Pilot study: Biomarkers for the diagnosis of severe neutrophil inflammatory asthma.

About 10% of all asthmatics have difficult-to-treat/refractory asthma (=severe
asthma), which is characterised by uncontrolled asthma, even when high doses of
inhaled corticosteroids and a second controller are administered. Severe asthma
is associated with increased neutrophils and T helper (Th) 17 chemokine
overexpression in bronchial biopsies.
Phenotyping of patients is important to optimize therapy and disease
outcome. Whereas eosinophilic asthma patients respond well to anti-inflammatory
treatment with steroids, severe eosinophilic and non-eosinophilic asthma
patients (including neutrophilic asthma patients) show inadequate response. For
non-eosinophilic/non-allergic (neutrophilic) asthma therapeutic options are
focused on targeting the Th17/IL-17 route.; i.e. treatment with macrolide
antibiotics. For severe eosinophilic (with or without allergy) anti-IL-5 or
anti-IgE might be therapeutic options.
In the Netherlands, asthma affects up to 3.5% of the adults and 4% of
the children below the age of 15 yrs. In 2007 the overall health expenditure
was ¤ 300 million. Half of these costs involve medication, i.e. combination
preparations (a long-acting-sympathomimetic agent with inhaled
corticosteroids). The annual direct medical expenditures and indirect
nonmedical costs range from around ¤500 for controlled asthma to ¤2281 for
uncontrolled asthma.
Since severe asthma burdens on overall health costs, early phenotypic
classification of severe asthma can guide the choice of the most appropriate
therapy with a reduction of exacerbations and health costs.
Until recently, cell analysis in sputum induction was used for asthma
phenotyping. However, this is an expensive technique, not widely available and
is uncomfortable for the patient. For eosinophilic/allergic asthma non-invasive
diagnostic test are available; e.g. blood eosinophils, serum IgE/Skin Prick
Test, exhaled nitric oxide. However, routine non-invasive laboratory tests are
not available for the diagnosis of patients with neutrophilic asthma.
Furthermore, it is unknown which patients with non-eosinophilic/non-allergic
asthma will respond well to macrolide therapy. Asthma is more and more
considered to be an inflammatory disease of both the lower and upper airways.
This is also reflected in multiple publications showing a significant
correlation between the cytokine profiles in induced sputum/bronchial biopsy
and nasal biopsy, showing that the nose may be considered the window of bronchi
in asthma. A recent study by Sorbello et al. (2015) showed for
uncontrolled/severe asthma, in contrast to mild asthma and controls, increased
neutrophils/IL-17 in nasal/bronchial biopsies. Furthermore, significant
correlations were observed between bronchial IL-17 and neutrophils, nasal IL-17
and bronchial neutrophil/IL-17, suggesting that nasal IL-17 might be
informative on bronchial IL-17-driven neutrophilia. Albano et al (2013) showed
that levels of IL-17 were higher in induced sputum and nasal lavage of children
with mild-moderate astma compared to intermittent asthma and healthy controls.
Little et al. (2014) showed that even saliva might be suitable for phenotyping
patients with asthma since salivary myeloid markers (like IL-8) were associated
with disease control and exacerbations. As nasal lavage and saliva collection
are less invasive procedures compared to sputum induction, these body fluids
are of particular interest for further research.

The aim of this pilot study is to validate potential biomarkers which diagnose
patients who will benefit from asthma treatment with macrolide therapy by using
non-invasively collected body fluid specimens. Th1/Th2/Th17 patterns in
patients suffering from severe eosinophilic and/or allergic asthma and
non-eosinophilic/non-allergic asthma are compared. The group of
non-eosinophilic/non-allergic asthma patients will be tested twice, before and
after treatment with macrolide therapy during 12 weeks (standard therapeutic
procedure). Th1/Th2/Th17 patterns are determined by analysing cytokine
concentrations (Th1: IL-2, TNF, IFNγ; Th2: IL-4, IL-6, IL-10; Th17: IL-17A) in
nasal fluid (nasal wash and cotton wool method), saliva and blood. The results
of these factors in the asthma subpopulations will also be compared to the
results found in the pilot study with healthy controls. The pilot study with
healthy controls (METC nr NL56960.091.16) was conducted recently.

The study is an observational pilot study with restricted invasive treatment.
The study involves 20 patients suffering from severe eosinophilic and/or
allergic asthma, and 20 patients suffering from severe
non-eosinophilic/non-allergic asthma (eligible for macrolide therapy). The
group of non-eosinophilic/non-allergic asthma patients will be tested twice,
before and after treatment with macrolide therapy during 12 weeks. All patients
are 18 years or older and mentally competent. Subjects will visit the
department of pulmonary medicine of the Rijnstate Hospital in Arnhem. The visit
will take 30 to 45 minutes. During the visit blood, nasal fluid (two different
methods) and saliva (one tube per fluid per time) will be collected. The fluids
will be processed and stored until analysis of the Th1/Th2/Th17 patterns. In
addition, C-reactive protein (CRP) will be measured in the blood to exclude an
acute phase and total protein in nasal fluid and saliva will be measured to
express cytokine levels on the total protein content.

Subjects have to donate a small amount of nasal fluid, saliva and blood in a
certified hospital environment (KHCL Rijnstate Arnhem). Risks of participation
include the regular risks involved in the sampling procedures; i.e. pain and
bruises (for blood sampling), irritation and a dry feeling in the nose (for the
collection of nasal fluid).