invention aims to provide new markers and / or combinations of biomarkers, capable of defining in simple samples to be analyzed, preferably derived from peripheral blood such as serum, plasma or proteins derived from PBMCs, as well as, sputum or other biological sample of the target organ (target organ means lung or any part of the pathways of a human subject), Differential patient groups of 3 clinical phenotypes: AA (Allergic Asthma), ANA (Non-Allergic Asthma) and Allergy without asthma (A), as well as potential markers capable of differentiating severity.
[0137]
[0138] In the present invention, therefore, it is proposed to study the expression of 8 proteins, 5 in serum and 3 by westem-blot (as described in the examples of the invention), as potential differential biomarkers of several clinical phenotypes: Non Allergic Asthma (ANA), Allergic Asthma (AA) and Allergy without asthma (A), as well as, of differential biomarkers of severity in asthma: Severe ANA vs Moderate / mild ANA (M / l) and Severe AA vs Moderate / mild AA (M / l). Also, the present invention provides a series of biomarkers capable of discriminating between different clinical phenotypes as illustrated throughout the description.
[0139]
[0140] To define the specific biomarkers of each condition, an analysis of ROC curves (quantitative, which allows us to define an appropriate threshold of discrimination and qualitative) of protein expression was made, comparing the AUC of the potential biomarkers individually and combining them two in two or three in three.
[0141]
[0142] The comparisons made are:
[0143]
[0144] 1. Each clinical phenotype (total ANA, total AA and A) with respect to the control group.
[0145] 2. Each sub-phenotype of asthmatics attending to severity:
[0146] to. Moderate / mild with respect to control
[0147] b. Serious with respect to control
[0148] c. Moderate / mild with respect to Grave
[0149]
[0150] 3. The clinical phenotypes have also been compared with each other: total ANA vs total AA, severe ANA vs severe AA, ANA M / L vs AA M / L, total AA vs A, AA Severe vs A, AA M / L vs A .
[0151]
[0152] We enclose the tables of results, of all biomarkers analyzed, noting only the results that give a good quantitative ROC curve (AUC: 0.75-0.9), very good (AUC: 0.9-0.97) or excellent (AUC: 0.97-1) . The criterion of the quantitative AUC value has been chosen because it allows us to define separation thresholds between groups, in an objective way and therefore, it would be useful for future Kits.
[0153] BIOMARKERS FOR NON-ALLERGIC ASTHMA (ANA)
[0154]
[0155] 1. Comparisons between healthy controls and non-allergic asthmatics (total population) (ANA)
[0156]
[0157] - N0 = Observations in Control group
[0158] - N1 = Observations in non-allergic asthmatic group (ANA)
[0159] - AUC: Area under the ROC Curve at a quantitative level. For the models with combinations of two or more variables, the AUC value has been split into two values: Quantitative AUC value denominated in the tables as "Quantitative variables" and Qualitative AUC value denominated in the tables as "Qualitative variables".
[0160]
[0161] Table 5. Individual ROC Curves
[0162]
[0163] Variable N0 N1 AUC (IC95%) Threshold
[0164] CHI3L1 30 30 0.60 (0.45 - 0.75) 13064
[0165] IL10 9 11 0.42 (0.15 -0.70) 102.9
[0166] IL8 9 14 0.50 (0.25 - 0.75) 39
[0167] PI3 30 29 0.50 (0.35 - 0.65) 6528
[0168] Periostin 30 30 0.62 (0.48 - 0.77) 13633
[0169] MSR1 upper 9 18 0.60 (0.38 -0.81) 0.208
[0170] Lower MSR1 9 18 0.96 (0.89 - 1.00) 0.148
[0171] PHLDA1 8 5 0.42 (0.07 - 0.78) 0.011
[0172] SERPINB2 6 11 0.91 (0.72 - 1.00) 0.404
[0173]
[0174] The threshold value is only detailed here, at the individual level, because it will be the same for each biomarker, even if combined.
[0175] Table 6. ROC curves for models with combinations of two variables Qualitative variables Combination N0 N1 qualitative quantitative CHI3L1 + IL10 9 11 0.48 (0.20 - 0.77) 0.63 (0.37 - 0.89) CHI3L1 + IL8 9 14 0.67 (0.44 -0.91) 0.64 (0.43 - 0.84) CHI3L1 + PI3 30 29 0.59 (0.44 - 0.74) 0.70 (0.57 - 0.82) CHI3L1 + Periostin 30 30 0.63 (0.49 - 0.78) 0.72 (0.60 - 0.85) CHI3L1 MSR1 higher 9 18 0.78 (0.60 - 0.95) 0.84 ( 0.71 - 0.98) CHI3L1 Lower MSR1 9 18 0.97 (0.91 - 1.00) 0.99 (0.97 - 1.00) CHI3L1 + PHLDA1 8 5 0.70 (0.39 - 1.00) 0.68 (0.37 - 0.98) CHI3L1 SERPINB2 6 11 0.92 (0.77 - 1.00) 0.92 ( 0.78 - 1.00) IL10 + PI3 9 11 0.59 (0.32 - 0.85) 0.58 (0.33 - 0.82) IL10 + Periostine 9 11 0.47 (0.19 -0.76) 0.64 (0.39 - 0.89) IL8 + PI3 9 14 0.61 (0.37 - 0.85) 0.70 (0.53 - 0.87) IL8 + Periostine 9 14 0.74 (0.52 - 0.95) 0.74 (0.59 - 0.89) PI3 + Periostin 30 29 0.67 (0.53 - 0.82) 0.69 (0.56 - 0.82) PI3 + MSR1 higher 9 17 0.63 (0.41 - 0.84) ) 0.73 (0.56 - 0.89) PI3 MSR1 lower 9 17 0.96 (0.88 - 1.00) 0.99 (0.98 - 1.00 ) PI3 + PHLDA1 8 5 0.47 (0.09 - 0.86) 0.75 (0.50 - 1.00) PI3 SERPINB2 6 11 0.91 (0.72 - 1.00) 0.96 (0.88 - 1.00) Periostine + higher MSRI 9 18 0.73 (0.54 - 0.93) 0.75 (0.63 - 0.63) 0.87) Periostin MSR1 lower 9 18 0.96 (0.89 - 1.00) 0.97 (0.92 - 1.00) Periostin PHLDA1 8 5 0.82 (0.59 - 1.00) 0.81 (0.50 - 1.00) Periostin SERPINB2 6 11 0.95 (0.85 - 1.00) 0.98 (0.95 - 1.00 ) Table 7. ROC curves for models with combinations of three variables
[0176]
[0177] Variable variables Variable N0 N1 qualitative quantitative
[0178] CHI3L1 IL10 Periostine 9 11 0.53 (0.25 - ■ 0.80) 0.73 (0.50 - 0.95)
[0179] CHI3L1 IL8 Periostine 9 14 0.72 (0.50 - ■ 0.94) 0.75 (0.55 - 0.94) CHI3L1 PI3 Periostin 30 29 0.66 (0.52 - 0.80) 0.78 (0.66 - 0.89) CHI3L1 PI3 Upper MSR1 9 17 0.77 (0.59 - ■ 0.96) 0.88 ( 0.76 - 1.00) CHI3L1 PI3 MSR1 lower 9 17 0.97 (0.91 - ■ 1.00)
[0180]
[0181] CHI3L1 Periostin MSR1 upper 9 18 0.83 (0.67 - 0.98) 0.86 (0.73 - 0.99) CHI3L1 Periostin MSR1 lower 9 18 0.97 (0.91 - ■ 1.00) 0.99 (0.97 -1.00) CHI3L1 Periostin PHLDA1 8 5
[0182] CHI3L1 Periostine SERPINB2 6 11 0.94 (0.81 - ■ 1.00) 0.99 (0.97 - 1.00) IL10 PI3 Periostine 9 11 0.57 0.83) 0.65 (0.39 - 0.90) IL8 PI3 Periostine 9 14 0.75 (0.53 - ■ 0.96) 0.76 (0.59 - 0.92) PI3 Periostin MSR1 upper 9 17 0.77 (0.58 - ■ 0.96) 0.80 (0.65 - 0.95) PI3 Periostin MSR1 lower 9 17 0.97 (0.90 - ■ 1.00) 0.99 (0.98 -1.00) PI3 Periostin PHLDA1 8 5 0.78 (0.51 - ■ 1.00) 0.89 (0.68 - 1.00) PI3 Periostin SERPINB2 6 11 0.94 (0.81 - ■ 1.00) 0.980.95 - 1.00)
[0183]
[0184] As a result of the information presented in Tables 5 to 7, we propose, as the first aspect of the invention, an in vitro method for selecting subjects at risk of suffering from non-allergic asthma, comprising: (a) measuring the concentration pattern or level of less one of the following proteins: SERPINB2, lower MSR1, PHLDA1, higher MSR1, CHI3L1, IL10, IL8, PI3 and / or Periostina, obtained from a biological sample isolated from the subjects to be selected; and (b) comparing said pattern or level of concentration of at least one of said protein biomarkers, of the subjects to be selected with a standard or level of concentration already established or with the concentration level of a healthy subject, where differences in concentration of at least one of said protein biomarkers is indicative that said subject presents a risk of suffering non-allergic asthma. It is noted that said concentration differences with respect to the healthy individual or with respect to the reference value, will correspond to an overexpression or an under-expression of the concentration depending on the type of biomarker. In this sense, table 5 indicates clearly when the overexpression or under-expression of each of the biomarkers is indicative that said subject presents a risk of suffering non-allergic asthma. On the other hand, tables 6 and 7 indicate specific combinations of biomarkers useful in determining the risk of suffering from non-allergic asthma. Each of said combinations forms a part and can be implemented in the method proposed in the first aspect of the invention.
[0185]
[0186] A preferred embodiment of the first aspect of the invention relates to an in vitro method for diagnosis / prognosis of a subject suspected of having non-allergic asthma, comprising steps a) and b) of the first aspect of the invention, and optionally (c) confirming the presence of asthma through a clinical examination.
[0187]
[0188] Another preferred embodiment of the first aspect of the invention relates to an in vitro method for monitoring the response to a therapy or to monitor the progression of non-allergic asthma, in a subject suffering from said disease, comprising steps a) and b) of the first aspect of the invention.
[0189]
[0190] Another preferred embodiment of the first aspect of the invention relates to a method for treating subjects suffering from non-allergic asthma, comprising steps a) and b) of the first aspect of the invention, and (c) treating the patient who has been diagnosed asthma not allergic. It is noted that possible therapies are expressly mentioned throughout the description, in particular control or maintenance drugs are particularly useful, which must be administered daily for prolonged periods, include inhaled glucocorticoids (GCI) or systemic, receptor antagonists of leukotrienes (ARLT), long-acting p2-adrenergic agonists (LABA), tiotropium and anti-IgE monoclonal antibodies (omalizumab). The chromones and the delayed release theophylline could also be useful therapy in this type of pathologies. Relief medications could also be used on demand to treat or prevent bronchoconstriction quickly, including inhaled short-acting p2-adrenergic agonists (SABAs) (of choice) and inhaled anticholinergics (ipratropium bromide). ). In a case of severe asthma, there may be a need to specify multiple drugs at high doses for treatment.
[0191] Another preferred embodiment of the first aspect of the invention relates to the method according to any of the preceding embodiments, wherein the biological sample is selected from the group consisting of a minimally invasive biological sample of the subjects to be selected, such as a blood sample. (such as a whole blood sample) or derived from peripheral blood such as serum, plasma or proteins derived from PBMCs, as well as, sputum or other biological sample from the target organ (per target organ means lung or any part of the pathway areas of a human subject). Preferably, the subject is a human subject.
[0192]
[0193] Additionally, and again, as a result of the information set forth in tables 5 to 7, we propose as a second aspect of the invention, the use of a kit comprising reagents that detect biomarkers to determine a differential expression level of at least one of the biomarkers mentioned in the first aspect of the invention, where differences in expression of at least one of the biomarkers mentioned is indicative of the risk of having non-allergic asthma, to diagnose in vitro the risk of having non-allergic asthma.
[0194]
[0195] A preferred embodiment of the second aspect of the invention relates to the use of the kit, wherein the kit described in the preceding paragraph can be implemented or used to carry out the present invention using any suitable technique for this. In this regard, various techniques capable of implementing or implementing the present invention are well known in the state of the art. Below I detail some of them: formation of immuno-precipitates alone or combined with diffusion and / or electrophoresis (Western-blot), colorimetric techniques, or direct measurement of the binding of the antibody to the antigen (ELISAS). All of these principles are used in one way or another in kits that are commercially available for the diagnosis of diseases and can be easily implemented in the present invention.
[0196]
[0197] 2. Comparisons between controls and mild or moderate nonallergic asthmatics (ANA)
[0198]
[0199] - N0 = Observations in Control group
[0200] - N1 = Observations in ANA Group Moderate / slight
[0201] - AUC: Area Under the ROC Curve. For models with combinations of two or more variables, the AUC value has been split into two values: Quantitative AUC value denominated in the tables as "Quantitative variables" and Qualitative AUC value denominated in the tables as "Qualitative variables".
[0202]
[0203] Table 8. Individual ROC Curves
[0204] Variable N0 N1 AUC (IC95%) Threshold
[0205] CHI3L1 30 15 0.56 (0.39 - 0.73) 13064
[0206] IL10 9 6 0.41 (0.08 - 0.74) 102.9
[0207] IL8 9 7 0.62 (0.30 - 0.94) 677
[0208] PI3 30 15 0.51 (0.34 - 0.69) 3074
[0209] Periostin 30 15 0.69 (0.52 - 0.86) 15787
[0210] MSR1 upper 9 8 0.60 (0.29 - 0.90) 0.208
[0211] Lower MSR1 9 8 1.00 (1.00 - 1.00) 0.148
[0212] PHLDA1 8 7 0.48 (0.15 -0.81) 0.014
[0213] SERPINB2 6 6 0.89 (0.66 - 1.00) 0.404
[0214]
[0215] Table 9. ROC curves for models with combinations of two variables
[0216] Variables Variables Combination N0 N1 qualitative quantitative CHI3L1 + IL10 9 6 0.41 (0.08 - 0.74) 0.73 (0.45 - 1.00) CHI3L1 IL8 9 7 0.63 (0.32 - 0.95) 0.77 (0.54 - 1.00) CHI3L1 + PI3 30 15 0.56 (0.39 - 0.73 ) 0.71 (0.60 -0.81) CHI3L1 Periostine 30 15 0.70 (0.53 - 0.86) 0.81 (0.68 - 0.94) CHI3L1 MSR1 higher 9 8 0.83 (0.63 - 1.00) 0.90 (0.78 - 1.00) CHI3L1 SERPINB2 6 6 0.97 (0.90 - 1.00) 0.92 (0.75 - 1.00) IL10 + PI3 9 6 0.67 (0.35 - 0.99) 0.67 (0.40 - 0.94) IL10 + Periostin 9 6 0.63 (0.32 - 0.94) 0.74 (0.49 - 0.99) IL8 PI3 9 7 0.76 (0.50 - 1.00) 0.90 (0.79 - 1.00) IL8 Periostine 9 7 0.87 (0.70 - 1.00) 0.90 (0.79 - 1.00) PI3 + Periostin 30 15 0.69 (0.52 - 0.86) 0.73 (0.61 - 0.86) PI3 Upper MSR1 9 8 0.61 (0.32 - 0.90) 0.81 (0.60 - 1.00) PI3 SERPINB2 6 6 0.89 (0.66 - 1.00) 0.97 (0.91 - 1.00) Periostina MSR1 higher 9 8 0.81 (0.58 - 1.00) 0.80 (0.60 - 1.00) Periostin SERPINB2 6 6 0.97 (0.90 - 1.00) 0.99 (0.95 - 1.00) Table 10. ROC curves for models with combinations of three variables
[0217] Variable variables Variable N0 N1 qualitative quantitative
[0218] CHI3L1 IL10 Periostine 9 6 0.63 (0.31 - 0.95) 0.80 (0.57 - 1.00)
[0219] CHI3L1 IL8 Periostine 9 7 0.87 (0.70 - 1.00) 0.91 (0.79 - 1.00) CHI3L1 PI3 Periostin 30 15 0.68 (0.51 - 0.85) 0.82 (0.70 - 0.94) CHI3L1 PI3 Upper MSR1 9 8 0.83 (0.63 - 1.00) 0.92 (0.81 - 0.81) 1.00)
[0220] CHI3L1 Periostin MSR1 upper 9 8 0.88 (0.71 - 1.00) 0.98 (0.93 - 1.00) CHI3L1 Periostin SERPINB2 6 6 0.99 (0.95 -1.00) IL10 PI3 Periostin 9 6 0.61 (0.29 - 0.93) 0.77 (0.52 - 1.00) IL8 PI3 Periostin 9 7 0.84 (0.64 - 1.00) 0.97 (0.90 - 1.00) PI3 Periostin MSR1 higher 9 8 0.81 (0.58 - 1.00) 0.88 (0.74 - 1.00) PI3 Periostin SERPINB2 6 6 0.97 (0.90 - 1.00) 0.99 (0.95 -1.00)
[0221]
[0222] Following the information presented in tables 8 to 10, we propose as third aspect of the invention, an in vitro method to select subjects at risk of suffering from non-allergic asthma with mild or moderate persistence that comprises: (a) measuring the pattern or level of concentration of at least one of the following proteins: SERPINB2, lower MSR1, PHLDA1, higher MSR1, CHI3L1, IL10, IL8, PI3 and / or Periostina, obtained from a biological sample isolated from the subjects to be selected; and (b) comparing said pattern or level of concentration of at least one of said protein biomarkers, of the subjects to be selected with a standard or level of concentration already established or with the concentration level of a healthy subject, where differences in concentration of at least one of said protein biomarkers is indicative that said subject presents a risk of suffering from non-allergic asthma with mild or moderate persistence. It is noted that said concentration differences with respect to the healthy individual or with respect to the reference value, will correspond to an overexpression or an under-expression of the concentration depending on the type of biomarker. In this sense, Table 8 clearly indicates when the overexpression or under-expression of each of the biomarkers is indicative that said subject presents a risk of suffering from non-allergic asthma with mild or moderate persistence. On the other hand, tables 9 and 10 indicate specific combinations of biomarkers useful in determining the risk of suffering from non-asthma. allergic with mild or moderate persistence. Each of said combinations forms a part and can be implemented in the method proposed in the third aspect of the invention.
[0223]
[0224] A preferred embodiment of the third aspect of the invention relates to an in vitro method for diagnosis / prognosis of a subject suspected of having non-allergic asthma with mild or moderate persistence, comprising steps a) and b) of the first aspect of the invention, and optionally (c) confirming the presence of non-allergic asthma with mild or moderate persistence by means of a clinical examination.
[0225]
[0226] Another preferred embodiment of the third aspect of the invention relates to an in vitro method for monitoring the response to a therapy or to monitor the progression of non-allergic asthma with mild or moderate persistence, in a subject suffering from said disease, comprising the steps a) and b) of the third aspect of the invention.
[0227]
[0228] Another preferred embodiment of the third aspect of the invention relates to a method for treating subjects suffering from non-allergic asthma with mild or moderate persistence, comprising steps a) and b) of the third aspect of the invention, and (c) treating the patient who has been diagnosed with non-allergic asthma with mild or moderate persistence. It is noted that possible therapies are expressly mentioned throughout the description, in particular in the first aspect of the invention.
[0229]
[0230] Another preferred embodiment of the third aspect of the invention relates to the method according to any of the preceding embodiments, wherein the biological sample is selected from the group of biological samples detailed in the first aspect of the invention.
[0231]
[0232] Additionally and, again, following the information set forth in tables 8 to 10, we propose as a fourth aspect of the invention, the use of a kit comprising reagents that detect biomarkers to determine a differential expression level of at least one of the biomarkers mentioned in the third aspect of the invention, where differences of expression of at least one of the mentioned biomarkers is indicative of risk of having non-allergic asthma with mild or moderate persistence, to diagnose in vitro the risk of having non-allergic asthma with mild or moderate persistence.
[0233] A preferred embodiment of the fourth aspect of the invention relates to the use of the kit, wherein said kit can be implemented by means of any technique described in the second aspect of the invention.
[0234]
[0235] ^ Comparisons between controls and severe non-allergic asthmatics (ANA)
[0236]
[0237] - N0 = Observations in Control group
[0238] - N1 = Observations in Grupo ANA Grave
[0239] - AUC: Area Under the ROC Curve. For the models with combinations of two or more variables, the AUC value has been split into two values: Quantitative AUC value denominated in the tables as "Quantitative variables" and Qualitative AUC value denominated in the tables as "Qualitative variables".
