EVALUATION OF EFFECTIVENESS OF ALLERGEN-SPECIFIC SUBCUTANEOUS IMMUNOTHERAPY WITH HOUSE DUST MITES FOR PATIENTS WITH ALLERGIC AIRWAY DISEASES

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EVALUATION OF EFFECTIVENESS OF ALLERGEN-SPECIFIC

SUBCUTANEOUS IMMUNOTHERAPY WITH HOUSE DUST MITES FOR

PATIENTS WITH ALLERGIC AIRWAY DISEASES

by Long Chen Sun

A Thesis Presented to The Faculty of Medicine

Department of Immunology and Allergology supervised by Simona Kašinskaitė, Doctor of Medicine

consulted by Ieva Bajoriūnienė, Doctor of Science prepared in Kaunas, Lithuania 2018-2020.

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1. SUMMARY

Author´s name and surname: Long Chen Sun

Research title: Evaluation of effectiveness of allergen-specific subcutaneous immunotherapy with house dust mites for patients with allergic airway diseases.

Aim: To evaluate the effectiveness of house dust mite allergen-specific subcutaneous immunotherapy for patients diagnosed with allergic airway diseases caused by house dust mites and compare effectiveness between different groups of patients.

Objectives:

1) To evaluate the effectiveness of SCIT with HDM for different allergic airway diseases caused by HDM and compare it between patients of different demographic characteristics.

2) To evaluate the responsiveness to SCIT with HDM for patients with AR with and without AA caused by HDM.

3) To determine the time point of SCIT effectiveness with HDM in patients with allergic airway diseases caused by HMD.

4) To analyze and compare the effectiveness between single (HDM) allergen SCIT for monosensitized and polysensitized patients, and multiple allergen SCIT for polysensitized patients with allergic airway diseases.

Methodology and study participants: This retrospective observational study was performed in the Immunology and Allergology Department of Hospital of Lithuanian University of Health Sciences, Kaunas Clinics. A total of 40 patients were included in this research. Patients’ data was used to evaluate the effectiveness of SCIT by comparing medication scores before and during the process of undergoing SCIT with HMD.

Results: Most of the patients after undergoing HDM SCIT experienced a significant improvement on AR MS (p<0.05). Benefits on AR MS were mostly seen in patients being under HDM SCIT for more than 7 months. HDM SCIT benefits on AR MS were found to be superior on male patients over female patients. No significant differences were found between adults and children or between patients with AA and patients without AA. On the contrary, HMD SCIT did not reduce AA medication scores significantly (p>0.05) and no differences were found between subgroups.

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Conclusions:

HDM SCIT is effective on reducing AR MS for patients with AR (especially on male patients over female patients), but is not effective on reducing AA MS on patients with AA.

There are no significant differences in terms of HDM SCIT effectiveness for allergic rhinitis treatment between patients with allergic rhinitis only and patients with co-existing asthma.

HDM SCIT starts to be effective on reducing AR MS after 7-12 months of immunotherapy.

Single-allergen (HDM) SCIT is equally effective for monosensitized and polysensitized patients with allergic airway diseases.

Key words: House dust mite; subcutaneous allergen-specific immunotherapy; allergic asthma; allergic rhinitis; effectiveness.

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2. ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to my supervisor Simona Kašinskaitė for her guidance and suggestions throughout the process of writing this final master thesis.

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3. CONFLICT OF INTEREST

The author reports no conflicts of interest.