[0240]
[0241] Table 11. Individual ROC Curves
[0242] Variable N0 N1 AUC (IC95%) Threshold
[0243] CHI3L1 30 15 0.63 (0.45 -0.81) 14367
[0244] IL10 9 5 0.56 (0.23 - 0.88) 32.5
[0245] IL8 9 7 0.62 (0.28 - 0.96) 262
[0246] PI3 30 14 0.52 (0.33 -0.71) 6528
[0247] Periostin 30 15 0.55 (0.37 - 0.74) 13633
[0248] MSR1 upper 9 10 0.60 (0.31 - 0.89) 0.254
[0249] Lower MSR1 9 10 0.93 (0.80 - 1.00) 0.137
[0250] PHLDA1 8 7 0.48 (0.15 -0.81) 0.014
[0251] SERPINB2 6 5 0.93 (0.78 - 1.00) 0.359
[0252] Table 12. ROC curves for models with combinations of two variables
[0253] Variables Variables Combination qualitative quantitative N0 N1 CHI3L1 + IL10 9 5 0.62 (0.30 - 0.94) 0.77 (0.54 - 0.99) CHI3L1 + IL8 9 7 0.67 (0.37 - 0.96) 0.78 (0.55 - 1.00) CHI3L1 + PI3 30 14 0.63 (0.45 - 0.81) 0.68 (0.53 - 0.84) CHI3L1 + Periostine 30 15 0.64 (0.46 - 0.82) 0.72 (0.57 - 0.88) CHI3L1 Upper MSR1 9 10 0.76 (0.50 - 1.00) 0.83 (0.66 - 1.00) CHI3L1 Lower MSR1 9 10 0.94 (0.83) - 1.00) 0.99 (0.96 - 1.00) CHI3L1 SERPINB2 6 5 0.93 (0.78 - 1.00) 0.93 (0.80 - 1.00) IL10 + PI3 9 5 0.51 (0.18 -0.84) 0.70 (0.44 - 0.96) IL10 Periostine 9 5 0.60 (0.23 - 0.97) 0.93 (0.80 - 1.00) IL8 PI3 9 7 0.46 (0.15 -0.77) 0.77 (0.54 - 1.00) IL8 + Periostin 9 7 0.57 (0.27 - 0.88) 0.76 (0.51 - 1.00) PI3 + Periostin 30 14 0.65 (0.47 - 0.84) 0.71 (0.56 - 0.86) PI3 + higher MSR1 9 9 0.64 (0.35 - 0.93) 0.78 (0.56 - 1.00) PI3 Lower MSR1 9 9 0.95 (0.85 - 1.00) 0.99 (0.96 - 1.00) PI3 SERPINB2 6 5 0.93 (0.78) - 1.00) 0.97 (0.89 - 1.00) Periostina MSR1 superior 9 10 0.66 (0.38 - 0.93) 0.75 (0.59 -0.91) Periostina MSR1 lower 9 10 0.93 (0.80 - 1.00) 0.95 (0.85 - 1.00) Periostin SERPINB2 6 5 0.97 (0.87 - 1.00) 0.98 (0.94 - 1.00) Table 13. ROC curves for models with combinations of three variables
[0254] Variable variables Variable N0 N1 qualitative quantitative
[0255] CHI3L1 IL10 Periostine 9 5 0.64 (0.33 - 0.96) 0.97 (0.89 - 1.00)
[0256] CHI3L1 IL8 Periostine 9 7 0.67 (0.37 - 0.96) 0.84 (0.63 - 1.00) CHI3L1 PI3 Periostin 30 14 0.62 (0.43 - 0.81) 0.80 (0.65 - 0.94) CHI3L1 PI3 MSR1 higher 9 9 0.64 (0.34 - 0.94) 0.94 (0.84 - 1.00) CHI3L1 PI3 MSR1 lower 9 9 0.95 (0.86 - 1.00)
[0257]
[0258] CHI3L1 Periostin MSR1 upper 9 10 0.76 (0.50 - 1.00) 0.84 (0.68 - 1.00) CHI3L1 Periostin MSR1 lower 9 10 0.94 (0.84 - 1.00) 0.99 (0.96 -1.00) IL10 PI3 Periostin 9 5 0.60 (0.22 - 0.98) 0.97 (0.89) - 1.00) IL8 PI3 Periostine 9 7 0.62 (0.32 - 0.92) 0.76 (0.51 - 1.00) PI3 Periostin MSR1 higher 9 9 0.77 (0.54 - 0.99) 0.87 (0.70 - 1.00) PI3 Periostin MSR1 lower 9 9 0.96 (0.88 - 1.00) 0.99 (0.96 -1.00 )
[0259]
[0260] Following the information presented in Tables 11 to 13, we propose, as the fifth aspect of the invention, an in vitro method for selecting subjects at risk of suffering from non-allergic asthma with severe persistence, comprising: (a) measuring the pattern or level of concentration of at least one of the following proteins: SERPINB2, lower MSR1, PHLDA1, higher MSR1, CHI3L1, IL10, IL8, PI3 and / or Periostina, obtained from a biological sample isolated from the subjects to be selected; and (b) comparing said pattern or level of concentration of at least one of said protein biomarkers, of the subjects to be selected with a standard or level of concentration already established or with the concentration level of a healthy subject, where differences in concentration of at least one of said protein biomarkers is indicative that said subject presents a risk of suffering from non-allergic asthma with severe persistence. It is noted that said concentration differences with respect to the healthy individual or with respect to the reference value, will correspond to an overexpression or an under-expression of the concentration depending on the type of biomarker. In this sense, Table 11 clearly indicates when the overexpression or under-expression of each of the biomarkers is indicative that said subject presents a risk of suffering from non-allergic asthma with severe persistence. On the other hand, Tables 12 and 13 indicate specific combinations of biomarkers useful in determining risk of suffering non-allergic asthma with severe persistence. Each of said combinations forms part and can be implemented in the method proposed in the fifth aspect of the invention.
[0261]
[0262] A preferred embodiment of the fifth aspect of the invention relates to an in vitro method for diagnosis / prognosis of a subject suspected of suffering from non-allergic asthma with severe persistence, comprising steps a) and b) of the fifth aspect of the invention, and optionally (c) confirming the presence of non-allergic asthma with severe persistence by means of a clinical examination.
[0263]
[0264] Another preferred embodiment of the fifth aspect of the invention relates to an in vitro method for monitoring the response to a therapy or to monitor the progression of non-allergic asthma with severe persistence, in a subject suffering from said disease, comprising the steps a ) and b) of the fifth aspect of the invention.
[0265]
[0266] Another preferred embodiment of the fifth aspect of the invention relates to a method for treating subjects suffering from non-allergic asthma with severe persistence, comprising steps a) and b) of the fifth aspect of the invention, and (c) treating the patient to whom He has been diagnosed with non-allergic asthma with severe persistence. It is noted that possible therapies are expressly mentioned throughout the description, in particular in the first aspect of the invention.
[0267]
[0268] Another preferred embodiment of the fifth aspect of the invention relates to the method according to any of the preceding embodiments, wherein the biological sample is selected from the group of biological samples provided in the first aspect of the invention.
[0269]
[0270] Additionally and, again, following the information set forth in tables 11 to 13, we propose as the sixth aspect of the invention, the use of a kit comprising reagents that detect biomarkers to determine a differential expression level of at least one of the biomarkers mentioned in the fifth aspect of the invention, where differences in expression of at least one of the biomarkers mentioned is indicative of risk of having non-allergic asthma with severe persistence, to diagnose in vitro the risk of having non-allergic asthma with severe persistence .
[0271] A preferred embodiment of the sixth aspect of the invention relates to the use of the kit, wherein said kit can be implemented by means of any technique of those described in the second aspect of the invention.
[0272]
[0273] 4. Comparisons between mild / moderate and severe nonallergic asthmatics (ANA)
[0274]
[0275] - N0 = Observations in light / moderate ANA group
[0276] - N1 = Observations in Grupo ANA Grave
[0277] - AUC: Area Under the ROC Curve. For the models with combinations of two or more variables, the AUC value has been split into two values: Quantitative AUC value denominated in the tables as "Quantitative variables" and Qualitative AUC value denominated in the tables as "Qualitative variables".
[0278]
[0279] Table 14. Individual ROC curves
[0280] Variable N0 N1 AUC (IC95%) Threshold
[0281] CHI3L1 15 15 0.62 (0.40 - 0.83) 18500
[0282] IL10 6 5 0.47 (0.07 - 0.86) 105.2
[0283] IL8 7 7 0.76 (0.49 - 1.00) 841
[0284] PI3 15 14 0.55 (0.33 - 0.78) 4845
[0285] Periostin 15 15 0.65 (0.44 - 0.86) 17419
[0286] MSR1 upper 8 10 0.46 (0.17 -0.75) 0.257
[0287] Lower MSR1 8 10 0.55 (0.27 - 0.83) 0.056
[0288] PHLDA1 8 7 0.48 (0.15 -0.81) 0.014
[0289] SERPINB2 6 5 0.70 (0.32 - 1.00) 0.132
[0290] Table 15. ROC curves for models with combinations of two variables
[0291] Variables Variables Combination N0 N1 qualitative quantitative CHI3L1 + IL10 6 5 0.43 (0.05 -0.81) 0.65 (0.29 - 1.00) CHI3L1 IL8 7 7 0.76 (0.49 - 1.00) 0.85 (0.65 - 1.00) CHI3L1 + PI3 15 14 0.56 (0.33 - 0.79 ) 0.68 (0.48 - 0.87) CHI3L1 Periostine 15 15 0.77 (0.59 - 0.96) 0.75 (0.57 - 0.94) CHI3L1 + higher MSR1 8 10 0.68 (0.41 - 0.94) 0.73 (0.51 - 0.94) CHI3L1 + lower MSR1 8 10 0.65 (0.37) - 0.93) 0.73 (0.51 - 0.94) CHI3L1 + SERPINB2 6 5 0.63 (0.25 - 1.00) 0.90 (0.75 - 1.00) IL10 + PI3 6 5 0.63 (0.25 - 1.00) 0.83 (0.60 - 1.00) IL10 Periostina 6 5 0.77 (0.43) - 1.00) 0.90 (0.73 - 1.00) IL8 PI3 7 7 0.82 (0.58 - 1.00) 0.96 (0.87 - 1.00) IL8 Periostine 7 7 0.78 (0.52 - 1.00) 0.96 (0.87 - 1.00) PI3 + Periostin 15 14 0.57 (0.34 - 0.80) 0.73 (0.55 - 0.92) PI3 + higher MSR1 8 9 0.60 (0.30 - 0.90) 0.67 (0.42 - 0.93) PI3 + lower MSR1 8 9 0.60 (0.30 - 0.90) 0.74 (0.49 - 1.00) PI3 SERPINB2 6 5 0.73 ( 0.39 - 1.00) 0.93 (0.82 - 1.00) Periostina + MSR1 superior 8 10 0.64 (0.36 - 0.92) 0.72 (0.50 - 0.95) Periost ina lower MSR1 8 10 0.62 (0.35 - 0.90) 0.81 (0.60 - 1.00) Periostin SERPINB2 6 5 0.90 (0.71 - 1.00) 0.97 (0.89 - 1.00) higher MSR1 + lower MSR1 8 10 0.55 (0.26 - 0.84) 0.66 (0.41 - 0.41) 0.92) Table 16. ROC curves for models with combinations of three variables
[0292] Variable variables Variable N0 N1 qualitative quantitative
[0293] CHI3L1 IL10 Periostine 6 5 0.77 (0.46 - 1.00) 0.92 (0.76 - 1.00)
[0294] CHI3L1 IL8 Periostine 7 7 0.98 (0.92 -1.00) 0.99 (0.96 -1.00) CHI3L1 PI3 Periostine 15 14 0.76 (0.57 - 0.95) 0.76 (0.58 - 0.94) CHI3L1 PI3 MSR1 higher 8 9 0.78 (0.53 - 1.00) 0.82 (0.63 - 1.00) CHI3L1 PI3 MSR1 lower 8 9 0.68 (0.40 - 0.96) 0.82 (0.62 - 1.00)
[0295] CHI3L1 Periostin MSR1 upper 8 10 0.80 (0.57 - 1.00) 0.88 (0.72 - 1.00) CHI3L1 Periostin MSR1 lower 8 10 0.80 (0.57 - 1.00) 0.89 (0.73 - 1.00) CHI3L1 Upper MSR1 Lower MSR1 8 10 0.68 (0.41 - 0.94) 0.76 (0.54 - 0.98) IL10 PI3 Periostin 6 5 0.77 (0.43 - 1.00) 0.97 (0.89 - 1.00) IL8 PI3 Periostin 7 7 0.82 (0.58 - 1.00)
[0296] PI3 Periostin MSR1 upper 8 9 0.60 (0.30 - 0.89) 0.72 (0.48 - 0.97) PI3 Periostin MSR1 lower 8 9 0.60 (0.30 - 0.89) 0.82 (0.61 - 1.00) PI3 Periostin SERPINB2 6 5 0.93 (0.78 - 1.00)
[0297] PI3 MSR1 upper MSR1 lower 8 9 0.60 (0.30 - 0.89) 0.78 (0.52 - 1.00) Periostin MSR1 upper MSR1
[0298] lower 8 10 0.62 (0.35 - 0.90) 0.81 (0.61 - 1.00)
[0299]
[0300] Based on the information set forth in Tables 14 to 16, we propose as the seventh aspect of the invention, an in vitro method for selecting subjects at risk of suffering from non-allergic asthma with mild / moderate or severe persistence, comprising: (a) measuring the pattern or level of concentration of at least one of the following proteins: SERPINB2, lower MSR1, PHLDA1, higher MSR1, CHI3L1, IL10, IL8, PI3 and / or Periostina, obtained from a biological sample isolated from the subjects to be selected; and (b) comparing said pattern or level of concentration of at least one of said protein biomarkers, of the subjects to be selected with a standard or level of concentration already established, where differences in concentration of at least one of said protein biomarkers is indicative of that said subject presents a risk of suffering from non-allergic asthma with different levels of severity depending on the type of biomarker and its level of differential expression (in terms of concentration levels). It is noted that such differences in concentration with respect to the reference value, will correspond to an overexpression or an under-expression of the concentration depending on the type of biomarker. In this sense, Table 14 clearly indicates when the overexpression or under-expression of each of the biomarkers is indicative that said subject presents a risk of suffering from non-allergic asthma with different levels of persistence. On the other hand, Tables 15 and 16 indicate specific combinations of biomarkers useful in determining the risk of suffering non-allergic asthma with different levels of persistence. Each of said combinations forms part and can be implemented in the method proposed in the seventh aspect of the invention.
[0301]
[0302] A preferred embodiment of the seventh aspect of the invention relates to an in vitro method for the diagnosis / prognosis of a subject suspected of suffering from non-allergic asthma with different levels of persistence, comprising steps a) and b) of the seventh aspect of the invention, and optionally (c) confirming the presence of non-allergic asthma with different persistence levels by means of a clinical examination.
[0303]
[0304] Another preferred embodiment of the seventh aspect of the invention relates to an in vitro method for monitoring the response to a therapy or to monitor the progression of non-allergic asthma, in a subject suffering from said disease, comprising steps a) and b) of the seventh aspect of the invention.
[0305]
[0306] Another preferred embodiment of the seventh aspect of the invention relates to a method for treating subjects suffering from non-allergic asthma, comprising steps a) and b) of the fifth aspect of the invention, and (c) treating the patient who has been diagnosed non-allergic asthma, taking into account the type of severity or persistence that presents. It is noted that possible therapies are expressly mentioned throughout the description, in particular in the first aspect of the invention.
[0307]
[0308] Another preferred embodiment of the seventh aspect of the invention relates to the method according to any of the preceding embodiments, wherein the biological sample is selected from the group of samples provided in the first aspect of the invention.
[0309]
[0310] Additionally and, again, following the information set forth in Tables 14 to 16, we propose as the eighth aspect of the invention, the use of a kit comprising reagents that detect biomarkers to determine a level of differential expression of at least one of the biomarkers mentioned in the seventh aspect of the invention, where differences in expression of at least one of the aforementioned biomarkers is indicative of the risk of having non-allergic asthma with different levels of persistence, to diagnose in vitro the risk of having non-allergic asthma with a certain persistence, be it mild / moderate or severe.
[0311]
[0312] A preferred embodiment of the eighth aspect of the invention relates to the use of the kit, wherein said kit can be implemented by means of any technique described in the second aspect of the invention.
[0313]
[0314] BIOMARKERS FOR ALLERGIC ASTHMA (AA)
[0315]
[0316] 1. Comparisons between Controls and Allergic Asthmatics (AA) (Total population)
[0317] - N0 = Observations in Control group
[0318] - N1 = Observations in Allergic Asthmatic Group
[0319] - AUC: Area Under the ROC Curve. For the models with combinations of two or more variables, the AUC value has been split into two values: Quantitative AUC value denominated in the tables as "Quantitative variables" and Qualitative AUC value denominated in the tables as "Qualitative variables".
[0320]
[0321] Table 17. Individual ROC curves
[0322] Variable N0 N1 AUC (IC95%) Threshold
[0323] CHI3L1 30 30 0.78 (0.66 -0.91) 20065
[0324] IL10 9 21 0.37 (0.14 -0.60) 101.4
[0325] IL8 9 14 0.76 (0.56 - 0.97) 64
[0326] PI3 30 30 0.44 (0.29 - 0.59) 8196
[0327] Periostin 30 30 0.51 (0.36 - 0.66) 13544
[0328] PHLDA1 8 6 0.50 (0.16 -0.84) 0.005
[0329] SERPINB2 6 11 0.97 (0.90 - 1.00) 0.375
[0330] Table 18. ROC curves for models with combinations of two variables Quantitative variables Qualitative Combination N0 N1 qualitative
[0331] CHI3L1 Periostine 30 30 0.79 (0.66 - 0.91) 0.80 (0.69 - 0.91)
[0332] IL10 Periostine 9 21 0.85 (0.69 - 1.00) 0.85 (0.73 - 0.98)
[0333] IL8 Periostine 9 14 0.74 (0.52 - 0.95) 0.77 (0.58 - 0.95) PI3 + Periostin 30 30 0.56 (0.41 - 0.71) 0.60 (0.46 - 0.73)
[0334] Periostine PHLDA1 8 6 0.79 (0.51 - 1.00) 0.90 (0.76 - 1.00) Periostin SERPINB2 6 11 0.95 (0.85 - 1.00) 0.98 (0.93 -1.00)
[0335]
[0336] Table 19. ROC curves for models with combinations of three variables
[0337] Variable variables Variable N0 N1 qualitative quantitative
[0338] CHI3L1 IL10 Periostin 9 21 0.83 (0.64 - 1.00) 0.90 (0.80 - 1.00)
[0339] CHI3L1 IL8 Periostine 9 14 0.91 (0.79 - 1.00) 0.92 (0.82 - 1.00) CHI3L1 PI3 Periostin 30 30 0.78 (0.66 - 0.91) 0.81 (0.70 - 0.92)
[0340] CHI3L1 Periostine PHLDA1 8 6 0.71 (0.40 - 1.00) 0.93 (0.80 - 1.00) CHI3L1 Periostin SERPINB2 6 11 0.99 (0.97 -1.00) IL10 PI3 Periostin 9 21 0.86 (0.72 - 1.00) 0.86 (0.74 - 0.99) IL8 PI3 Periostine 9 14 0.67 (0.40 - 0.93) 0.87 (0.73 - 1.00) PI3 Periostina PHLDA1 8 6 0.79 (0.51 - 1.00) 0.95 (0.84 - 1.00) PI3 Periostin SERPINB2 6 11 0.97 (0.90 - 1.00) 0.99 (0.97 -1.00) Following the information set forth in Tables 17 to 19, we propose as the ninth aspect of the invention, an in vitro method for selecting subjects at risk of suffering from allergic asthma comprising: (a) measuring the pattern or level of concentration of at least one of the following proteins: SERPINB2, lower MSR1, PHLDA1, higher MSR1, CHI3L1, IL10, IL8, PI3 and / or Periostina, obtained from a biological sample isolated from the subjects to be selected; and (b) comparing said pattern or level of concentration of at least one of said protein biomarkers, of the subjects to be selected with a standard or level of concentration already established or with the concentration level of a healthy subject, where differences in concentration of at least one of said protein biomarkers is indicative that said subject presents a risk of suffering from allergic asthma. It is noted that said concentration differences with respect to the healthy individual or with respect to the reference value, will correspond to an overexpression or an under-expression of the concentration depending on the type of biomarker. In this sense, Table 17 clearly indicates when the overexpression or under-expression of each of the biomarkers is indicative that said subject presents a risk of suffering from allergic asthma. On the other hand, tables 18 and 19 indicate specific combinations of biomarkers useful in determining the risk of suffering from allergic asthma. Each of said combinations forms a part and can be implemented in the method proposed in the ninth aspect of the invention.
[0341]
[0342] A preferred embodiment of the ninth aspect of the invention relates to an in vitro method for diagnosis / prognosis of a subject suspected of having allergic asthma, comprising steps a) and b) of the ninth aspect of the invention, and optionally (c) confirm the presence of allergic asthma through a clinical examination.
[0343]
[0344] Another preferred embodiment of the ninth aspect of the invention relates to an in vitro method for monitoring the response to a therapy or to monitor the progression of allergic asthma, in a subject suffering from said disease, comprising steps a) and b) of the ninth aspect of the invention.
[0345]
[0346] Another preferred embodiment of the ninth aspect of the invention relates to a method for treating subjects suffering from allergic asthma, comprising steps a) and b) of the ninth aspect of the invention, and (c) treating the patient who has been diagnosed Allergic asthma. It is noted that possible therapies are expressly mentioned throughout the description, in particular in tables 3 and 4, in the first aspect of the invention and directing the therapy towards the underlying allergy that is the cause of the asthma (for example with allergen-specific immunotherapy or vaccines with the allergens causing the allergy or of allergen sensitization etc ...).
[0347]
[0348] Another preferred embodiment of the ninth aspect of the invention relates to the method according to any of the preceding embodiments, wherein the biological sample is selected from the group of samples provided in the first aspect of the invention.
[0349]
[0350] Additionally and, again, following the information set forth in Tables 17 to 19, we propose as the tenth aspect of the invention, the use of a kit comprising reagents that detect biomarkers to determine a differential expression level of at least one of the biomarkers mentioned in the ninth aspect of the invention, where the overexpression or differences of expression of at least one of the mentioned biomarkers is indicative of the risk of having allergic asthma, to diagnose in vitro the risk of having allergic asthma.
[0351]
[0352] A preferred embodiment of the tenth aspect of the invention relates to the use of the kit, wherein said kit can be implemented by means of any technique described in the second aspect of the invention.
[0353]
[0354] 2. Comparisons between Controls and Allergic Asthmatics (AA) mild or moderate
[0355]
[0356] - N0 = Observations in Control group
[0357] - N1 = Observations in Group AA slight / moderate
[0358] - AUC: Area Under the ROC Curve. For the models with combinations of two or more variables, the AUC value has been split into two values: Quantitative AUC value denominated in the tables as "Quantitative variables" and Qualitative AUC value denominated in the tables as "Qualitative variables".
[0359] Table 20. Individual ROC Curves
[0360] Variable N0 N1 AUC (IC95%) Threshold
[0361] CHI3L1 30 15 0.78 (0.64 -0.91) 14426
[0362] IL10 9 13 0.36 (0.11 - 0.61) 101.4
[0363] IL8 9 7 0.59 (0.24 - 0.93) 121
[0364] PI3 30 15 0.46 (0.28 - 0.64) 6519
[0365] Periostin 30 15 0.52 (0.34 - 0.69) 7067
[0366] PHLDA1 8 3
[0367] SERPINB2 6 6 1.00 (1.00 - 1.00) 0.097
[0368]
[0369] Table 21. ROC curves for models with combinations of two variables Quantitative variables Qualitative Combination N0 N1 qualitative
[0370] CHI3L1 Periostine 30 15 0.78 (0.64 -0.91) 0.78 (0.69 - 0.87)
[0371] IL10 Periostine 9 13 0.83 (0.66 - 1.00) 0.82 (0.65 - 0.98)
[0372] IL8 Periostine 9 7 0.54 (0.17 -0.91) 0.75 (0.52 - 0.99) PI3 + Periostin 30 15 0.53 (0.35 -0.71) 0.64 (0.50 - 0.79)
[0373]
[0374] Table 22. ROC curves for models with combinations of three variables Qualitative Variables Variable N0 N1 qualitative variables
[0375] CHI3L1 IL10 Periostine 9 13 0.82 (0.64 - 1.00) 0.88 (0.75 - 1.00)
[0376] CHI3L1 IL8 Periostine 9 7 0.86 (0.66 - 1.00) 0.95 (0.86 - 1.00) CHI3L1 PI3 Periostin 30 15 0.78 (0.65 - 0.92) 0.83 (0.72 - 0.94) IL10 PI3 Periostin 9 13 0.85 (0.68 - 1.00) 0.83 (0.67 - 1.00 ) IL8 PI3 Periostine 9 7 0.65 (0.36 - 0.94) 0.82 (0.59 - 1.00) Following the information set forth in tables 20 to 22, we propose as an eleventh aspect of the invention, an in vitro method for selecting subjects at risk of suffering from allergic asthma with mild or moderate persistence, comprising: (a) measuring the pattern or level of concentration of at least one of the following proteins: SERPINB2, lower MSR1, PHLDA1, higher MSR1, CHI3L1, IL10, IL8, PI3 and / or Periostina, obtained from a biological sample isolated from the subjects to be selected; and (b) comparing said pattern or level of concentration of at least one of said protein biomarkers, of the subjects to be selected with a standard or level of concentration already established or with the concentration level of a healthy subject, where differences in concentration of at least one of said protein biomarkers is indicative that said subject presents a risk of suffering from allergic asthma with mild or moderate persistence. It is noted that said concentration differences with respect to the healthy individual or with respect to the reference value, will correspond to an overexpression or an under-expression of the concentration depending on the type of biomarker. In this sense, Table 20 clearly indicates when the overexpression or under-expression of each of the biomarkers is indicative that said subject presents a risk of suffering from allergic asthma with mild or moderate persistence. On the other hand, Tables 21 and 22 indicate specific combinations of biomarkers useful in determining the risk of suffering from allergic asthma with mild or moderate persistence. Each of said combinations forms part and can be implemented in the method proposed in the eleventh aspect of the invention.
[0377]
[0378] A preferred embodiment of the eleventh aspect of the invention relates to an in vitro method for the diagnosis / prognosis of a subject suspected of having allergic asthma with mild or moderate persistence, comprising steps a) and b) of the eleventh aspect of the invention, and optionally (c) confirming the presence of allergic asthma with mild or moderate persistence by means of a clinical examination.
[0379]
[0380] Another preferred embodiment of the eleventh aspect of the invention relates to an in vitro method for monitoring the response to a therapy or to monitor the progression of allergic asthma with mild or moderate persistence in a subject suffering from said disease, comprising the steps a) and b) of the eleventh aspect of the invention.
[0381]
[0382] Another preferred embodiment of the eleventh aspect of the invention relates to a method for treating subjects suffering from allergic asthma with mild or moderate persistence, comprising steps a) and b) of the eleventh aspect of the invention, and (c) treating the patient who has been diagnosed with allergic asthma with mild or moderate persistence. It is noted that possible therapies are expressly mentioned throughout the description, in particular in the ninth aspect of the invention.
[0383]
[0384] Another preferred embodiment of the eleventh aspect of the invention relates to the method according to any of the preceding embodiments, wherein the biological sample is selected from the group of biological samples provided in the first aspect of the invention.
[0385]
[0386] Additionally and, again, following the information set forth in tables 20 to 22, we propose as the twelfth aspect of the invention, the use of a kit comprising reagents that detect biomarkers to determine a differential expression level of at least one of the biomarkers mentioned in the eleventh aspect of the invention, where differences in expression of at least one of the aforementioned biomarkers is indicative of risk of having allergic asthma with mild or moderate persistence, to diagnose in vitro the risk of having allergic asthma with mild persistence or moderate.
[0387]
[0388] A preferred embodiment of the twelfth aspect of the invention relates to the use of the kit, wherein said kit can be implemented by means of any technique described in the second aspect of the invention.
[0389]
[0390] 3. Comparisons between Controls and Serious Allergic Asthmatics (AA)
[0391]
[0392] - N0 = Observations in Control group
[0393] - N1 = Observations in Group AA serious
[0394] - AUC: Area Under the ROC Curve. For the models with combinations of two or more variables, the AUC value has been split into two values: Quantitative AUC value denominated in the tables as "Quantitative variables" and Qualitative AUC value denominated in the tables as "Qualitative variables".
[0395] Table 23. ROC curves
[0396] Variable N0 N1 AUC (IC95%) Threshold
[0397] CHI3L1 30 15 0.72 (0.57 - 0.87) 20202
[0398] IL10 11 13 0.45 (0.20 - 0.69) 457.4
[0399] IL8 14 7 0.52 (0.22 - 0.82) 122
[0400] PI3 29 15 0.46 (0.28 - 0.64) 3269
[0401] Periostin 30 15 0.62 (0.45 - 0.79) 22785
[0402] MSR1 top
[0403] MSR1 lower
[0404] PHLDA1 8 3
[0405] SERPINB2 11 6 0.77 (0.54 - 1.00) 0.086
[0406]
[0407] Table 24. ROC curves for models with combinations of two variables
[0408] Qualitative Quantitative N0 N1 Combination Variables
[0409] CHI3L1 Periostine 30 15 0.80 (0.67 - 0.92) 0.86 (0.75 - 0.96)
[0410] IL10 Periostine 9 8 0.86 (0.68 - 1.00) 0.98 (0.93 - 1.00)
[0411] IL8 Periostine 9 7 0.95 (0.85 - 1.00) 0.94 (0.85 - 1.00) PI3 + Periostin 30 15 0.58 (0.40 - 0.76) 0.68 (0.54 -0.81)
[0412] Periostin SERPINB2 6 5 0.97 (0.87 - 1.00) 0.98 (0.94 - 1.00) Table 25. ROC curves for models with combinations of three variables
[0413] Variable variables Variable N0 N1 qualitative quantitative
[0414] CHI3L1 IL10 Periostine 9 8 0.86 (0.68 - 1.00) 0.99 (0.95 - 1.00) CHI3L1 PI3 Periostin 30 15 0.81 (0.68 - 0.94) 0.88 (0.79 - 0.98) IL10 PI3 Periostine 9 8 0.89 (0.73 - 1.00) 0.99 (0.95 - 1.00 ) IL8 PI3 Periostin 9 7 0.94 (0.82 - 1.00) 0.99 (0.97 - 1.00)
[0415]
[0416] Following the information set forth in Tables 23 to 25, we propose as the 13th aspect of the invention, an in vitro method for selecting subjects at risk of suffering from allergic asthma with severe persistence comprising: (a) measuring the pattern or level of concentration of at least one of the following proteins: SERPINB2, lower MSR1, PHLDA1, higher MSR1, CHI3L1, IL10, IL8, PI3 and / or Periostina, obtained from a biological sample isolated from the subjects to be selected; and (b) comparing said pattern or level of concentration of at least one of said protein biomarkers, of the subjects to be selected with a standard or level of concentration already established or with the concentration level of a healthy subject, where differences in concentration of at least one of said protein biomarkers is indicative that said subject presents a risk of suffering from allergic asthma with severe persistence. It is noted that said concentration differences with respect to the healthy individual or with respect to the reference value, will correspond to an overexpression or an under-expression of the concentration depending on the type of biomarker. In this sense, Table 23 clearly indicates when the overexpression or under-expression of each of the biomarkers is indicative that said subject presents a risk of suffering from allergic asthma with severe persistence. On the other hand, Tables 24 and 25 indicate specific combinations of biomarkers useful in determining the risk of suffering from allergic asthma with severe persistence. Each of said combinations forms part and can be implemented in the method proposed in the 13th aspect of the invention.
[0417]
[0418] A preferred embodiment of the 13th aspect of the invention relates to an in vitro method for diagnosis / prognosis of a subject suspected of suffering from allergic asthma with severe persistence, comprising steps a) and b) of the 13th aspect of the invention, and optionally (c) confirming the presence of allergic asthma with severe persistence by means of a clinical examination.
[0419] Another preferred embodiment of the 13th aspect of the invention relates to an in vitro method for monitoring the response to a therapy or for monitoring the progression of allergic asthma with severe persistence, in a subject suffering from said disease, comprising the steps a ) and b) of the 13th aspect of the invention.
[0420]
[0421] Another preferred embodiment of the 13th aspect of the invention relates to a method for treating subjects suffering from allergic asthma with severe persistence, comprising steps a) and b) of the 13th aspect of the invention, and (c) treating the patient at who has been diagnosed with nonallergic asthma with severe persistence. It is noted that possible therapies are expressly mentioned throughout the description, in particular in the ninth aspect of the invention.
[0422]
[0423] Another preferred embodiment of the 13th aspect of the invention relates to the method according to any of the preceding embodiments, wherein the biological sample is selected from the group of biological samples provided in the first aspect of the invention.
[0424]
[0425] Additionally and, again, following the information set forth in Tables 23 to 25, we propose as the 14th aspect of the invention, the use of a kit comprising reagents that detect biomarkers to determine a differential expression level of at least one of the biomarkers mentioned in the 13th aspect of the invention, where differences in expression of at least one of the biomarkers mentioned is indicative of the risk of having allergic asthma with severe persistence, to diagnose in vitro the risk of having allergic asthma with severe persistence .
[0426]
[0427] A preferred embodiment of the 14th aspect of the invention relates to the use of the kit, wherein said kit can be implemented by means of any technique of those described in the second aspect of the invention.
[0428]
[0429] 4. Comparisons between mild or moderate and severe allergic asthmatics (AA)
[0430]
[0431] - N0 = Observations in group AA Light / mod
[0432] - N1 = Observations in Group AA Serious
[0433] - AUC: Area Under the ROC Curve. For models with combinations of two or more variables, the AUC value has been split into two values: Quantitative AUC value denominated in the tables as "Quantitative variables" and Qualitative AUC value denominated in the tables as "Qualitative variables".
[0434]
[0435] Table 26. Individual ROC Curves
[0436] Variable N0 N1 AUC (95% CI) Threshold
[0437] CHI3L1 15 15 0.50 (0.27 - 0.72) 24144
[0438] IL10 13 8 0.46 (0.20 - 0.72) 57.1
[0439] IL8 7 7 0.82 (0.58 - 1.00) 17
[0440] PI3 15 15 0.56 (0.35 - 0.78) 5524
[0441] Periostin 15 15 0.52 (0.30 - 0.74) 19119
[0442] MSR1 top
[0443] MSR1 lower
[0444] PHLDA1 3 3
[0445] SERPINB2 6 5 0.77 (0.44 - 1.00) 0.095
[0446]
[0447] Table 27. ROC curves for models with combinations of two variables Quantitative variables Qualitative Combination N0 N1
[0448] CHI3L1 + Periostin 15 15 0.50 (0.28 - 0.72) 0.72 (0.55 - 0.89)
[0449] IL10 + Periostin 13 8 0.62 (0.37 - 0.88) 0.73 (0.55 - 0.90)
[0450] IL8 + Periostin 7 7 0.84 (0.62 - 1.00) 0.85 (0.69 - 1.00) PI3 + Periostin 15 15 0.54 (0.32 - 0.75) 0.66 (0.48 - 0.84)
[0451] Periostin + SERPINB2 6 5 0.93 (0.78 - 1.00) 0.90 (0.75 - 1.00) Table 28. ROC curves for models with combinations of three variables
[0452] Qualitative Quantitative N0 N1 Combination Variables
[0453] CHI3L1 IL10 Periostine 13 8 0.63 (0.38 - 0.88) 0.83 (0.67 - 1.00)
[0454] CHI3L1 IL8 Periostine 7 7 0.84 (0.62 - 1.00) 0.92 (0.78 - 1.00) CHI3L1 PI3 Periostin 15 15 0.56 (0.35 - 0.78) 0.76 (0.58 - 0.93)
[0455] CHI3L1 Periostine
[0456]
[0457] IL10 PI3 Periostine 13 8 0.74 (0.51 - 0.97) 0.82 (0.65 - 1.00) IL8 PI3 Periostine 7 7 0.82 (0.55 - 1.00) 0.97 (0.90 - 1.00) PI3 Periostin SERPINB2 6 5 0.93 (0.78 - 1.00) 0.95 (0.84 - 1.00 )
[0458]
[0459] Following the information presented in Tables 26 to 28, we propose as 15th aspect of the invention, an in vitro method to select subjects at risk of suffering from allergic asthma with mild / moderate or severe persistence that comprises: (a) measuring the pattern or level of concentration of at least one of the following proteins: SERPINB2, lower MSR1, PHLDA1, higher MSR1, CHI3L1, IL10, IL8, PI3 and / or Periostina, obtained from a biological sample isolated from the subjects to be selected; and (b) comparing said pattern or level of concentration of at least one of said protein biomarkers, of the subjects to be selected with a standard or level of concentration already established, where differences in concentration of at least one of said protein biomarkers is indicative of that said subject presents a risk of suffering from allergic asthma with different levels of severity depending on the type of biomarker and its level of differential expression (in terms of concentration levels). It is noted that said differences in concentration with respect to the reference value, will correspond to an overexpression or an under-expression of the concentration depending on the type of biomarker. In this sense, Table 26 clearly indicates when the overexpression or under-expression of each of the biomarkers is indicative that said subject presents a risk of suffering from allergic asthma with different levels of persistence. On the other hand, Tables 27 and 28 indicate specific combinations of biomarkers useful in determining the risk of suffering from allergic asthma with different levels of persistence. Each of these combinations is part of and can be implemented in the method proposed in the 15th aspect of the invention.
[0460]
[0461] A preferred embodiment of the 15th aspect of the invention relates to an in vitro method for diagnosis / prognosis of a subject suspected of suffering from allergic asthma with different levels of persistence, comprising steps a) and b) of ° aspect of the invention, and optionally (c) confirming the presence of allergic asthma with different levels of persistence by means of a clinical examination.
[0462]
[0463] Another preferred embodiment of the 15th aspect of the invention relates to an in vitro method for monitoring the response to a therapy or to monitor the progression of allergic asthma, in a subject suffering from said disease, comprising steps a) and b) of the 15th aspect of the invention.
[0464]
[0465] Another preferred embodiment of the 15th aspect of the invention relates to a method for treating subjects suffering from allergic asthma, comprising steps a) and b) of the 15th aspect of the invention, and (c) treating the patient who is has diagnosed allergic asthma, taking into account the type of severity or persistence that presents. It is noted that possible therapies are expressly mentioned throughout the description, in particular in the ninth aspect of the invention.
[0466]
[0467] Another preferred embodiment of the 15th aspect of the invention relates to the method according to any of the preceding embodiments, wherein the biological sample is selected from the group of biological samples provided in the first aspect of the invention.
[0468]
[0469] Additionally and, again, following the information set forth in Tables 26 to 28, we propose as the 16th aspect of the invention, the use of a kit comprising reagents that detect biomarkers to determine a differential expression level of at least one. of the biomarkers mentioned in the 15th aspect of the invention, where differences in expression of at least one of the biomarkers mentioned is indicative of the risk of having allergic asthma with different levels of persistence, in order to diagnose in vitro the risk of having allergic asthma with a certain persistence, whether it is mild / moderate or severe.
[0470] A preferred embodiment of the 16th aspect of the invention relates to the use of the kit, wherein said kit can be implemented by means of any technique described in the second aspect of the invention.
[0471]
[0472] BIOMARKERS FOR ALLERGY ( A) WITHOUT ASTHMA
[0473]
[0474] 1. Comparisons between Controls and Allergy (Total Population) (A)
[0475]
[0476] - N0 = Observations in Control group
[0477] - N1 = Observations in Allergic Group
[0478] - AUC: Area Under the ROC Curve. For the models with combinations of two or more variables, the AUC value has been split into two values: Quantitative AUC value denominated in the tables as "Quantitative variables" and Qualitative AUC value denominated in the tables as "Qualitative variables".