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4. ETHICS COMITTE APPROVAL

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5. ABBREVIATIONS

AIT Allergen-specific immunotherapy

SCIT Allergen-specific subcutaneous immunotherapy SLIT Allergen-specific sublingual immunotherapy AR Allergic rhinitis

AA Allergic asthma

ARC Allergic Rhinoconjunctivitis TLSP Thymic stromal lymphopoietin AECs Airway epithelial cells

ILC2s Type 2 innate lymphoid cells Bregs Regulatory B cells

Tregs Regulatory T cells

TGF- Transforming growth factor beta Th1 Type 1 T helper

Th2 Type 2 T helper LPR Late phase response EPR Early phase response

HDM House dust mites (D. pteronyssimus + D.farinae)

D. pteronyssinus Dermatophagoides pteronyssimus D. farinae Dermatophagoides farinae

RCTs Randomized controlled trials

SS Symptom score

MS Medication score

CSMS Combined symptom medication score HRQoL Health-related quality of life

ICS Inhaled corticosteroids OCS Oral corticosteroids LABA Long-acting beta2-agonist SABA Short-acting beta2-agonist LTRA Leukotriene receptor antagonist SD Standard deviation

µ Mean

WSRT Wilcoxon Signed-Rank test MWUT Mann-Whitney U test

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6. TERMS

 Endotype: subtype of a condition defined by a distinct pathophysiologic mechanism 1.

 Phenotype: observable clinical manifestations that result from a combination of hereditary and environmental influences 1.

 Alarmins: epithelial-derived mediators that initiate multiple type 2 signaling pathways in response to infection and allergen-driven inflammation. Examples are thymic stromal lymphopoietin, IL-25, and IL-33 [2].

 Type 2 innate lymphoid cells: group cells of the innate immune system that belong to the lymphoid lineage. These cells do not have antigen specific receptors nor surface makers, but when they are activated, they produce large amounts of IL-5 and IL-13, stimulating type-2 immune responses [2].

 Monosensitized: State of testing positive in allergy skin tests to one allergen. In this study, patients testing positive for D. pteronyssinus and/or D. farinae were considered to be monosensitized.

 Polysensitized: State of testing positive in allergy skin tests to more than one allergen. In this study, patients who tested positive for HDM and any other allergen(s) were considered polysensitized.

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7. INTRODUCTION

Allergic asthma (AA) and allergic rhinitis (AR) are allergic airway diseases that have been increasing in prevalence over the past few years 3, thus it is important to find effective measures for their management and prevention.

AA and AR management and prevention involves allergen avoidance, pharmacotherapy and allergen specific immunotherapy (AIT). Some allergens, like house dust mites, are very difficult to avoid. Drugs used for these diseases (such as antihistamines and corticosteroids) only reduce symptoms and do not change the course of the disease. This makes patients with perennial allergies completely dependent on these medications during the whole year. AIT remains as the only disease-modifying therapy for these allergic diseases, being able to reduce symptoms and the need of medications 4]. Furthermore, AIT also provides other benefits such as improving quality of life, prevention of asthma development and other long-term benefits [5]. Despite these facts, it is still relatively underused probably due to reasons such as lack of awareness of general population or not enough evidence supporting its cost-effectiveness [6,7,8].

AIT is a type of treatment that involves the administration of gradually increased allergens to desensitize the immune system and decrease symptoms caused by subsequent exposure to allergens 9. There are two routes of administration of AIT: allergen-specific subcutaneous immunotherapy (SCIT) and allergen-specific sublingual immunotherapy (SLIT).

The aim of this study is to evaluate the effectiveness of HDM allergen-specific subcutaneous immunotherapy for patients diagnosed with allergic airway diseases caused by house dust mites and compare the effectiveness between different groups of patients.

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8. AIMS AND OBJECTIVES OF THE THESIS

Aim: To evaluate the effectiveness of HDM allergen-specific subcutaneous immunotherapy for patients diagnosed with allergic airway diseases caused by house dust mites and compare the effectiveness between different groups of patients.

Objectives:

1) To evaluate the effectiveness of allergen-specific subcutaneous immunotherapy with house dust mites for different allergic airway diseases caused by house dust mites and compare it between patients of different demographic characteristics.

2) To evaluate and compare the responsiveness to allergen-specific subcutaneous immunotherapy with house dust mites for patients with allergic rhinitis with and without allergic asthma caused by house dust mites.

3) To determine the time point of allergen-specific subcutaneous immunotherapy efficacy with house dust mites in patients with allergic airway diseases caused by house dust mites.