[0479]
[0480] Table 29. Individual ROC Curves
[0481] Variable N0 N1 AUC (IC95%) Threshold
[0482] CHI3L1 30 14 0.63 (0.45 - 0.82) 9953
[0483] IL10 9 4
[0484] IL8 9 7 0.78 (0.48 - 1.00) 627
[0485] PI3 30 14 0.68 (0.49 - 0.87) 7232
[0486] Periostin 30 14 0.74 (0.60 - 0.89) 14505
[0487] PHLDA1 8 7 0.48 (0.15 -0.81) 0.014
[0488] SERPINB2 6 5 0.70 (0.32 - 1.00) 0.554
[0489] Table 30. ROC curves for models with combinations of two variables
[0490] Qualitative Quantitative N0 N1 Combination Variables
[0491] CHI3L1 Periostine 30 14 0.75 (0.61 - 0.90) 0.85 (0.76 - 0.94)
[0492] IL8 Periostine 9 7 0.90 (0.75 - 1.00) 0.95 (0.88 - 1.00) PI3 Periostin 30 14 0.80 (0.65 - 0.94) 0.90 (0.83 - 0.98)
[0493] Periostine + PHLDA1 8 7 0.39 (0.08 - 0.70) 0.70 (0.42 - 0.97) Periostin SERPINB2 6 5 0.80 (0.50 - 1.00) 0.95 (0.84 - 1.00)
[0494]
[0495] Table 31. ROC curves for models with combinations of three variables
[0496] Variables Variables N0 N1 quantitative qualitative CHI3L1 PI3 Periostine 30 14 0.80 (0.66 - 0.95) 0.93 (0.87 - 1.00)
[0497] CHI3L1 Periostine PHLDA1 8 7 0.66 (0.35 - 0.97) 0.85 (0.57 - 1.00) CHI3L1 Periostine
[0498] SERPINB2 6 5 0.77 (0.46 - 1.00)
[0499] PI3 Periostine PHLDA1 8 7 0.82 (0.57 - 1.00) 0.93 (0.81 - 1.00) PI3 Periostin SERPINB2 6 5 0.90 (0.69 - 1.00)
[0500]
[0501] Following the information set forth in Tables 29 to 31, we propose as the 17th aspect of the invention, an in vitro method for selecting allergic subjects without asthma comprising: (a) measuring the pattern or level of concentration of at least one of the following proteins: SERPINB2, lower MSR1, PHLDA1, higher MSR1, CHI3L1, IL10, IL8, PI3 and / or Periostina, obtained from a biological sample isolated from the allergic subjects to be selected; Y (b) comparing said standard or level of concentration of at least one of said protein biomarkers, of the subjects
[0502]
[0503] A preferred embodiment of the 17th aspect of the invention relates to an in vitro method for the diagnosis of an allergic subject suspected of not having asthma, comprising steps a) and b) of the 17th aspect of the invention, and optionally (c) confirm the absence of asthma through a clinical examination.
[0504]
[0505] Another preferred embodiment of the 17th aspect of the invention relates to a method for treating allergic subjects who do not suffer from asthma, comprising steps a) and b) of the 17th aspect of the invention, and (c) treating the patient who is not He has been diagnosed with asthma.
[0506]
[0507] Another preferred embodiment of the 17th aspect of the invention relates to the method according to any of the preceding embodiments, wherein the biological sample is selected from the group detailed in the first aspect of the invention.
[0508]
[0509] Additionally and, again, following the information set forth in Tables 29 to 31, we propose as 18th aspect of the invention, the use of a kit comprising reagents that detect biomarkers to determine a differential expression level of at least one of the biomarkers mentioned in the first aspect of the invention, where the overexpression or differences of expression of at least one of the mentioned biomarkers is indicative that the allergic subject does not suffer from asthma.
[0510] A preferred embodiment of the 18th aspect of the invention relates to the use of the kit, wherein said kit can be implemented by means of any technique of those described in the second aspect of the invention.
[0511]
[0512] In addition to comparing with the control group comparisons were made between the clinical groups, and based on the analysis of individual and grouped ROC curves of the 8 protein biomarkers studied (see data at the end example 3), the best biomarker options to discriminate each Clinical condition analyzed are detailed below.
[0513]
[0514] BIOMARKERS FOR DISCRIMINATION BETWEEN ASTHMA CLINICAL PHENOTYPES
[0515]
[0516] 1. Biomarkers capable of discriminating non-allergic asthmatics (ANA)
[0517] of Allergic Asthmatics (AA). Total population
[0518]
[0519] AUC CHI3L1 0.74
[0520] IL10 + Periostin 0.87
[0521] CHI3L1 + SERPINB2 0.83
[0522] CHI3L1 + IL8 0.82
[0523] IL8 + SERPINB2 0.82
[0524] PHLDA1 + SERPINB2 0.80
[0525] IL8 + Periostin 0.76
[0526] CHI3L1 + IL10 + Periostin 0.92
[0527] CHI3L1 + IL8 + Periostin 0.88
[0528] PI3 + PHLDA1 + SERPINB2 0.84
[0529] CHI3L1 + PI3 + Periostin 0.80
[0530]
[0531] Based on the information presented above, we propose as the 19th aspect of the invention, an in vitro method to discriminate between subjects at risk of suffering ANA (non-allergic asthmatics) from Allergic Asthmatics (AA), which comprises: (a) measuring the pattern or level of expression of at least one of the biomarkers listed above, obtained from a biological sample isolated from the subjects to be selected; and (b) comparing said pattern or level of expression of at least one of said biomarkers, of the subjects to be selected with a pattern or level of expression already established, where differences of expression of at least one of said biomarkers is indicative that said subject presents a risk of suffering one of said clinical phenotypes. It is noted that said differences of expression with respect to the reference value, will correspond to an overexpression or an under expression of the expression depending on the type of biomarker. In this sense, the information provided throughout the present invention clearly indicates when the overexpression or under-expression of each of the biomarkers is indicative that said subject presents a risk of suffering from one of said clinical phenotypes.
[0532]
[0533] A preferred embodiment of the 19th aspect of the invention refers to an in vitro method for the differential diagnosis between subjects at risk of suffering one of the clinical phenotypes mentioned in the previous paragraph, comprising steps a) and b) of the 19th aspect of the invention, and optionally (c) confirming the absence of allergy by means of a clinical examination.
[0534]
[0535] Another preferred embodiment of the 19th aspect of the invention relates to a method for treating subjects with a certain clinical phenotype of asthma, comprising steps a) and b) of the 19th aspect of the invention, and (c) treating the patient according to the type of asthma diagnosed. Possible treatments are reflected in the first and ninth aspects of the invention according to the clinical phenotype of the disease.
[0536]
[0537] Another preferred embodiment of the 19th aspect of the invention relates to the method according to any of the preceding embodiments, wherein the biological sample is selected from the group detailed in the first aspect of the invention.
[0538]
[0539] Additionally and, again, following the information set forth above, we propose as the 20th aspect of the invention, the use of a kit comprising reagents that detect biomarkers to determine a level of differential expression of at least one of the biomarkers mentioned in the 19th aspect of the invention, where differences of expression of at least one of the aforementioned biomarkers is able to discriminate between subjects at risk of suffering from ANA (non-allergic asthmatics) of Allergic Asthmatics (AA).
[0540]
[0541] A preferred embodiment of the 20 th aspect of the invention relates to the use of the kit, wherein said kit can be implemented by means of any technique described in the second aspect of the invention.
[0542] 2. Biomarkers capable of discriminating non-allergic asthmatics (ANA)
[0543] severe Allergic Asthmatics (AA).
[0544]
[0545] AUC CHI3L1 0.82
[0546] SERPINB2 0.78
[0547] IL10 + Periostin 0.94
[0548] PI3 + SERPINB2 0.92
[0549] CHI3L1 + IL8 0.86
[0550] CHI3L1 + Periostin 0.86
[0551] IL8 + Periostin 0.86
[0552] CHI3L1 + IL8 + Periostin 0.96
[0553]
[0554] Based on the information presented above, we propose as an 21st aspect of the invention, an in vitro method to discriminate between subjects at risk of suffering from serious ANA (nonallergic asthmatic) allergic asthmatic (AA), comprising: (a) measure the pattern or level of expression of at least one of the biomarkers collected above, obtained from a biological sample isolated from the subjects to be selected; and (b) comparing said pattern or level of expression of at least one of said biomarkers, of the subjects to be selected with an established pattern or level of expression, where expression differences of at least one of said biomarkers is indicative that said subject presents a risk of suffering one of these clinical phenotypes. It is noted that said differences of expression with respect to the reference value, will correspond to an overexpression or an under expression of the expression depending on the type of biomarker. In this sense, the information provided throughout the present invention clearly indicates when the overexpression or under-expression of each of the biomarkers is indicative that said subject presents a risk of suffering from one of said clinical phenotypes.
[0555]
[0556] A preferred embodiment of the 21st aspect of the invention refers to an in vitro method for the differential diagnosis between subjects at risk of suffering one of the clinical phenotypes mentioned in the paragraph above, comprising steps a) and b) of 21 ° aspect of the invention, and optionally (c) confirming the absence of allergy by means of a clinical examination.
[0557] Another preferred embodiment of the 21st aspect of the invention relates to a method for treating subjects with a certain clinical phenotype of asthma, comprising steps a) and b) of the 21st aspect of the invention, and (c) treating the patient according to the type of asthma diagnosed. Possible treatments are reflected in the first and ninth aspects of the invention according to the clinical phenotype of the disease.
[0558]
[0559] Another preferred embodiment of the 21st aspect of the invention relates to the method according to any of the preceding embodiments, wherein the biological sample is selected from the group detailed in the first aspect of the invention.
[0560]
[0561] Additionally, and again, as a result of the information presented above, we propose as the 22nd aspect of the invention, the use of a kit comprising reagents that detect biomarkers to determine a level of differential expression of at least one of the biomarkers mentioned in the 21st aspect of the invention, wherein differences in expression of at least one of the aforementioned biomarkers is able to discriminate between subjects at risk of severe ANA (non-allergic asthmatics) of severe Allergic Asthmatics (AA),
[0562]
[0563] A preferred embodiment of the 20 th aspect of the invention relates to the use of the kit, wherein said kit can be implemented by means of any technique described in the second aspect of the invention.
[0564]
[0565] 3. Biomarkers capable of discriminating Non-allergic asthmatics (ANA) Mod / mild Allergic asthmatics (AA) Mod / mild.
[0566]
[0567] AUC CHI3L1 + IL8 0.86
[0568] IL10 + Periostin 0.82
[0569] IL8 + PI3 0.82
[0570] IL8 + Periostin 0.82
[0571] CHI3L1 + IL10 + Periostin 0.90
[0572] CHI3L1 + PI3 + Periostin 0.80
[0573]
[0574] As a result of the information presented above, we propose as an 23rd aspect of the invention, an in vitro method to discriminate between subjects at risk of suffering from ANA (Asthmatics not allergic) Mod / mild Allergic Asthmatics (AA) Mod / mild, comprising: (a) measuring the pattern or level of expression of at least one of the biomarkers listed above, obtained from a biological sample isolated from subjects select; and (b) comparing said pattern or level of expression of at least one of said biomarkers, of the subjects to be selected with an established pattern or level of expression, where expression differences of at least one of said biomarkers is indicative that said subject presents a risk of suffering one of these clinical phenotypes. It is noted that said differences of expression with respect to the reference value, will correspond to an overexpression or an under expression of the expression depending on the type of biomarker. In this sense, the information provided throughout the present invention clearly indicates when the overexpression or under-expression of each of the biomarkers is indicative that said subject presents a risk of suffering from one of said clinical phenotypes.
[0575]
[0576] A preferred embodiment of the 23rd aspect of the invention refers to an in vitro method for the differential diagnosis between subjects at risk of suffering one of the clinical phenotypes mentioned in the previous paragraph, comprising steps a) and b) of the 23rd aspect of the invention, and optionally (c) confirming the absence of allergy by means of a clinical examination.
[0577]
[0578] Another preferred embodiment of the 23rd aspect of the invention relates to a method for treating subjects with a determined clinical phenotype of asthma, comprising steps a) and b) of the 23rd aspect of the invention, and (c) treating the patient according to the type of asthma diagnosed. Possible treatments are reflected in the first and ninth aspects of the invention according to the clinical phenotype of the disease.
[0579]
[0580] Another preferred embodiment of the 23rd aspect of the invention relates to the method according to any of the preceding embodiments, wherein the biological sample is selected from the group detailed in the first aspect of the invention.
[0581]
[0582] Additionally, and again, as a result of the information set forth above, we propose as the 24th aspect of the invention, the use of a kit comprising reagents that detect biomarkers to determine a level of differential expression of at least one of the biomarkers mentioned in the 23rd aspect of the invention, where differences of Expression of at least one of the aforementioned biomarkers is able to discriminate between subjects at risk of suffering ANA (Non-allergic asthmatics) Mod / mild of Allergic Asthmatics (AA) Mod / mild.
[0583]
[0584] 4. Biomarkers capable of discriminating Allergic Asthmatics (AA) from
[0585] Allergic (A). Total population.
[0586]
[0587] AUC IL8 0.88
[0588] Periostin 0.8
[0589] SERPINB2 0.93
[0590] CHI3L1 + SERPINB2 1
[0591] PI3 + SERPINB2 0.96
[0592] CHI3L1 + IL8 0.91
[0593] CHI3L1 + Periostin 0.81
[0594] IL8 + PI3 + Periostin 0.93
[0595]
[0596] Following the information presented above, we propose as an 25th aspect of the invention, an in vitro method to discriminate between subjects at risk of suffering from AA (Allergic Asthmatics) of Allergic (A), which comprises: (a) measuring the pattern or level of expression of at least one of the biomarkers listed above, obtained from a biological sample isolated from the subjects to be selected; and (b) comparing said pattern or level of expression of at least one of said biomarkers, of the subjects to be selected with an established pattern or level of expression, where expression differences of at least one of said biomarkers is indicative that said subject presents a risk of suffering one of these clinical phenotypes. It is noted that said differences of expression with respect to the reference value, will correspond to an overexpression or an under expression of the expression depending on the type of biomarker. In this sense, the information provided throughout the present invention clearly indicates when the overexpression or under-expression of each of the biomarkers is indicative that said subject presents a risk of suffering from one of said clinical phenotypes.
[0597]
[0598] A preferred embodiment of the 25th aspect of the invention refers to an in vitro method for differential diagnosis among subjects at risk of suffering one of the clinical phenotypes mentioned in the preceding paragraph, comprising steps a) and b) of the 25th aspect of the invention, and optionally (c) confirming the absence of asthma by means of a clinical examination.
[0599]
[0600] Another preferred embodiment of the 25th aspect of the invention relates to a method for treating subjects with a certain clinical phenotype of allergy, with and without asthma, comprising steps a) and b) of the 25th aspect of the invention, and (c) ) treat the patient who has been diagnosed with asthma. Possible treatments are reflected in the first and ninth aspects of the invention according to the clinical phenotype of the disease.
[0601]
[0602] Another preferred embodiment of the 25th aspect of the invention relates to the method according to any of the preceding embodiments, wherein the biological sample is selected from the group detailed in the first aspect of the invention.
[0603]
[0604] Additionally and, again, following the information presented above, we propose as the 26th aspect of the invention, the use of a kit comprising reagents that detect biomarkers to determine a differential expression level of at least one of the biomarkers mentioned in the 25th aspect of the invention, where differences in expression of at least one of the aforementioned biomarkers is able to discriminate between subjects at risk of suffering from AA (Allergic Asthmatics) of Allergic (A).
[0605]
[0606] 5. Biomarkers capable of discriminating Allergic Asthmatics (AA)
[0607] Serious of Allergic (A).
[0608] AUC IL8 0.80
[0609] Periostin 0.77
[0610]
[0611] IL8 + Periostin 0.90
[0612] IL8 + PI3 + Periostin 0.98
[0613]
[0614] Following the information presented above, we propose as an 27th aspect of the invention, an in vitro method to discriminate between subjects at risk of suffering from severe AA (Allergic Allergy) allergic (A), which comprises: (a) measuring the pattern or level of expression of at least one of the biomarkers collected above, obtained from a biological sample isolated from the subjects to be selected; and (b) comparing said pattern or level of expression of at least one of said biomarkers, of the subjects to be selected with an established pattern or level of expression, where expression differences of at least one of said biomarkers is indicative that said subject presents a risk of suffering one of these clinical phenotypes. It is noted that said differences of expression with respect to the reference value, will correspond to an overexpression or an under expression of the expression depending on the type of biomarker. In this sense, the information provided throughout the present invention clearly indicates when the overexpression or under-expression of each of the biomarkers is indicative that said subject presents a risk of suffering from one of said clinical phenotypes.
[0615]
[0616] A preferred embodiment of the 27th aspect of the invention relates to an in vitro method for the differential diagnosis between subjects at risk of suffering one of the clinical phenotypes mentioned in the previous paragraph, comprising steps a) and b) of the 27th aspect of the invention, and optionally (c) confirming the absence of asthma by means of a clinical examination.
[0617]
[0618] Another preferred embodiment of the 27th aspect of the invention relates to a method for treating subjects with a particular allergic clinical phenotype, comprising steps a) and b) of the 27th aspect of the invention, and (c) treating the patient at who has been diagnosed with asthma. Possible treatments are reflected in the first and ninth aspects of the invention according to the clinical phenotype of the disease.
[0619]
[0620] Another preferred embodiment of the 27th aspect of the invention relates to the method according to any of the preceding embodiments, wherein the biological sample is selected from the group detailed in the first aspect of the invention.
[0621]
[0622] Additionally and, again, following the information set forth above, we propose as the 28th aspect of the invention, the use of a kit comprising reagents that detect biomarkers to determine a level of differential expression of at least one of the biomarkers mentioned in the 27th aspect of the invention, where differences in expression of at least one of the aforementioned biomarkers is able to discriminate between subjects at risk of suffering from severe AA (Allergic Asthmatics) allergic (A).
[0623] 6. Biomarkers capable of discriminating Allergic Asthmatics (AA)
[0624] Mod / mild of Allergic (A).
[0625] AUC IL8 0.96
[0626] Periostin 0.83
[0627] SERPINB2 0.84
[0628] PI3 + SERPINB2 0.92
[0629]
[0630] Based on the information presented above, we propose as an 29th aspect of the invention, an in vitro method to discriminate between subjects at risk of suffering from AA (Allergic Asthmatics) Moderate / mild Allergic (A), which comprises: (a) measuring the pattern or level of expression of at least one of the biomarkers listed above, obtained from a biological sample isolated from the subjects to be selected; and (b) comparing said pattern or level of expression of at least one of said biomarkers, of the subjects to be selected with an established pattern or level of expression, where expression differences of at least one of said biomarkers is indicative that said subject presents a risk of suffering one of these clinical phenotypes. It is noted that said differences of expression with respect to the reference value, will correspond to an overexpression or an under expression of the expression depending on the type of biomarker. In this sense, the information provided throughout the present invention clearly indicates when the overexpression or under-expression of each of the biomarkers is indicative that said subject presents a risk of suffering from one of said clinical phenotypes.
[0631]
[0632] A preferred embodiment of the 29th aspect of the invention refers to an in vitro method for the differential diagnosis between subjects at risk of suffering one of the clinical phenotypes mentioned in the previous paragraph, comprising steps a) and b) of the 29th aspect of the invention, and optionally (c) confirming the absence of asthma by means of a clinical examination.
[0633]
[0634] Another preferred embodiment of the 29th aspect of the invention relates to a method for treating subjects with a particular allergic clinical phenotype, comprising steps a) and b) of the 29th aspect of the invention, and (c) treating the patient at who has been diagnosed with asthma. Possible treatments are reflected in the first and ninth aspects of the invention according to the clinical phenotype of the disease.
[0635] Another preferred embodiment of the 29th aspect of the invention relates to the method according to any of the preceding embodiments, wherein the biological sample is selected from the group detailed in the first aspect of the invention.
[0636]
[0637] Additionally and, again, following the information set forth above, we propose as a 30th aspect of the invention, the use of a kit comprising reagents that detect biomarkers to determine a level of differential expression of at least one of the biomarkers mentioned in the 29th aspect of the invention, where differences in expression of at least one of the mentioned biomarkers is able to discriminate between subjects at risk of suffering from AA (Allergic Asthmatics) Moderate / mild Allergic (A).
[0638] Table 32. Gene Biomarkers. Ranking of the 33 gene biomarkers in Allergic Asthmatics (AA) compared to healthy controls taking into account the data of the ROC curves
[0639]
[0640]
[0641]
[0642]
[0643] Following the information set forth in Table 32, we propose as 30th aspect of the invention, an in vitro method to select subjects at risk of suffering from allergic asthma as well as to determine the severity of said asthma, such as severe asthma or moderate asthma. or mild, which comprises: (a) measuring the pattern or level of expression of at least one of the genes listed in Table 32, obtained from a biological sample isolated from the subjects to be selected; and (b) comparing said pattern or level of expression of at least one of said gene biomarkers, of the subjects to be selected with an established pattern or level of expression, where expression differences of at least one of said biomarkers is indicative that said subject presents a risk of suffering from allergic asthma, as well as an indication of the severity of allergic asthma. It is noted that said differences of expression with respect to the reference value, will correspond to an overexpression or an under expression of the expression depending on the type of biomarker. In this sense, Table 32 clearly indicates when the overexpression or under-expression of each of the biomarkers is indicative that said subject presents a risk of suffering from allergic asthma.
[0644]
[0645] A preferred embodiment of the 30th aspect of the invention relates to an in vitro method for the diagnosis / prognosis of a subject suspected of suffering from allergic asthma, as well as for the diagnosis / prognosis of the severity of said asthma, which comprises steps a) and b) of the 30th aspect of the invention, and optionally (c) confirming the absence of asthma by means of a clinical examination.
[0646]
[0647] Another preferred embodiment of the 30th aspect of the invention relates to a method for treating allergic subjects suffering from asthma, comprising steps a) and b) of the 30th aspect of the invention, and (c) treating the patient who is He has diagnosed asthma. Possible treatments are reflected in the ninth aspect of the invention.
[0648]
[0649] Another preferred embodiment of the 30th aspect of the invention relates to the method according to any of the preceding embodiments, wherein the biological sample is selected from the group detailed in the first aspect of the invention.
[0650]
[0651] Additionally and, again, following the information set forth in Table 32, we propose as a 31st aspect of the invention, the use of a kit comprising reagents that detect biomarkers to determine a differential expression level of at least one of the biomarkers mentioned in the 30th aspect of the invention, where overexpression or expression differences of at least one of the aforementioned biomarkers is indicative of risk of a subject suffering from allergic asthma, to diagnose in vitro the presence of risk of having allergic asthma.
[0652] Table 33. Ranking of the 9 gene biomarkers in non-allergic asthma with the ROC curve data comparing with controls.
[0653]
[0654] Following the information set forth in Table 33, we propose as an 32nd aspect of the invention, an in vitro method to select subjects at risk of suffering from non-allergic asthma as well as to determine the severity of said asthma, such as severe asthma or moderate asthma. or mild, which comprises: (a) measuring the pattern or level of expression of at least one of the genes listed in Table 33, obtained from a biological sample isolated from the subjects to be selected; and (b) comparing said pattern or level of expression of at least one of said gene biomarkers, of the subjects to be selected with an established pattern or level of expression, where expression differences of at least one of said biomarkers is indicative that said subject presents a risk of suffering non-allergic asthma as well as an indication of the severity of non-allergic asthma. It is noted that said differences of expression with respect to the reference value, will correspond to an overexpression or an under expression of the expression depending on the type of biomarker. In this sense, Table 33 clearly indicates when the overexpression or under-expression of each of the biomarkers is indicative that said subject presents a risk of suffering non-allergic asthma.
[0655]
[0656] A preferred embodiment of the 32nd aspect of the invention relates to an in vitro method for the diagnosis / prognosis of a subject suspected of having non-allergic asthma, as well as for the diagnosis / prognosis of the severity of said asthma, comprising steps a) and b) of the 32nd aspect of the invention, and optionally (c) confirming the absence of asthma by means of a clinical examination.
[0657]
[0658] Another preferred embodiment of the 32nd aspect of the invention relates to a method for treating subjects suffering from non-allergic asthma, comprising steps a) and b) of the 32nd aspect of the invention, and (c) treating the patient to whom it is intended. He has been diagnosed with asthma. Possible treatments are reflected in the first aspect of the invention.
[0659]
[0660] Another preferred embodiment of the 32nd aspect of the invention relates to the method according to any of the preceding embodiments, wherein the biological sample is selected from the group detailed in the first aspect of the invention.
[0661]
[0662] Additionally and, again, following the information set forth in Table 33, we propose as 33 ° aspect of the invention, the use of a kit comprising reagents that detect biomarkers to determine a level of differential expression of at least one of the biomarkers mentioned in the 32nd aspect of the invention, where the overexpression or differences of expression of at least one of the aforementioned biomarkers is indicative of risk that a subject suffers non-allergic asthma, to diagnose in vitro the presence of risk of having asthma not allergic
[0663]
[0664] It is noted that the biomarkers cited throughout the 33 aspects of the invention would allow to improve the classification or diagnosis of the different phenotypes studied, which will have a positive effect on limiting and improving the specific treatment guidelines to be carried out. Additionally, in the examples of the present invention and, in particular, in the tables of ROC curve results, the individual values of each protein marker, capable of discerning one condition of another, are detailed according to the comparison analyzed. The threshold value is the quantitative parameter capable of differentiating one condition from another. Overall, in this invention we propose different combinations of biomarkers that would allow to be able to discriminate with very good sensitivity and specificity the three clinical phenotypes, as well as the severity in the case of asthmatic phenotypes. All the combinations indicated, are combinations that could allow to make good, excellent or excellent diagnostic tests. The choice in each case, we propose to perform it based on the best value of quantitative AUC (which allows to objectify the data), analyzed in the largest number of cases and preferably, performed by the simplest technique, the ELISA (although the combination is not ruled out) of ELISA and Western), which would be performed with the patient's serum as a routine clinical test. In the case that genetic biomarkers are analyzed, appropriate techniques for such use would be used, such as measuring gene expression through qRT-PCR techniques.
[0665]
[0666] Additionally, one of the mentioned biomarkers such as the PHLDA1 protein biomarker; or the protein biomarker SERPINB2 in peripheral samples, are the first time they are described for use as potential biomarkers of asthma. For this reason, a 34th aspect of the invention relates to the use of the PHLDA1 protein biomarker, or the SERPINB2 protein biomarker in peripheral samples for the detection or diagnosis of asthma in a subject, preferably in a human subject. Also, a 35th aspect of the invention relates to the use of a kit or device capable of detecting any of the protein biomarkers PHLDA1, or SERPINB2 for the detection or diagnosis of asthma in a subject, preferably in a human subject.
[0667] It is noted that each of the aspects described throughout the present description are to be interpreted by virtue of the results provided in the examples and in the figures in order to be able to determine in a specific way those biomarkers or combinations of biomarkers, as well as its levels with respect to certain patterns, capable of discriminating between subjects at risk of suffering from asthma, in particular distinguishing the severity of asthma, more particularly distinguishing between different asthma phenotypes.
[0668]
[0669] The present invention is described below by means of a series of examples, whose sole pretension consists in illustrating the present invention and in no case should be understood that they limit it.
[0670]
[0671] Examples
[0672]
[0673] Materials and methods
[0674]
[0675] STUDY POPULATION
[0676]
[0677] The study population consisted of 104 subjects from four clinically defined groups:
[0678]
[0679] 1. Subject controls, defined as non-allergic, non-asthmatic subjects and without respiratory diseases (n = 30).
[0680] 2. Allergic asthmatic subjects (n = 30).
[0681] 3. Allergic, non-asthmatic subjects (n = 14).
[0682] 4. Asthmatic, non-allergic subjects (n = 30).
[0683]
[0684] The asthmatic subjects (allergic and non-allergic) came from the CIBERES Asthma Samples Bank (CIBER for Respiratory Diseases), composed of more than 300 subjects, of which there is an associated clinical data base and it is estimated that approximately 40% have allergic asthma. The bank has PBMC (peripheral blood mononuclear cells), serum and DNA.
[0685]
[0686] Control subjects and non-asthmatic allergic subjects have been selected expressly for this Project, following the protocols established in the Project original. Specifically, all the subjects were selected and clinically diagnosed by a Specialized Allergist (specifically from Seville and Granada), through clinical history and skin tests against a battery of allergens among which olive pollen was included. All the study participants (patients and controls) were informed and proposed to participate in the study, after which they signed an Informed Consent, previously approved by the Ethics Committee for Research (CEI) of the IIS-Jiménez Díaz Foundation. Aliquots of whole blood were extracted, with anticoagulant and without it, which were sent to the Department of Immunology of the IIS-Fundación Jiménez Díaz, to be processed.
[0687]
[0688] Selection criteria:
[0689]
[0690] ■ Non-allergic asthmatic patients with a severe, moderate or mild diagnosis according to the criteria of the Spanish Guide to Asthma Management (GEMA), and without any allergic symptoms.
[0691] ■ Allergic asthmatic patients with a severe, moderate or mild diagnosis according to GEMA criteria.
[0692] ■ Non-asthmatic allergic patients with allergies to aerial allergens.
[0693] ■ Not having received treatment before or during the collection of samples
[0694] ■ Maximum availability of serum samples and PBMC.
[0695]
[0696] Exclusion criteria: Be in treatment at the time of collection of samples.
[0697]
[0698] Table 34 summarizes the characteristics of the population studied. As can be observed, the average age between the groups is different, with allergic asthmatic subjects being significantly older than the rest of the groups. The non-asthmatic allergic subjects were the youngest, this difference being statistically significant with respect to the groups of control subjects and patients with non-allergic asthma. The distribution of smokers was similar among the different groups. In addition, the table reflects the severity of asthmatic patients, who in half of the cases have a diagnosis of severe asthma, while the other of moderate-mild asthma. This table also includes the total serum IgE value measured by the Immunocap method . This assessment is important, since a high total IgE value is characteristic of patients suffering from allergy. In fact, in our population we observed that the mean of the total IgE levels is significantly increased in the two groups with allergy compared with the rest of the groups. further Functional lung parameters are included, the FVC percentages ( percentage of forced vital capacity) and FVE1 ( percentage of forced expiratory volume in 1 second) whose values lower than 80% suppose an indicative of asthma disease. Thus, we see how the values of these parameters are similar between the two groups of asthma studied, although if they are analyzed considering the severity of the disease there are significant differences within the group with non-allergic asthma between the serious patients and those of moderate diagnosis and mild (66.33 ± 16.62 vs 85.38 ± 21.03, p = 0.0127 of% FEV1, 69.93 ± 19.94 vs 94 ± 19.52, p = 0.0031 of% FVC, respectively).
[0699] Table 34: Study population
[0700]
[0701]
[0702] % FVC: percentage of forced vital capacity ; % FEV1: percentage of forced expiratory volume in 1 seconó. * Statistically significant comparisons (p <0.05) between the control group and the patient group. # Statistically significant comparisons (p <0.05) between the non-allergic asthma group and the selected group.
[0703] OBTAINING SAMPLES
[0704]
[0705] The group of subjects with asthma were selected from the asthma biobank, which has samples of PBMC (stored in liquid nitrogen), serum and DNA (stored at -80 ° C). From the PBMC, RNA and total protein were extracted, according to the protocol detailed below.
[0706]
[0707] Samples from the 30 healthy subjects and 30 non-asthmatic allergic subjects were obtained from whole blood, which were processed according to the following protocol.
[0708]
[0709] 40ml of peripheral blood was extracted from each study subject: 10ml without anticoagulant (to obtain serum) and 30ml with heparin (to obtain DNA, plasma and PBMC).
[0710] The samples were processed according to the final product required and always working in laminar flow chambers:
[0711]
[0712] ■ Obtaining serum (by centrifugation under standard conditions): Five aliquots were stored at -80 ° C of each study subject, for further determinations.
[0713] ■ Extraction of PBMC and plasma : To do this, a fractionation technique from whole blood was performed: the differential centrifugation in Ficol gradient ( Linfoprep ), using the protocol of the commercial house ( Comercial Rafer). Five aliquots of plasma were collected for each subject (preservation at -80 ° C) and approximately ten of PBMC (6x106 cells / vial) that were frozen in freezing medium (RPMI-1640 medium 10% inactive fetal calf serum (STFi) 10% DMSO) at -80 ° C and subsequently stored in liquid nitrogen.
[0714] ■ RNA extraction: For the study of differential expression, RNA was extracted from PBMC samples from all study subjects (n = 104), using the Trizol ( TriReagent RNA isolation) method , from 1x106 PBMC, following the specifications of the commercial house ( Vitro). The quantification and purity of the RNA was carried out by measuring its optical density in a Nanodrop. Samples are stored at -80 ° C until use.
[0715] ■ Protein extraction : Using the Trizol method, the protein fraction was also obtained from 1x106 PBMC. The protein was quantified by the BCA method of Thermo Fisher Scientific.
[0716]
[0717] STUDY OF GENE EXPRESSION
[0718]
[0719] To decide the genes of interest in our study, we established the following criteria: 1. Relevant genes for differential expression in relation to asthma and studies of polymorphic variants (SNPs) in asthma, obtained in more than one independent work after an exhaustive bibliographic search.
[0720] 2. Genes previously defined by our group as significant by means of analysis of gene expression ( Aguerrí M. et al., J. Biol. Reg. Homeost, Ag. 2013 Vol.27 ( 2): 329-341.).
[0721] 3. Candidate genes of interest in cellular plasticity, inflammation and / or regulation that could have been excluded by previous criteria.
[0722]
[0723] Based on these criteria, we selected 94 genes to make a microfluidic card design.
[0724]
[0725] The preliminary study of the expression data by PCA (Principal Component Assay) shows how the expression of these 94 genes perfectly separates subjects by groups, confirming the good selection of the genes studied and encourages us to look for specific biomarkers depending on the clinical phenotype in samples such as blood (Figure 1)
[0726]
[0727] We studied 94 genes and two endogenous genes in 384-well microfluidic cards ( Applied Biosystem) for the analysis of gene expression in the RNA extracted from the 104 subjects of the study, by quantitative polymerase chain reaction in real time (qRT- PCR).
[0728]
[0729] In the analysis of expression by qRT-PCR, the expression of a certain gene is evaluated in a relative way, ie its expression is compared with that of a gene that is expressed stably or constitutively (endogenous genes). For our microfluidic plates we chose two endogenous genes usually used with PBMC: 18S and GADPH. The qRT-PCR data were analyzed with the StatMiner program , with the help of experts from the Genomics Unit of the Cantoblanco Science Park, Madrid.
[0730]
[0731] For the expression analysis, the value of Ct or threshold cycle obtained in the qRT-PCR is compared with the Ct of the endogenous by the expression:
[0732]
[0733] ACt = Ct gene analyzed-Ct endogenous gene.
[0734] A higher value of ACt implies a lower gene expression and a lower ACt determines greater expression of the gene. The data obtained from the microfluidic cards were analyzed by comparing the AACt (average ACt of each group AACt = [ACt (eXperimental)] - [ACt (control)]) between the control group and group of each clinical condition. The level of significance of the differential expression was established by a relative quantification or RQ (relative quantification) <-2 or> 2 and a pajustada value of less than 0.05 by the Benjamini-Hochberg FDR method .
[0735]
[0736] Ten genes ( CLCA1, IL17A, IL25, IL33, IL5, IL9, MUC5AC, POSTN, SERPINB4, and TCF21) were undetermined in all samples, and 10 genes ( ADAM33, CCL17, IL13, MUC5B, MUC2, NOS2, TSLP, CCL11 , DRB1, and IL4) could not be studied because they were detected only in some conditions or the data were inconsistent.
[0737]
[0738] To define specific gene profiles related to different clinical conditions, the gene expression of the 3 clinical groups was compared with the group of healthy control subjects. After the detailed analysis of the results of the qRT-PCR, the results summarized in Table 35 were obtained. Of all the genes differentially expressed in the clinical conditions compared with the healthy control group, 22 genes were common in the three clinical groups, 42 were common in the two groups of allergic subjects and one, MSR1, was common in the 2 groups of subjects with asthma (allergic and non-allergic). On the other hand, one gene, SERPINB2 was only significantly overexpressed in the group of subjects with non-allergic asthma. In contrast, ADRB1, ALOX15, CTSG and CX3CR1 were significantly repressed only in the group of allergic subjects with asthma. None of the genes analyzed was exclusively modulated differentially in the group of allergic subjects without asthma.
[0739] Table 35: Results of differential gene expression among the three groups of patients with respect to the control group.
[0740]
[0741]
[0742]
[0743]
[0744] In order to find genes specifically related to asthma and / or allergy, we analyzed the statistical differences in gene expression between the three clinical conditions studied. The comparison between non-allergic asthmatic subjects vs allergic asthmatic subjects showed that 74 genes were overexpressed in the group of nonallergic asthma, being the 5 genes with greater statistical differences CCL5, CHI3L1, CTSG, GZMH and IL1-R2. The most significant genes were those with a change of expression or RQ greater than 10. Comparing the groups with non-allergic asthma vs subjects with allergy without asthma we found 66 differentially expressed genes, 64 overexpressed in the asthma group and only 2 genes decreased CHI3L1 and PI3. Selecting genes with a stricter criterion (RQ> 10), 10 genes were differentially overexpressed in subjects with asthma: CCL5, CRTAP, GPX3, HLA-DQB1, IL-10, IL2RB, MSR1, NLRP3, PHLDA1 and SERPINB2. PI3 was the only gene repressed with this criterion. Finally, the comparison between the groups of allergic subjects with asthma vs allergic without asthma, showed that 14 genes were differentially expressed, 4 genes over-expressed ( IL-10, MSR1, PHLDA1 and SERPINB2) and 10 repressed genes ( ALOX15, CHI3L1, CPA3, CTSG, IL1R2, IL8, NKKB1Z, PI3, SVIL and TNF) in allergic subjects with asthma. The selection with a stricter criterion (RQ> 10), showed that 4 genes were extremely diminished in subjects with allergic asthma: CHI3L1, CPA3, CTSG and PI3.
[0745]
[0746] In order to select the most differential genes in the comparison study of the groups of sick patients and control, we established as criteria a RQ greater than 4 or less than 0.25. Thus we reduced the genes to 9 in the group with non-allergic asthma, 33 in the group with allergic asthma and 37 in the group with non-asthmatic allergy (Table 35). By having a population of asthmatic patients with 50% with diagnosis of severe asthma and the other 50% of moderate and mild asthma, a comparison of the expression of the genes that we highlighted as more significant was made to find out if there could be any marker of severity. This is reflected in Table 36A and 36B in which it is observed that in most genes the weight of the expression falls in the group of subjects with severe asthma. For example in Table 37A, MSR1 has a 4-fold expression in non-allergic asthmatic patients of severe diagnosis, or PHLDA1 in which expression is doubled. On the other hand, in the case of CPA3 it is in the moderate-mild pathology in which there is a greater change in the differential expression. The comparison of the severity of the disease is not evident in the expression of these genes in the allergic asthma group (Table 36B), but we see how at least CHI3L1, CPA3, IL8 and PI3 are shared with the group with non-allergic asthma ( Table 36A).
[0747]
[0748] Table 36: List of genes with significant differential expression ( p aj. <0.05) per group of patients with respect to the control group according to the criterion of RQ> 4 or <0.25.
[0749]
[0750]
[0751]
[0752]
[0753]
[0754]
[0755]
[0756]
[0757]
[0758] A) Genes with differential expression of the group with non-allergic asthma. B) Genes with differential expression of the group with allergic asthma. C) Genes with differential expression of the group with non-asthmatic allergy. In the two groups with asthma, the comparison of severe asthma and moderate-mild asthma is added. In bold, the common genes with the group with non-allergic asthma are highlighted in Tables 37B and 37C.
[0759]
[0760] In summary, the comparisons with respect to the control subjects of the clinical groups defined a group of genes associated with severity. Specifically, in the group of severe non-allergic asthma, and using stricter criteria of gene expression, we were allowed to define 9 genes. The analysis of the allergic asthma group showed a larger group of differential genes, even with stricter criteria, being 33 (all repressed) genes associated with severity.
[0761]
[0762] Example 1. STUDY OF PROTEIN EXPRESSION
[0763]
[0764] In view of the results of gene expression, we decided to study at the protein level the expression of the most relevant genes, MSR1 (which codes for the class A macrophage junk receptor), SERPINB2 (whose transcription gives rise to a member of the family of inhibitors of the serine protease) and PHLDA1 (which gives rise to a nuclear protein rich in proline and histidine) by the western blot technique and, the genes that give rise to soluble proteins IL8, IL10, CHI3L1 or chitinase3 (a glycoprotein member of the family 18 of the glycosyl hydrolase) and PI3 (which encodes the peptidase inhibitor 3) by ELISA, since they are secreted and found in the serum easily. We also quantified POSTN or Periostin, an extracellular matrix protein induced by IL-4 and IL-13 in the epithelium of the airway and fibroblasts and that has a role in subepithelial fibrosis and accelerates the tissue infiltration of eosinophils. Although it was one of the genes that we could not assess, since its gene expression was not titrable in our samples, serum periostin has been proposed as a biomarker of Th2 asthma, which is why we decided to measure its protein expression (by ELISA).