4) To analyze and compare the effectiveness between single-allergen (House dust mite) specific subcutaneous immunotherapy for monosensitized and polysensitized patients, and multiple allergen-specific subcutaneous immunotherapy for polysensitized patients.

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9. LITERATURE REVIEW

9.1. Pathogenesis of allergic rhinitis

The pathogenesis of AR is complex and consists of several steps 10. Firstly, it requires a sensitization, which happens during the first time an allergic person is exposed to an allergen (pollen, mites, animal dander, pollutants and fungal allergens being the most prevalent 10).

This allergen is uptaken and processed by an antigen presenting cell (dendritic cell) which presents it to a naïve T cell. This cell activates and differentiates into Type 2 T-helper (Th2) cell through the release of Th2-driving cytokines (IL-4, IL-13) from dendritic cells. Other cells and cytokines are also involved in the stimulation of Th2 immune response (mentioned in the section “pathogenesis of asthma”). Th2 cells stimulate B lymphocytes to undergo immunoglobulin isotype switching resulting in their differentiation into allergen-specific IgE-producing plasma cells. Released allergen-specific IgE will attach to basophils and mast cells waiting for a secondary exposure to the sensitized allergen. Upon reexposure, allergen is bound to ¨IgE-coated cells¨, resulting in their activation and release of inflammatory mediators such as histamine and leukotrienes. These mediators can directly activate sensory nerve-endings, blood vessels and glands, causing increased vascular permeability, vasodilation, pruritus and nasal gland secretion 10. This results in typical symptoms of the disease (sneezing, itching, nasal congestion, rhinorrhea and watery eyes) 11.

9.2. Pathogenesis of asthma

Regarding asthma, traditionally its pathogenesis was accepted to be due to excessive Th2 cell response and specific-IgE caused airway hyperresponsiveness. Until a few years ago, all asthma patients were treated universally. However, a small percentage of these patients were found to not respond despite high-intensity therapy 2.

According to recent studies the term ¨asthma¨ is now being considered an umbrella diagnosis of different endotypes and phenotypes as opposed to being a single disease. All of them manifest with symptoms of wheezing, shortness of breath, cough, chest tightness and with a variable airflow obstruction2.

Currently there are two endotypes and each of them can be subclassified according to their phenotypes: 2, 12

 Th2-high (eosinophilic): atopic (AA), late onset, aspirin exacerbated respiratory diseases.  Th2-low (non-eosinophilic): non-atopic, smokers, obesity, elderly.

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13 Th2-high endotype involves Type 2 immune pathways and is similar to AR pathogenesis.

Most asthmatics are predisposed to suffer from the disease by having a pathological epithelial barrier with reduced amount of proteins (E-cadherin and claudin-18) that regulate tight junctions, which facilitates the invasion of allergens to our stroma tissues. Once the allergen is detected by airway epithelial cells (innate immune system) through pattern recognition receptors, they release alarmins (thymic stromal lymphopoietin, IL-25, and IL-33) which participates in the activation of Th2 immune pathways.

Thymic stromal lymphopoietin (TSLP) activates and orients dendritic cells to stimulate a Th2 immune response. Activated dendritic cell presents the allergen to a naïve T cell which differentiates into Th2 cell and proliferates. Th2 cells promotes the differentiation of B lymphocytes into allergen-specific IgE producing plasma cells. Secreted IgE will be attached to mast cells, basophils waiting for a reexposure of the allergen.

On the other hand, IL-25 and IL-33 can activate type 2 innate lymphoid cells (ILC2s), which are a group of cells that once activated produce IL-4, IL-5 and IL-13 to stimulate a Th2 immune response. IL-5 promotes eosinophil differentiation and survival. The set of IL-13, IL-4, and inflammatory mediators from mast cells, basophils, and eosinophils stimulates airway hyperresponsiveness, smooth muscle hypertrophy, and airway remodeling 2.