[0765]
[0766] 1. Expression of MSR1, SERPINB2 and PHLDA1
[0767]
[0768] The analysis of the expression of these three proteins was carried out by the western blot technique . This technique separates a mixture of proteins based on their molecular weight by means of an acrylamide gel. The separated proteins in the gel are transferred to a membrane, giving rise to a pattern of protein bands. The membrane is incubated with antibodies labeled with an enzyme and specific for the protein of interest. The antibodies that are bound are detected by exposure to ultraviolet light , since the enzyme bound to the antibody reacts with a substrate that we supply to it. The development system used in our tests was that of ECL. The thickness of the band gives an idea of the amount of protein, so it can be standardized to obtain the amount of protein in the sample.
[0769]
[0770] The protein extract obtained from the PBMC of the study subjects was quantified by the BCA technique and we performed the electrophoresis in PAGE-SDS. 12% gels were prepared in Novex cassettes ( Life Technologies) formed by a separating lower part (gel buffer 1M Tris-HCl pH 8.8, 50% acrylamide / bisacrylamide (29: 1), 10% SDS, 10% APS and TEMED) and another top concentrator (0.375M Tris-HCl gel buffer, pH 6.8, 50% acrylamide / bisacrylamide (29: 1), 10% SDS, 10% APS and TEMED). The electrophoresis buffer was also made in the laboratory with the following protocol: 25mM of Trizma base, 0.2M of glycine and 0.1% SDS. The protein fraction is loaded in different wells (the amount depends on the primary antibody used), being previously denatured at 70 ° C for 10 minutes. The electrophoresis was performed at 125V for 1 hour and a half. The Blot Dry Bloting system from Invitmgen ( ThermoFisherScientific) was used to transfer proteins to nitrocellulose membranes. For the blocking and washing of the membrane, and the detection of the bands, the Western Breeze Chemiluminescent kit ( Invitrogen, Thermo Fisher Scientific) was used. After blocking the membrane (approximately 1 hour), it was incubated with the primary antibody at 4 ° C overnight with different dilutions according to the one being studied and after a series of washes, it was incubated with the secondary antibody (at room temperature for 30 minutes). After the development in the Amersham Imager 6000 ( GE Healthcare Life Sciences) the bands corresponding to each protein were obtained. After the development of these antibodies, each one had its optimal dilution and adequate amount of protein.
[0771]
[0772] For the study of MSR1, we studied the differential protein expression between the group with severe nonallergic asthma and the control group, since it is in these groups where a greater expression change is observed and a greater association with the severity at the level of gene expression.
[0773] For MSR1, the polyclonal rabbit IgG antibody, specific for human CD204 (or MSR1) ( Thermo Fisher Scientific) was used at a dilution of 1: 2500 and loading 40 g of sample. Its molecular weight (MW) is 75kDa. Two main bands were observed in their profile, one between molecular weights 113 and 72.7 (upper band) and another between marker 72.7 and 46.7 (lower band). When analyzing the relative quantification of the bands of 9 control subjects and 18 severe non-allergic asthmatic patients, we saw significant differences only in the lower band that had higher expression in the healthy subjects than in the asthmatic ones (Figure 2)
[0774]
[0775] The MSR1 gene is one of the most differential in asthmatic disease and we also show its importance in the severity of non-allergic asthma. The expression of the MSR1 protein in PBMC manifests with a different protein profile in healthy subjects and non-allergic asthmatic patients. These data confirm the expression Protein of MSR1 in PBMC and points to interesting differences between healthy subjects and asthmatics.
[0776]
[0777] Rabbit polyclonal IgG antibody specific for human SERPINB2 ( R & D Systems) was diluted 1: 1000 and 10 ^ g sample was loaded into the gel. Its molecular weight is 49kDa. Figure 3 represents the results of the western blot of SERPINB2. We studied 6 control subjects, 11 non-allergic asthmatic subjects (6 with severe asthma and 5 with moderate-mild asthma), 11 allergic asthmatic subjects (6 with severe asthma and 5 with moderate-mild asthma) and 5 subjects with non-asthmatic allergy. The relative quantification of the band around 43kDa shows significant differences between the different groups. The control group (0.66 ± 0.31) and the group with non-asthmatic allergy (0.41 ± 0.33) differed statistically from the two groups with asthma (0.11 ± 0.05 without allergy, and 0.08 ± 0.04 with allergy), with p <0.0001 and p <0.05, respectively.
[0778]
[0779] The rabbit polyclonal IgG antibody specific for human PHLDA1 ( Invitrogen, Thermo Fisher Scientific) was diluted 1: 500 and 10 ^ g sample was loaded (the band height should be 48 kDa). Figure 4 shows the western blot results of PHLDA1 from 8 control subjects, 5 non-allergic asthmatic subjects (3 with severe asthma and 2 with moderate-mild asthma), 6 allergic asthmatic subjects (3 with severe asthma and 3 with moderate asthma) and 7 subjects with non-asthmatic allergy. Although the non-allergic asthmatic group shows the highest mean expression of this protein, the relative quantification of the band around 43kDa does not show significant differences between the different groups.
[0780]
[0781] 2. Protein expression of IL8, IL10, CHI3L1, PI3 and POSTN
[0782]
[0783] The study and quantification of these soluble proteins was carried out with the ELISA technique. The method of detection by ELISA ( Enzyme-Linked Immuno Sorbent Assay) is an immunoassay technique in which a primary antibody immobilized on a plate detects the antigen, which is recognized by a biotinylated secondary antibody, which in turn is joined an antibody with a linked enzyme, which is capable of generating a detectable product by changing color by reacting with the appropriate chromogenic substrate. The intensity of the color allows indirectly measuring the antigen in the sample by spectrophotometry. With this technique we measured the amount of IL8, IL10, CHI3L1, PI3 and POSTN in the serum of our patients using the kits: human IL8 ELISA kit (detection range: 62.50-2000pg / ml) of Diaclone, human Chitinase 3- Like1 DuoSet ELISA (range of detection: 31.20-2000pg / ml), human Trappin-2 / Elafin ( PI3) DuoSet ELISA (detection range: 31.20-2000pg / ml) and human Periostin / OSF-2 Duoset ELISA (detection range: 62 , 50-4000pg / ml) of R & D systems, and human Interleukin-10 ELISA (detection range: 16-1000pg / ml) of ImmunoTools, whose color is measured at 450nm. The average results per group and the statistical comparisons are summarized in Figure 5.
[0784]
[0785] The data indicated significant differences between the JL8 levels of the group of control subjects and allergic asthmatics, compared with that of non-asthmatic allergic patients (283.69 ± 167.69pg / ml, 207.02 ± 328.90pg / ml, 912.84 ± 608.74pg / ml, respectively ) (Figure 5A). There were 68.97% of control subjects with negative values, 53.33% in non-allergic asthmatic patients, 56.67% in allergic asthmatic patients and 46.15% in non-asthmatic allergic patients. When performing the more detailed analysis of patients with severe diagnosis and with moderate-mild diagnosis within the group of non-allergic asthmatics and allergic asthmatics, this showed no significant differences between the IL8 concentrations of each asthmatic group (505.49 ± 387.80pg / ml subjects severe asthmatics and 377.79 ± 338.27pg / ml moderate-mild asthmatic patients: 342.91 ± 406.96pg / ml severe allergic asthmatic subjects and 48.47 ± 69.77pg / ml moderate-mild allergic asthmatic patients). We emphasize that there is a tendency for the more severe asthmatic patients of the two pathological conditions to have higher levels. In gene expression we obtained the opposite. This is possibly due to the fact that IL8 produced by other cell types different from PBMC is released in blood (serum).
[0786]
[0787] The concentrations of CHI3L1 in serum also showed significant differences between the control group and allergic asthma (15729.18 ± 8576.85pg / ml and 22812.55 ± 3573.46pg / ml, respectively) (Figure 5C). None of the groups presented negative values. Analyzing the patients according to severity within the asthma groups, no significant differences were found between the CHI3L1 concentrations of each asthmatic group (17026.99 ± 4845.20pg / ml severe asthmatic subjects and 21702.56 ± 11589.97pg / ml moderate asthmatic patients- mild; 22708.69 ± 4301.77pg / ml severe allergic asthmatic subjects and 22916.40 ± 2814.77pg / ml moderate-mild allergic asthmatic patients).
[0788]
[0789] Mean serum levels of IL10 and PI3 did not show significant differences between any of the groups (Figure 5B and 5D). However, the analysis of IL10 according to severity in the groups of asthmatic patients showed that although there was no Statistically significant difference, subjects with severe asthma in both cases, showed increased levels of IL10 (200 ± 219 vs 105.27 ± 79pg / ml in severe non-allergic asthmatic vs moderate-moderate asthmatic patients: 211.40 ± 193 vs 168 ± 136pg / ml in severe allergic asthmatics vs moderate-mild). The results of gene expression agree with the tendency of the non-allergic asthma group.
[0790]
[0791] The mean levels of POSTN (or Periostina) did show differences between the different groups (Figure 5E). The group of patients with non-asthmatic allergy (11190.29 ± 2310.13pg / ml) was the only group that showed significant differences with the control group (15487.71 ± 6532.85pg / ml). Significant differences were also observed with the two groups with asthma (18679.59 ± 8086.07pg / ml non-allergic asthma, 15199.93 ± 4263.88pg / ml allergic asthma), with the rhinitic group having the lowest levels of POSTN. When comparing the groups with asthma, asthma without allergy had higher levels of this protein than asthma with allergy, showing significant differences. When studying these groups according to the severity of the asthma, statistically significant differences were obtained between the control group and the group with severe nonallergic asthma (15487.71 ± 6532.85pg / ml and 20198.91 ± 7859.24pg / ml, respectively). In addition, it was observed, significantly, that severe nonallergic asthma had higher levels of POSTN than severe allergic asthma (15305.71 ± 4607.65pg / ml).
[0792]
[0793] Example 2. ANALYSIS BIOMARKERS BY ROC CURVES
[0794]
[0795] To emphasize the good selection of genes that we have defined as possible biomarkers and to assess the specificity and sensitivity of these, we perform ROC curves of gene expression in the asthma groups. We obtained a large number of genes with a moderate or high significance, with an area under the curve (AUC) greater than 0.80. To interpret the ROC curves, the following intervals are established for the AUC values: 0.50-0.60 bad test, 0.61-0.75 regular test, 0.76-0.90 good test, 0.91-0.96 very good test, and 0.97-1 excellent test. This analysis has been carried out on the gene and protein expression of the most differential markers in the 3 study groups. The markers were studied individually and combined with each other.
[0796]
[0797] Table 37 (A, B and C) summarizes the data obtained from the ROC curve analysis of the two groups with asthma studying the markers at the gene and protein level individually.
[0798] Most analyzes fall into the category of good or very good. At the protein level, IL10 in the total group of asthma without allergy and the moderate-mild group do not fall into any category according to our criteria. MSR1 was not assessed at the protein level in the moderate-mild non-allergic asthmatic group. PHLDA1 at the protein level could not be classified into any criteria since there was not enough data on asthma without allergy. In the total group of asthma with allergy and in particular the moderate-mild group, protein expression of PI3 and IL10 did not fall into any category according to our criteria. MSR1 was not evaluated at the protein level in the allergic asthmatic group. PHLDA1 at the protein level could not be classified into asthma groups without moderate-mild or severe allergy. Periostine could only be classified with the total group and moderate-mild asthma with allergy. In the group with only allergy, at the MSR1 protein level it was not studied and the result of the PHLDA1 analysis does not fall into any category of asthma.
[0799]
[0800] Table 38 summarizes the results of the combination of analysis by ROC curves of the biomarkers according to their gene and protein expression, respectively
[0801]
[0802] Table 39 summarizes the results of the combination of three analysis markers by ROC curves according to their gene and protein expression.
[0803] Table 37: Classification by the analysis of ROC curves of the biomarkers according to their individual gene and protein expression.
[0804]
[0805] A) Non-allergic asthmatic group (ANA).
[0806]
[0807]
[0808]
[0809] Summary of the analysis by ROC curves of the important biomarkers of the group with non-allergic asthma, indicating the markers analyzed according to the ROC curve value, indicating in each biomarker the comparison to which it refers. C: Group of Control subjects. Total: total group of subjects in the group. ML: group of subjects with moderate-mild asthma. G: group of subjects with severe asthma.
[0810]
[0811] B) Allergic asthmatic group (AA).
[0812]
[0813]
[0814]
[0815]
[0816] Summary of the analysis by ROC curves of the important biomarkers of the group with allergic asthma, indicating the markers analyzed according to the value of the ROC curve, indicating in each biomarker the comparison to which it refers. C: Group of Control subjects. Total: total group of subjects in the group. ML: group of subjects with moderate asthma. G: group of subjects with severe asthma.
[0817]
[0818] C) Non-asthmatic allergy group (A).
[0819]
[0820]
[0821]
[0822]
[0823] Summary of the analysis by ROC curves of the important biomarkers of the group with allergy without asthma, indicating the markers analyzed according to the value of the ROC curve, indicating in each biomarker the comparison to which it refers. C: Group of Control subjects. Total: total group of subjects in the group.
[0824]
[0825] Table 38: AUC values of the ROC curve analysis by combining the protein expression of two biomarkers.
[0826]
[0827] A) Total non-allergic asthmatic group (ANA) compared to the control group
[0828]
[0829]
[0830]
[0831] All values are indicated with criteria of AUC> 0.75, good, very good or excellent. Of these, an improvement or synergy is observed between: CHI3L1 and MSR1sup and minimally between CHI3L1 and MSR1inf, CHI3L1 and SERPINB2; POSTN (Periostina) and PHLDA1; POSTN and SERPINB2.
[0832] B) Moderate-mild non-allergic asthmatic group (ANA) compared with the control group
[0833]
[0834]
[0835]
[0836] All values are indicated with criteria of AUC> 0.75 (good, very good or excellent). Of these, an improvement or synergy is observed between: CHI3L1 and MSR1sup; CHI3L1 and SERPINB2; IL8 and PI3; IL8 and POSTN (Periostina) and PHLDA1; POSTN and SERPINB2.
[0837] C) Severe non-allergic asthmatic group (ANA) compared to the control group.
[0838]
[0839]
[0840]
[0841] All values are indicated with criteria of AUC> 0.75 (good, very good or excellent). Of these, an improvement or synergy is observed between: CHI3L1 and MSR1sup; minimally between CHI3L1 and MSR1 inf; minimally between PI3 and MSR1 inf; POSTN (Periostina) and SERPINB2.
[0842]
[0843] D) Moderate-mild non-allergic asthmatic group (ANA) compared to the severe ANA group
[0844]
[0845]
[0846]
[0847] All values are indicated with criteria of AUC> 0.75 (good, very good or excellent). Of these, an improvement or synergy is observed between: CHI3L1 and POSTN (Periostina); IL10 and POSTN; IL8 and PI3; minimally between IL8 POSTN; SERPINB2 and POSTN.
[0848] E) Allergic asthmatic group (AA) total compared to the control group
[0849]
[0850]
[0851]
[0852] All values are indicated with criteria of AUC> 0.75 (good, very good or excellent). Of these, there is an improvement or synergy between: CHI3L1 and IL8; minimally between CHI3L1 and POSTN (Periostina); CHI3L1 and SERPINB2; IL10 and POSTN; POSTN and PHLDA1.
[0853]
[0854] F) Moderate-mild allergic asthmatic group (AA) compared with the control group
[0855]
[0856]
[0857]
[0858] All values are indicated with criteria of AUC> 0.75 (good, very good or excellent). Of these, there is an improvement or synergy between: CHI3L1 and IL8; IL10 and POSTN.
[0859] G) Severe allergic asthmatic group (AA) compared to the control group
[0860]
[0861]
[0862]
[0863] All values are signaled with criteria of AU C> 0.75 (good, very good or excellent). Of these, there is an improvement or synergy between: CHI3L1 and IL8; CHI3L1 and PI3; CHI3L1 and POSTN (Periostina); CHI3L1 and SERPINB2; IL10 and POSTN; IL8 and PI3; IL8 and POSTN; PI3 and SERPINB2; POSTN and SERPINB2.
[0864]
[0865] H) Moderate-mild allergic asthmatic group (AA) compared to severe AA group
[0866]
[0867]
[0868]
[0869] In bold all values are indicated with AUC> 0.75 criteria (good, very good or excellent). Of these, an improvement or synergy is observed between: CHI3L1 and SERPINB2; minimally between IL8 and POSTN (Periostina); PI3 and SERPINB2; POSTN and SERPINB2.
[0870] I) Non-asthmatic allergic group (A) compared to the control group
[0871]
[0872]
[0873]
[0874] Bold is the AUC values> 0.75, whose combinations of biomarkers give rise to a good, very good or excellent category test. Of these, an improvement or synergy is observed between: CHI3L1 and IL8; minimally between CHI3L1 and POSTN (Periostina); IL8 and PI3; IL8 and POSTN; minimally between POSTN and SERPINB2.
[0875] Table 39: AUC values of the ROC curve analysis by combining the protein expression of three biomarkers.
[0876]
[0877] A) Non-allergic asthmatic group (ANA) compared to the control group.
[0878]
[0879]
[0880]
[0881]
[0882] Bold is the AUC values> 0.75, whose combinations of biomarkers give rise to a good, very good or excellent category test. Of these, an improvement or synergy is observed in: 1. Total ANA group compared with controls between: CHI3L1 PI3 MSR1 superior; minimally between CHI3L1 PI3 lower MSR1; minimally between CHI3L1 PI3 SERPINB2; CHI3L1 POSTN MSR1 higher; minimally between CHI3L1 POSTN MSR1 lower; CHI3L1 POSTN PHLDA1; CHI3L1 POSTN SERPINB2; PI3 POSTN MSR1 upper; minimally PI3 POSTN MSR1 lower; PI3 POSTN PHLDA1; PI3 POSTN SERPINB2. 2. In the moderate / mild ANA group compared with controls between: CHI3L1 IL8 PI3; CHI3L1 IL8 POSTN; CHI3L1 PI3 MSR1 upper; IL8 PI3 POSTN; PI3 POSTN MSR1 upper; PI3 POSTN SERPINB2. 3. In the severe ANA group compared with controls between: CHI3L1 IL8 PI3; minimally between CHI3L1 PI3 lower MSR1; PI3 POSTN MSR1 upper; PI3 POSTN MSR1 lower. Total: total group of subjects in the group. ML: group of subjects with moderate-mild asthma. G: group of subjects with severe asthma.
[0883] C) Moderate-mild non-allergic asthmatic group (ANA) compared to the severe ANA group
[0884]
[0885]
[0886]
[0887]
[0888] Bold is the AUC values> 0.75, whose combinations of biomarkers give rise to a good, very good or excellent category test. Of these, in all there is an improvement or synergy.
[0889] D) Allergic asthmatic group (AA) compared to the control group
[0890]
[0891]
[0892]
[0893]
[0894] Bold is the AUC values> 0.75, whose combinations of biomarkers give rise to a good, very good or excellent category test. Of these, an improvement or synergy is observed in: 1. Total AA group compared with controls in all, except the combination CHI3L1 + PI3 + POSTN. 2. Moderate / mild AA group compared with controls in all, except the combination CHI3L1 + PI3 + POSTN. 3. Severe AA group compared with controls in all. Total: group of group subjects. ML: group of subjects with moderate-mild asthma. G: group of subjects with severe asthma.
[0895] E) Moderate-mild allergic asthmatic group (AA) compared to the severe (AA) group.
[0896]
[0897]
[0898]
[0899]
[0900] Bold is the AUC values> 0.75, whose combinations of biomarkers give rise to a good, very good or excellent category test. Of these, an improvement or synergy is observed in all except: CHI3L1 IL8 PI3; IL8 PI3 POSTN.
[0901] F) Non-asthmatic allergic group (A) compared to the control group.
[0902]
[0903]
[0904]
[0905]
[0906] Bold is the AUC values whose combinations of biomarkers result in a good, very good or excellent category test. Of these, all show an improvement or synergy. Total: total group of subjects in the group.
[0907]
[0908] Example 3. Global Assessment of Protein Biomarker Results
[0909]
[0910] Based on the analysis of individual and grouped ROC curves of the 8 protein biomarkers studied, the best Biomarker options for each condition analyzed would be:
[0911]
[0912] to. Non-allergic asthma (ANA)
[0913]
[0914] 1. Biomarkers capable of discriminating ANA (Total Population) from
[0915] control subjects
[0916] AUC MSR1 lower 0.96
[0917] SERPINB2 0.91
[0918] Lower MSR1 + MSR1 sup 1
[0919] CHI3L1 + MSR1inf 0.97
[0920] POSTN + SERPINB2 0.95
[0921] CHI3L1 + SERPINB2 0.92
[0922] POSTN + PHLDA1 0.82
[0923] CHI3L1 + MSR1sup 0.78
[0924] PI3 + POSTN + MSR1inf 0.97
[0925] CHI3L1 + POSTN + MSR1sup 0.83
[0926] PI3 + POSTN + MSR1sup 0.77
[0927]
[0928] 2. Biomarkers capable of discriminating ANA M / L from control subjects
[0929]
[0930] AUC MSR1 lower 1
[0931] SERPINB2 0.89
[0932] CHI3L1 + SERPINB2 0.97
[0933] POSTN + SERPINB2 0.97
[0934] IL8 + POSTN 0.87
[0935] CHI3L1 + MSR1sup 0.83
[0936] POSTN + MSR1sup 0.81
[0937] IL8 + PI3 0.76
[0938] CHI3L1 + IL8 + POSTN 0.87
[0939] CHI3L1 + IL8 + PI3 0.79
[0940]
[0941] 3. Biomarkers capable of discriminating severe ANA from control subjects
[0942]
[0943] AUC MSR1 lower 0.93
[0944] SERPINB2 0.93
[0945] Lower MSR1 + MSR1 sup 1
[0946] POSTN + SERPINB2 0.97
[0947] PI3 + MSR1inf 0.95
[0948] CHI3L1 + MSR1inf 0.94
[0949] CHI3L1 + MSR1sup 0.76
[0950] PI3 + POSTN + MSR1inf 0.96
[0951] CHI3L1 + PI3 + MSR1inf 0.95
[0952] CHI3L1 + IL8 + PI3 0.79
[0953] PI3 + POSTN + MSR1sup 0.77
[0954] 4. Biomarkers capable of discriminating ANA M / L from ANA Grave
[0955]
[0956] AUC IL8 0.76
[0957] POSTN + SERPINB2 0.90
[0958] IL8 + PI3 0.82
[0959] IL8 + POSTN 0.78
[0960] CHI3L1 + POSTN 0.77
[0961] POSTN + IL10 0.77
[0962] CHI3L1 + IL8 + POSTN 0.98
[0963] PI3 + POSTN + SERPINB2 0.93
[0964] CHI3L1 + IL8 + PI3 0.88
[0965] CHI3L1 + POSTN + MSR1sup 0.80
[0966] CHI3L1 + POSTN + MSR1inf 0.80
[0967] CHI3L1 + PI3 + MSR1sup 0.78
[0968]
[0969] b. Allergic asthma (AA)
[0970]
[0971] 1. Biomarkers capable of discriminating AA (Total population) of control subjects
[0972]
[0973] AUC SERPINB2 0.97
[0974] CHI3L1 0.78
[0975] IL8 0.76
[0976] CHI3L1 + SERPINB2 1
[0977] CHI3L1 + IL8 0.92
[0978] IL10 + POSTN 0.85
[0979] CHI3L1 + POSTN 0.79
[0980] POSTN + PHLAD1 0.79
[0981] IL10 + POSTN + PI3 0.86
[0982] 2. Biomarkers capable of discriminating AA M / L from control subjects AUC SERPINB2 1
[0983] CHI3L1 0.78
[0984] CHI3L1 + IL8 0.87
[0985] IL10 + POSTN 0.83
[0986] CHI3L1 + IL8 + PI3 0.89
[0987] IL10 + POSTN + PI3 0.85
[0988]
[0989] 3. Biomarkers capable of discriminating AA Graves from control subjects
[0990]
[0991] AUC SERPINB2 0.77
[0992] CHI3L1 + IL8 1
[0993] POSTN + SERPINB2 0.97
[0994] IL8 + PI3 0.95
[0995] IL8 + POSTN 0.95
[0996] PI3 + SERPINB2 0.93
[0997] IL10 + POSTN 0.86
[0998] CHI3L1 + PI3 0.80
[0999] CHI3L1 + POSTN 0.80
[1000] IL10 + PI3 + POSTN 0.89
[1001] CHI3L1 + PI3 + POSTN 0.81
[1002]
[1003] 4. Biomarkers capable of discriminating AA M / L from AA Grave
[1004]
[1005] AUC IL8 0.82
[1006] SERPINB2 0.77
[1007] POSTN + SERPINB2 0.93
[1008] CHI3L1 + SERPINB2 0.90
[1009] PI3 + SERPINB2 0.87
[1010] IL8 + POSTN 0.84
[1011] c. Allergy (A) without asthma
[1012]
[1013] 1. Biomarkers capable of discriminating A from control subjects
[1014] AUC IL8 0.82
[1015] CHI3L1 + IL8 1
[1016] IL8 + POSTN 0.90
[1017] IL8 + PI3 0.87
[1018] PI3 + SERPINB2 0.83
[1019] PI3 + POSTN 0.80
[1020] POSTN + SERPINB2 0.80
[1021] PI3 + POSTN + SERPINB2 0.90
[1022] PI3 + POSTN + PHLDA1 0.82
[1023]
[1024] d. Comparisons of protein expression between clinical phenotypes
[1025]
[1026] 1. Comparisons between total non-allergic asthmatics (ANA) and total allergic asthmatics (AA).
[1027] - NO: Non-allergic asthmatics (ANA)
[1028] - N1: Allergic asthmatics (AA)
[1029] - AUC: Area Under the ROC Curve.
[1030]
[1031] ROC curves
[1032] Variable N0 N1 AUC (IC95%) Threshold
[1033] CHI3L1 30 30 0.74 (0.61 - 0.88) 20202
[1034] IL10 11 21 0.45 (0.23 - 0.67) 457.4
[1035] IL8 14 14 0.68 (0.46 - 0.89) 221
[1036] PI3 29 30 0.57 (0.42 - 0.72) 5126
[1037] Periostin 30 30 0.62 (0.48 - 0.77) 22785
[1038] MSR1 top
[1039] MSR1 lower
[1040] PHLDA1 5 6 0.40 (0.00 - 0.80) 0.012
[1041] SERPINB2 11 11 0.64 (0.39 - 0.88) 0.116
[1042] ROC curves for models with combinations of two variables
[1043] Variables Variables Combination N0 N1 quantitative qualitative CHI3L1 IL10 11 21 0.73 (0.49 - 0.96) 0.81 (0.65 - 0.96) CHI3L1 + IL8 14 14 0.82 (0.64 - 0.99) 0.90 (0.79 - 1.00) CHI3L1 + PI3 29 30 0.75 (0.62 - 0.89 ) 0.84 (0.75 - 0.94) CHI3L1 + Periostine 30 30 0.76 (0.64 - 0.89) 0.87 (0.79 - 0.95) CHI3L1 + PHLDA1 5 6 0.43 (0.02 - 0.85) 0.85 (0.59 - 1.00) CHI3L1 + SERPINB2 11 11 0.83 (0.64 - 1.00) 0.88 (0.74 - 1.00) IL10 + IL8 5 11 0.71 (0.42 - 0.99) 0.85 (0.65 - 1.00) IL10 + PI3 11 21 0.46 (0.24 - 0.68) 0.60 (0.40 - 0.79) IL10 + Periostin 11 21 0.87 (0.74) - 1.00) 0.74 (0.59 - 0.89) IL8 + PI3 14 14 0.67 (0.45 - 0.89) 0.74 (0.56 - 0.92) IL8 + Periostin 14 14 0.76 (0.57 - 0.94) 0.75 (0.57 - 0.93) IL8 + SERPINB2 7 7 0.82 ( 0.58 - 1.00) 0.81 (0.56 - 1.00) PI3 + Periostin 29 30 0.67 (0.53 -0.81) 0.75 (0.63 - 0.86) PI3 + PHLDA1 5 6 0.60 (0.19 - 1.00) 0.67 (0.32 - 1.00) PI3 + SERPINB2 11 11 0.69 (0.45 - 0.92) 0.77 (0.57 - 0.96) Periostina + PHLDAI 5 6 0.70 (0.34 - 1.00) 0.83 (0.57 - 1.00) Periostina + SERPINB2 11 11 0.75 (0.52 - 0.98) 0.85 (0.70 - 1.00) PHLDA1 + SERPINB2 5 5 0.80 (0.50 - 1.00) 0.82 (0.51 - 1.00) ROC curves for models with combinations of three variables
[1044] Variables Variables Qualitative quantitative N0 N1 CHI3L1 IL10 IL8 5 11 0.76 (0.47 - 1.00) 0.92 (0.79 - 1.00) CHI3L1 IL10 PI3 11 21 0.71 (0.48 - 0.93) 0.82 (0.65 - 0.99) CHI3L1 IL10 Periostin 11 21 0.92 (0.81 - 1.00) 0.90 (0.80 - 1.00) CHI3L1 IL8 PI3 14 14 0.82 (0.64 - 0.99) 0.93 (0.84 - 1.00) CHI3L1 IL8 Periostine 14 14 0.88 (0.76 - 1.00) 0.92 (0.82 - 1.00) CHI3L1 PI3 Periostin 29 30 0.80 (0.68) -0.91) 0.93 (0.87 - 0.99) CHI3L1 PI3 PHLDA1 5 6 0.63 (0.25 - 1.00) 0.87 (0.60 - 1.00) CHI3L1 PI3 SERPINB2 11 11 0.84 (0.65 - 1.00) 0.92 (0.78 - 1.00) CHI3L1 Periostin PHLDA1 5 6 0.77 ( 0.46 - 1.00) 0.98 (0.94 - 1.00) CHI3L1 Periostin SERPINB2 11 11 0.85 (0.69 - 1.00) 0.96 (0.89 - 1.00) CHI3L1 PHLDA1 SERPINB2 5 5 0.84 (0.52 - 1.00) 0.90 (0.70 - 1.00) IL10 IL8 PI3 5 11 ( 0.57 - 1.00) 0.95 (0.85 - 1.00) IL10 IL8 Periostin 5 11 0.87 (0.69 - 1.00) (0.70 - 1.00) IL10 PI3 Periostin 11 21 0.87 (0.74 - 0.99) 0.77 (0.58 - 0.95) IL8 PI3 Periostin 14 14 0.76 ( 0.58 - 0.94) 0.78 (0.60 - 0.96) IL8 PI3 SERPINB2 7 7 0.80 (0.54 - 1.00) (0.70 - 1.00) IL8 Periostine SERPINB2 7 7 0.82 (0.56 - 1.00) 0.87 (0.65 - 1.00) PI3 Periostine PHLDA1 5 6 0.67 (0.30 - 1.00) 0.92 (0.76 - 1.00) PI3 Periostin SERPINB2 11 11 0.77 (0.56 - 0.98) 0.89 (0.75 - 1.00) PI3 PHLDA1 SERPINB2 5 5 (0.57 - 1.00) 0.92 (0.74 - 1.00) Comparison table. Total non-allergic asthmatic group (ANA) compared to the total allergic asthmatic group (AA)
[1045]
[1046]
[1047]
[1048] All values are signaled with criteria of AU C> 0.75, good, very good or excellent. Of these, there is an improvement or synergy between: CHI3L1 and IL8; CHI3L1 and SERPINB2; IL10 and POSTN (Periostina), IL8 and POSTN; IL8 and SERPINB2; SERPINB2 and PHLDA1 and minimally between CHI3L1 and POSTN.
[1049] 2. Comparisons between severe non-allergic asthmatics (ANA) and severe allergic asthmatics (AA)
[1050]
[1051] - NO: Serious non-allergic asthmatics (ANA)
[1052] - N1: Serious allergic asthmatics (AA)
[1053] - AUC: Area Under the ROC Curve
[1054]
[1055] ROC curves
[1056] Variable N0 N1 AUC (IC95%) Threshold
[1057] CHI3L1 15 15 0.82 (0.67 - 0.98) 20202
[1058] IL10 6 13 0.49 (0.17 -0.81) 184.6
[1059] IL8 7 7 0.63 (0.30 - 0.97) 122
[1060] PI3 15 15 0.48 (0.26 - 0.70) 5758
[1061] Periostin 15 15 0.69 (0.50 - 0.89) 22785
[1062] MSR1 top
[1063] MSR1 lower
[1064] PHLDA1 3 3
[1065] SERPINB2 6 6 0.78 (0.47 - 1.00) 0.11
[1066]
[1067] ROC curves for models with combinations of two variables
[1068] Variables Quantitative Qualitative Combination N0 N1 CHI3L1 + IL10 6 13 0.69 (0.39 - 0.99) 0.81 (0.54 - 1.00) CHI3L1 + IL8 7 7 0.86 (0.64 - 1.00) 0.94 (0.83 - 1.00) CHI3L1 + PI3 15 15 0.83 (0.67 - 0.67) 0.98) 0.81 (0.65 - 0.97) CHI3L1 + Periostine 15 15 0.86 (0.72 - 1.00) 0.87 (0.76 - 0.99) IL10 + PI3 6 13 0.68 (0.40 - 0.96) 0.72 (0.51 - 0.94) IL10 + Periostin 6 13 0.94 (0.83) - 1.00) 0.91 (0.79 - 1.00) IL8 + PI3 7 7 0.69 (0.40 - 0.99) 0.71 (0.44 - 0.99) IL8 + Periostin 7 7 0.86 (0.65 - 1.00) 0.83 (0.62 - 1.00) PI3 + Periostin 15 15 0.69 ( 0.49 - 0.89) 0.73 (0.55 - 0.90) PI3 + SERPINB2 6 6 0.92 (0.76 - 1.00) 0.97 (0.91 - 1.00)
[1069] ROC curves for models with combinations of three variables
[1070] Variable variables Variable N0 N1 qualitative quantitative
[1071] CHI3L1 IL8 Periostine 7 7 0.96 (0.86 - 1.00) 0.95 (0.85 - 1.00) CHI3L1 PI3 Periostine 15 15 0.86 (0.72 - 1.00) 0.87 (0.74 - 1.00) IL10 PI3 Periostine 6 13 0.94 (0.83 - 1.00) 0.92 (0.78 - 1.00 ) IL8 PI3 Periostine 7 7 0.86 (0.65 - 1.00) 0.85 (0.63 - 1.00)
[1072]
[1073] Comparison table. Severe nonallergic asthmatic group (ANA) compared to severe allergic asthmatic group (AA)
[1074]
[1075]
[1076]
[1077] All values are signaled with criteria of AU C> 0.75, good, very good or excellent. Of these, there is an improvement or synergy between: CHI3L1 and IL8; CHI3L1 and POSTN; IL10 and POSTN (Periostina), IL8 and POSTN, PI3 and SERPINB2 and minimally between CHI3L1 and PI3.
[1078] Comparisons between mild nonallergic asthmatics (ANA) and mild allergic asthmatics (AA)
[1079]
[1080] to. NO: mild non-allergic asthmatics
[1081] b. N1: Mild allergic asthmatics
[1082] c. AUC: Area Under the ROC Curve
[1083] ROC curves
[1084] Variable N0 N1 AUC (IC95%) Threshold
[1085] CHI3L1 15 15 0.66 (0.43 - 0.89) 20227
[1086] IL10 5 8 0.60 (0.26 - 0.94) 167.9
[1087] IL8 7 7 0.73 (0.44 - 1.00) 221
[1088] PI3 14 15 0.62 (0.41 - 0.83) 5028
[1089] Periostin 15 15 0.53 (0.32 - 0.75) 24658
[1090] MSR1 top
[1091] MSR1 lower
[1092] PHLDA1 2 3
[1093] SERPINB2 5 5 0.60 (0.19 - 1.00) 0.134
[1094]
[1095] ROC curves for models with combinations of two variables
[1096] Variables Variables Combination quantitative qualitative N0 N1 CHI3L1 + IL10 5 8 0.60 (0.26 - 0.94) 0.75 (0.45 - 1.00) CHI3L1 + IL8 7 7 0.80 (0.51 - 1.00) 0.93 (0.79 - 1.00) CHI3L1 + PI3 14 15 0.63 (0.41 - 0.86) 0.86 (0.73 - 0.99) CHI3L1 + Periostine 15 15 0.72 (0.52 - 0.91) 0.87 (0.75 - 0.99) CHI3L1 + SERPINB2 5 5 0.72 (0.37 - 1.00) 0.88 (0.71 - 1.00) IL10 + PI3 5 8 0.68 (0.36) - 0.99) 0.70 (0.40 - 1.00) IL10 + Periostin 5 8 0.82 (0.59 - 1.00) 0.83 (0.62 - 1.00) IL8 + PI3 7 7 0.82 (0.53 - 1.00) 0.85 (0.65 - 1.00) IL8 + Periostin 7 7 0.82 ( 0.58 - 1.00) 0.84 (0.68 - 1.00) PI3 + Periostina 14 15 0.67 (0.46 - 0.87) 0.74 (0.57 -0.91) PI3 + SERPINB2 5 5 0.68 (0.30 - 1.00) 0.72 (0.40 - 1.00) Periostin + SERPINB2 5 5 0.40 (0.00 -0.81) 0.84 (0.59 - 1.00) ROC curves for models with combinations of three variables
[1097] Variable variables Variable N0 N1 qualitative quantitative
[1098]
[1099]
[1100]
[1101]
[1102] Comparison table. Mild non-allergic asthmatic group (ANA) compared with mild allergic asthmatic group (AA)
[1103]
[1104]
[1105]
[1106] All values are signaled with criteria of AU C> 0.75, good, very good or excellent. Of these, there is an improvement or synergy between: CHI3L1 and IL8; IL10 and POSTN (Periostina), IL8 and PI3; IL8 and POSTN.
[1107] 4. Comparisons between allergic asthmatics (AA) and allergic (A) non-asthmatic a. NO: Allergic asthmatics
[1108] b. N1: Non-asthmatic allergic.
[1109] c. AUC: Area Under the ROC Curve
[1110]
[1111] ROC curves
[1112] Variable N0 N1 AUC (IC95%) Threshold
[1113] CHI3L1 30 14 0.60 (0.36 - 0.84) 15719
[1114] IL10 21 4
[1115] IL8 14 7 0.88 (0.72 - 1.00) 67
[1116] PI3 30 14 0.63 (0.43 - 0.84) 7348
[1117] Periostin 30 14 0.80 (0.67 - 0.93) 14418
[1118] MSR1 top
[1119] MSR1 lower
[1120] PHLDA1 6 7 0.48 (0.12 - 0.83) 0.025
[1121] SERPINB2 11 5 0.93 (0.80 - 1.00) 0.116
[1122]
[1123] ROC curves for models with combinations of two variables
[1124] Variables Variables Combination N0 N1 qualitative quantitative CHI3L1 + IL8 14 7 0.91 (0.78 - 1.00) 0.95 (0.87 - 1.00) CHI3L1 + PI3 30 14 0.58 (0.35 - 0.80) 0.83 (0.70 - 0.96) CHI3L1 + Periostin 30 14 0.81 (0.68 - 0.94) 0.91 (0.84 - 0.97) CHI3L1 + PHLDA1 6 7 0.43 (0.09 - 0.77) 0.68 (0.40 - 0.96) CHI3L1 + SERPINB2 11 5 1.00 (1.00 - 1.00) 0.96 (0.90 - 1.00) IL8 + PI3 14 7 0.89 (0.75) - 1.00) 0.89 (0.76 - 1.00) IL8 + Periostina 14 7 0.90 (0.75 - 1.00) 0.94 (0.85 - 1.00) PI3 + Periostin 30 14 0.80 (0.66 - 0.93) 0.90 (0.81 - 0.98) PI3 + PHLDA1 6 7 0.76 ( 0.48 - 1.00) 0.77 (0.51 - 1.00) PI3 + SERPINB2 11 5 0.96 (0.88 - 1.00) 0.98 (0.94 - 1.00) ROC curves for models with combinations of three variables
[1125] Variable Variables
[1126] Quantitative variables N0 N1 qualitative
[1127]
[1128] CHI3L1 PI3 Periostin 30 14 0.80 (0.67 - 0.93) 0.95 (0.89 - 1.00)
[1129]
[1130] IL8 PI3 Periostin 14 7 0.93 (0.82 - 1.00) 0.94 (0.83 - 1.00)
[1131]
[1132] Comparison table. Asthmatic group allergic asthmatics (AA) and allergic (A) non-asthmatic
[1133]
[1134]
[1135]
[1136] All values are signaled with criteria of AU C> 0.75, good, very good or excellent. Of these, there is an improvement or synergy between: CHI3L1 and IL8; CHI3L1 and SERPINB2; PI3 and PHLDA1; PI3 and SERPINB2; minimum between CHI3L1 and POSTN; minimum between IL8 and PI3 and IL8 and POSTN.
[1137] 5. Comparisons between allergic asthmatics (AA) severe and allergic (A) non-asthmatic
[1138] to. NO: Serious allergic asthmatics
[1139] b. N1: Non-asthmatic allergic
[1140]
[1141] ROC curves
[1142] Variable N0 N1 AUC (IC95%) Threshold
[1143] CHI3L1 15 14 0.60 (0.36 - 0.84) 15719
[1144] IL10 13 4
[1145] IL8 7 7 0.80 (0.55 - 1.00) 1023
[1146] PI3 15 14 0.66 (0.44 - 0.88) 7348
[1147] Periostin 15 14 0.77 (0.58 - 0.95) 14732
[1148] MSR1 top
[1149] MSR1 lower
[1150] PHLDA1 3 7
[1151] SERPINB2 6 5
[1152]
[1153] ROC curves for models with combinations of two variables
[1154] Variables Variables Combination N0 N1 qualitative quantitative CHI3L1 + IL8 7 7 0.82 (0.58 - 1.00) 0.90 (0.73 - 1.00) CHI3L1 + PI3 15 14 0.62 (0.40 - 0.84) 0.83 (0.68 - 0.97) CHI3L1 + Periostin 15 14 0.78 (0.61 - 0.96) 0.90 (0.82 - 0.98) IL8 + PI3 7 7 0.82 (0.58 - 1.00) 0.93 (0.79 - 1.00) IL8 + Periostin 7 7 0.90 (0.73 - 1.00) 0.91 (0.79 - 1.00) PI3 + Periostin 15 14 0.80 (0.62) - 0.97) 0.91 (0.81 - 1.00) ROC curves for models with combinations of three variables
[1155] Variable Variables
[1156] Quantitative variables N0 N1 qualitative
[1157]
[1158] CHI3L1 PI3 Periostin 15 14 0.80 (0.63 - 0.98) 0.95 (0.89 - 1.00)
[1159] IL8 PI3 Periostin 7 7 0.98 (0.92 - 1.00) 0.97 (0.90 - 1.00)
[1160]
[1161] Comparison table. Asthmatic group allergic asthmatic (AA) severe and allergic (A) non-asthmatic
[1162]
[1163]
[1164]
[1165] All values are signaled with criteria of AU C> 0.75, good, very good or excellent. Of these, an improvement or synergy is observed between: IL8 and POSTN; Minimum between CHI3L1 and IL8; CHI3L1 and POSTN; IL8 and PI3; PI3 and POSTN.
[1166] 6. Comparisons between allergic asthmatics (AA) mild and allergic (A) non-asthmatic
[1167] to. NO: Mild allergic asthmatics.
[1168] b. N1: Non-asthmatic allergic.
[1169] c. AUC: Area Under the ROC Curve
[1170] ROC curves
[1171] Variable N0 N1 AUC (IC95%) Threshold
[1172] CHI3L1 15 14 0.60 (0.35 - 0.84) 16093
[1173] IL10 8 4
[1174] IL8 7 7 0.96 (0.86 - 1.00) 67
[1175] PI3 15 14 0.60 (0.38 - 0.83) 7256
[1176] Periostin 15 14 0.83 (0.67 - 1.00) 14418
[1177] MSR1 top
[1178] MSR1 lower
[1179] PHLDA1 3 7
[1180] SERPINB2 5 5 0.84 (0.57 - 1.00) 0.116
[1181]
[1182] ROC curves for models with combinations of two variables
[1183] Variables Variables Combination qualitative quantitative N0 N1 CHI3L1 + PI3 15 14 0.57 (0.33 -0.81) 0.83 (0.69 - 0.97) CHI3L1 + Periostin 15 14 0.82 (0.66 - 0.99) 0.92 (0.84 - 0.99) IL8 + PI3 7 7 0.96 (0.86 - 1.00) 0.96 (0.87 - 1.00) IL8 + Periostin 7 7 0.94 (0.82 - 1.00) 0.94 (0.82 - 1.00) PI3 + Periostin 15 14 0.80 (0.63 - 0.98) 0.89 (0.77 - 1.00) PI3 + SERPINB2 5 5 0.92 (0.74) - 1.00) 0.96 (0.87 - 1.00) ROC curves for models with combinations of three variables
[1184] Variables Variables N0 N1 quantitative qualitative CHI3L1 PI3 Periostine 15 14 0.81 (0.64 - 0.98) 0.94 (0.87- 1.00) IL8 PI3 Periostina 7 7 0.96 (0.86 - 1.00) 0.97 (0.90 - 1.00)
[1185]
[1186] Comparison table. Asthmatic group allergic asthmatic (AA) mild and allergic (A) non-asthmatic
[1187]
[1188]
[1189]
[1190] All values are signaled with criteria of AU C> 0.75, good, very good or excellent. Of these, only an improvement or synergy between PI3 and SERPINB2 is observed.
[1191] Comparisons of protein expression between clinical phenotypes. AUC values of the ROC curve analysis by combining the protein expression of three biomarkers.
[1192]
[1193] A) Non-allergic asthmatic group (ANA) compared with the allergic asthmatic group (AA).
[1194]
[1195]
[1196]
[1197]
[1198] Bold is the AUC values> 0.75, whose combinations of biomarkers give rise to a good, very good or excellent category test. Of these, an improvement or synergy is observed in: 1. Total ANA group compared to AA between: CHI3L1 IL10 POSTN; CHI3L1 IL8 PI3; CHI3L1 IL8 POSTN; CHI3L1 PI3 POSTN; CHI3L1 PI3 SERPINB2; CHI3L1 POSTN SERPINB2; IL10 IL8 + PI3; IL10 IL8 + POSTN; IL10 PI3 POSTN; IL8 PI3 POSTN; IL8 PI3 SERPINB2; IL8 POSTN + SERPINB2; PI3 POSTN SERPINB2; PI3 PHLDA1 + SERPINB2; Minimally between CHI3L1 POSTN PHLDA1, CHI3L1 IL10 IL8. 2. In the moderate / mild ANA group compared to the AA M / L group between: CHI3L1 IL10 POSTN; CHI3L1 PI3 POSTN; IL10 + PI3 + POSTN; IL8 + PI3 + POSTN. 3. In the severe ANA group compared to severe AA between: CHI3L1 IL8 PI3; CHI3L1 IL8 POSTN; CHI3L1 PI3 POSTN; IL10 + PI3 + POSTN; IL8 + PI3 POSTN. Total: total group of subjects in the group. ML: group of subjects with moderate-mild asthma. G: group of subjects with severe asthma
[1199]
[1200] B) Allergic asthmatic group (AA) compared with the non-asthmatic allergic group (A).
[1201]
[1202]
[1203]
[1204]
[1205] Bold is the AUC values> 0.75, whose combinations of biomarkers give rise to a good, very good or excellent category test. Of these, an improvement or synergy is observed in: 1. Total AA group compared to A between: CHI3L1 IL8 PI3; IL8 PI3 POSTN. 2. In the moderate / mild AA group compared with group A between: CHI3L1 PI3 POSTN; IL8 + PI3 + POSTN. 3. In the severe AA group compared to A: CHI3L1 IL8 PI3; CHI3L1 PI3 POSTN; IL8 + PI3 POSTN. Total: total group of subjects in the group. ML: group of subjects with moderate-mild asthma. G: group of subjects with severe asthma.
[1206] Global evaluation of results of protein biomarkers to discriminate clinical phenotypes from each other.
[1207]
[1208] Based on the analysis of individual and grouped ROC curves of the 8 protein biomarkers studied, the best Biomarker options for each clinical condition analyzed would be those detailed below.
[1209]
[1210] 1. Biomarkers capable of discriminating ANA (non-allergic asthmatics) from Allergic Asthmatics (AA)
[1211] AUC CHI3L1 0.74
[1212] IL10 + Periostin 0.87
[1213] CHI3L1 + SERPINB2 0.83
[1214] CHI3L1 + IL8 0.82
[1215] IL8 + SERPINB2 0.82
[1216] PHLDA1 + SERPINB2 0.80
[1217] IL8 + Periostin 0.76
[1218] CHI3L1 + IL10 + Periostin 0.92
[1219] CHI3L1 + IL8 + Periostin 0.88
[1220] PI3 + PHLDA1 + SERPINB2 0.84
[1221] CHI3L1 + PI3 + Periostin 0.80
[1222]
[1223] 2. Biomarkers capable of discriminating severe ANA (non-allergic asthmatic) from severe Allergic Asthmatics (AA).
[1224]
[1225] AUC CHI3L1 0.82
[1226] SERPINB2 0.78
[1227] IL10 + Periostin 0.94
[1228] PI3 + SERPINB2 0.92
[1229] CHI3L1 + IL8 0.86
[1230] CHI3L1 + Periostin 0.86
[1231] IL8 + Periostin 0.86
[1232] CHI3L1 + IL8 + Periostin 0.96
[1233] 3. Biomarkers capable of discriminating ANA (Non-allergic asthmatics) Mod / mild of Allergic asthmatics (AA) Mod / mild.
[1234] AUC CHI3L1 + IL8 0.86
[1235] IL10 + Periostin 0.82
[1236] IL8 + PI3 0.82
[1237] IL8 + Periostin 0.82
[1238] CHI3L1 + IL10 + Periostin 0.90
[1239] CHI3L1 + PI3 + Periostin 0.80
[1240]
[1241] 4. Biomarkers capable of discriminating AA (Allergic Asthmatics) from Allergic (A).
[1242] AUC IL8 0.88
[1243] Periostin 0.8
[1244] SERPINB2 0.93
[1245] CHI3L1 + SERPINB2 1
[1246] PI3 + SERPINB2 0.96
[1247] CHI3L1 + IL8 0.91
[1248] CHI3L1 + Periostin 0.81
[1249] IL8 + PI3 + Periostin 0.93
[1250]
[1251] 5. Biomarkers capable of discriminating AA (Serum Allergic) from Allergic (A).
[1252] AUC IL8 0.80
[1253] Periostin 0.77
[1254]
[1255] IL8 + Periostin 0.90
[1256] IL8 + PI3 + Periostin 0.98
[1257] 6. Biomarkers capable of discriminating AA (Allergic Asthmatics) Mod / mild Allergic (A).
[1258] AUC IL8 0.96
[1259] Periostin 0.83
[1260] SERPINB2 0.84
[1261] PI3 + SERPINB2 0.92

权利要求:
Claims (17)
[1]
1. In vitro use of the concentration levels of the SERPINB2 protein biomarker in a biological sample isolated from blood, serum or plasma to determine the risk of suffering from asthma in a human subject.
[2]
2. In vitro method to select subjects at risk of suffering from asthma, in particular distinguishing the severity of asthma, more particularly distinguishing between different asthma phenotypes, comprising: (a) measuring the pattern or concentration of at least the SERPINB2 protein, obtained from a biological sample isolated from blood, serum or plasma, of the subjects to be selected; and (b) comparing said pattern or concentration of at least the SERPINB2 protein of the subjects to be selected with a standard or concentration already established, where differences in concentration of at least the SERPINB2 protein is indicative that said subject presents a risk of suffering asthma, in particular distinguishing the severity of asthma, more particularly distinguishing between different asthma phenotypes.
[3]
3. In vitro method according to claim 2, wherein said method is for selecting subjects at risk of suffering from non-allergic asthma, comprising: (a) measuring the pattern or concentration of at least the SERPINB2 protein, obtained from a biological sample isolated from blood, serum or plasma, of the subjects to be selected; and (b) comparing said standard or concentration of at least the SERPINB2 protein, of the subjects to be selected with a standard or level of expression already established based on healthy controls subjects, where a reduced concentration level of at least the SERPINB2 protein, is indicative that said subject presents a risk of suffering from non-allergic asthma.
[4]
In vitro method according to claim 2, wherein said method is for selecting subjects at risk of suffering from allergic asthma, comprising: (a) measuring the pattern or concentration of at least the SERPINB2 protein, obtained from a sample biological isolation of blood, serum or plasma, of the subjects to be selected; and (b) comparing said pattern or concentration of at least the SERPINB2 protein, of the subjects to be selected with a standard or level of expression already established based on healthy controls subjects, where a concentration level reduced of at least the SERPINB2 protein, is indicative that said subject presents a risk of suffering allergic asthma.
[5]
5. In vitro method according to claim 2, wherein said method is for selecting subjects at risk of suffering intermittent or persistent mild / moderate non-allergic asthma of subjects at risk of suffering from severe non-allergic asthma, comprising: (a) measuring the pattern or concentration of at least the protein SERPINB2 and POSTN and / or PI3, obtained from a biological sample isolated from blood, serum or plasma, of the subjects to be selected; and (b) comparing said standard or concentration of at least the SERPINB2 protein, of the subjects to be selected with a standard or level of expression already established based on subjects suffering from severe non-allergic asthma, where differences in concentration of at least the protein SERPINB2 and POSTN and / or PI3, is indicative that said subject presents a risk of suffering one of said clinical subfenotypes of non-allergic asthma.
[6]
6. In vitro method according to claim 2, wherein said method is for selecting subjects at risk of suffering intermittent or persistent allergic mild / moderate asthma of subjects at risk of suffering from severe allergic asthma, comprising: (a) measuring the pattern or concentration of at least the protein SERPINB2 and POSTN and / or PI3 and / or CHI3L1, obtained from a biological sample isolated from blood, serum or plasma, of the subjects to be selected; and (b) comparing said pattern or concentration of at least the SERPINB2 and POSTN and / or PI3 and / or CHI3L1 protein of the subjects to be selected with an established pattern or level of expression based on subjects suffering from severe allergic asthma, where differences in concentration of at least the protein SERPINB2 and POSTN and / or PI3 and / or CHI3L1, is indicative that said subject presents a risk of suffering from one of said clinical subfenotypes of allergic asthma.
[7]
7. In vitro method according to claim 2, wherein said method is for selecting subjects at risk of suffering from non-allergic asthma of subjects at risk of suffering from allergic asthma, comprising: (a) measuring the pattern or concentration of at least the SERPINB2 protein and optionally periostin and CHI3L1 or PI3, PHLDA1 and IL8, obtained from a biological sample isolated from blood, serum or plasma, of the subjects to be selected; and (b) compare said pattern or concentration to minus the SERPINB2 protein and optionally periostin and CHI3L1 or PI3, PHLDA1 and IL8, of the subjects to be selected with a standard or level of expression already established based on subjects suffering from allergic asthma, where an increased level of concentration of at least the SERPINB2 protein in combination with a decreased level of the CHI3L1 or PI3 proteins, or in combination with an increased level of the PHLDA1 protein, or in combination with an increased level of the IL8 protein; it is indicative that said subject presents a risk of suffering non-allergic asthma; Y
where:
to. a reduced concentration level of at least the SERPINB2 protein and increased of the CHI3L1 or PI3 proteins, or a reduced concentration level of at least the SERPINB2 protein and the PHLDA1 protein; or b. a reduced concentration level of the SERPINB2 protein and decreased periostin and increased of the CHI3L1 or PI3 proteins, or a reduced concentration level of the SERPINB2 protein and decreased of periostin and decreased of the PHLDA1 protein;
it is indicative that said subject presents a risk of suffering from allergic asthma.
[8]
8. In vitro method according to claim 2, wherein said method is for selecting subjects at risk of suffering from severe non-allergic asthma of severe Allergic Asthmatics (AA), comprising: (a) measuring the pattern or level of expression of the minus the protein SERPINB2 and PI3, obtained from a biological sample isolated from blood, serum or plasma, of the subjects to be selected; and (b) comparing said pattern or level of expression of at least the protein SERPINB2 and PI3, of the subjects to be selected with a pattern or level of expression already established based on severe Allergic (AA) asthmatics, where an increased concentration level of the protein SERPINB2 and decreased PI3 with this pattern or level of expression already established is indicative of risk that the individual suffers from severe nonallergic asthma; and where a decreased concentration level of the SERPINB2 protein and increased PI3 with said standard or level of expression already established is indicative of the risk that the individual suffers from severe allergic asthma.
[9]
In vitro method according to claim 2, wherein said method is for selecting subjects with allergic asthma from allergic subjects without asthma comprising: (a) measuring the pattern or level of expression of at least the protein SERPINB2 and PI3 and / or CHI3L1, obtained from a biological sample isolated from blood, serum or plasma, of the subjects to be selected; and (b) comparing said pattern or level of expression of at least the protein SERPINB2 and PI3 or CHI3L1, of the subjects to be selected with a standard or level of expression already established based on allergic subjects without asthma, where a reduced concentration level of the protein SERPINB2 and decreased PI3 or increased CHI3L1 with such pattern or level of expression is indicative that the individual suffers from allergic asthma.
[10]
10. In vitro method according to claim 2, wherein said method is for selecting subjects with intermittent allergic asthma or mild / moderate persistence of allergic subjects without asthma comprising: (a) measuring the pattern or level of expression of at least the SERPINB2 and PI3 protein, obtained from a biological sample isolated from blood, serum or plasma, of the subjects to be selected; and (b) comparing said pattern or level of expression of at least the SERPINB2 protein and optionally PI3, of the subjects to be selected with a standard or level of expression already established based on allergic subjects without asthma, where a reduced concentration level of the SERPINB2 protein and optionally decreased PI3 with said pattern or level of expression is indicative that the individual suffers from intermittent allergic asthma or persist mild / moderate.
[11]
An in vitro method according to claim 2, wherein said method is for selecting subjects with severe allergic asthma from allergic subjects without asthma comprising: (a) measuring the pattern or level of expression of at least the protein SERPINB2 and CHI3L1 and / or PI3 and / or Periostina and / or IL8, obtained from a biological sample isolated from blood, serum or plasma, of the subjects to be selected; and (b) comparing said pattern or level of expression of at least the SERPINB2 protein and and CHI3L1 and / or PI3 and / or Periostin and / or IL8, of the subjects to be selected with an established pattern or level of expression based on allergic subjects. without asthma, where a reduced concentration level of the SERPINB2 protein and decreased of PI3 and / or increased of CHI3L1 and / or increased periostin and / or decreased of IL8 with said The pattern or level of expression is indicative that the individual suffers from severe allergic asthma.
[12]
12. In vitro method for the diagnosis / prognosis of a subject suspected of having asthma, comprising steps a) and b) of any of the methods of claims 2 to 11, and optionally (c) confirming the presence of a certain asthma phenotype and / or the severity of the disease by means of a clinical examination.
[13]
13. Method for obtaining data useful for the in vitro diagnosis / prognosis of asthma, comprising steps a) and b) of any of the methods of claims 2 to 11.
[14]
14. In vitro method for classifying subjects as healthy subjects or as subjects suffering from asthma or between different phenotypes of the disease, comprising steps a) and b) of any of the methods of claims 2 to 11.
[15]
15. In vitro method for monitoring the response to a therapy or for monitoring the progression of asthma, in a subject suffering from said disease, comprising steps a) and b) of any of the methods of claims 2 to 11.
[16]
16. Method according to any of claims 2 to 14, wherein the subject is a human subject.
[17]
17. Use of a kit comprising reagents that detect biomarkers to determine a level of differential expression of at least the SERPINB2 protein in a biological sample of blood, serum or plasma, where differences in expression of the SERPINB2 protein is indicative of risk of having asthma, in particular distinguishing the severity of asthma, more particularly distinguishing between different asthma phenotypes.

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同族专利:

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公开号 | 公开日

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ES2697299B2|2020-04-15|

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WO2019016426A1|2019-01-24|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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WO2009124090A1|2008-03-31|2009-10-08|Genentech, Inc.|Compositions and methods for treating and diagnosing asthma|

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PCT/ES2018/070515| WO2019016426A1|2017-07-19|2018-07-19|Differential biomarkers for asthma|