Conversely, Th2-low endotype is relatively under-studied. It is characterized by absence of markers of Th2-high disease (eosinophilia). Th2-low asthma can be classified into neutrophilic (sputum neutrophils>40-60%) and paucigranulocytic (normal sputum levels of both eosinophils and neutrophils) inflammation and a lack of response to corticosteroid therapy. This type of asthma has been linked with the activation of Th1 and Th17 cells.

Currently there is a lack of effective treatment for these patients and new treatment strategies are required for management 13.

9.3. Allergic rhinitis and asthma association

Patients with allergic rhinitis tend to develop asthma. In 2019, a meta-analysis that analyzed 22 cohort studies and 7 case-control studies with a total of 274489 subjects, concluded that allergic rhinitis is strongly associated with allergic asthma 14.

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• The ¨atopic march¨ hypothesis considers atopic diseases as set of consecutive clinical manifestations that begins with atopic dermatitis, and progresses with atopic rhinitis and asthma 15.

• The ¨one airway, one disease¨ hypothesis considers allergic rhinitis and asthma as an united airway disease. This concept justifies the strong association between both allergic diseases as a manifestation of a single disease in different parts of the airway. It also proposes that any disease process that affects the upper airway is more likely to affect the lower airway and vice versa 16, 17.

9.4. Allergen specific immunotherapy mechanism

Despite the recent progress in the field of immunology, the mechanism of AIT is not totally understood yet and still under investigation 9, 18. Currently, it is divided in several parts:

1) An early phase characterized by a rapid desensitization of mast cells and basophils to environmental allergens. This occurs several hours after the first injection 19.

2) Production and activation of B and T regulatory cells (Bregs, Tregs). These cells produce IL-10 which helps Tregs to differentiate and suppresses proinflammatory responses. The balance between these cells together with type 1 and type 2 T helper (Th1, Th2) cells directly influences in the type of immune response towards an allergen (therefore in the development or not of an allergy). In healthy individuals, the group that predominates against common environmental allergen is formed by Tregs and Bregs, whereas in allergic people, it is Th2. Besides IL-10 there are several factors that participate in the peripheral tolerance to allergens like TGF-, cytotoxic T lymphocyte antigen 4 and programmed cell death protein 1 9, 18, 20, 21.

3) Regulation of antibody isotypes. Initially, there is an early and transient increase in levels of IgE, which rarely manifests with any symptoms and it is followed by a progressive decrease after the next months-years of continued treatment. On the other hand, during the course of AIT, IgG4 levels also increases due to the secretion of IL-10 and TGF- that stimulates B cells to switch class, suppressing the synthesis of IgE and promoting the synthesis of IgG4. IgG4 competes with IgE to bind with allergens, thus having an appropriate IgE/IgG ratio is also a key factor in the prevention of activation of mast cells and basophils 9, 18, 19, 21.

4) Late-phase response (LPR) suppression. LPR differs from an early-phase response (EPR) in that LPR involves the activation, recruitment and maintenance of eosinophils and T cells at sites of exposure like eyes, skin, nasal or bronchial mucosa while EPR consists of quick release of mast

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15 9.5. AIT Efficacy

There is a large scientific evidence that supports the effectiveness of AIT (whether administered subcutaneously or sublingually) for treatment of AR and AA.

In 2017, Elliot et al. 22 published an umbrella review of 23 systematic reviews of randomized controlled trials (RCTs) of AIT for AR and AA. The patients receiving AIT (either SCIT or SLIT) showed markedly reduced symptom score (SS), medication score (MS) and combined symptom-medication score (CSMS) compared to those receiving placebo.

In 2017 Dhami et al. made a systematic review and meta-analysis of 134 RCTs including patients with allergic rhinitis or allergic rhinoconjunctivitis (ARC) that underwent AIT. This study stated that AIT (either SCIT or SLIT) is clearly effective for patients with ARC, improving their SS, MS and CSMS over the short term (long term benefits were not as clear) 23. On the same year, the authors repeated the same type of study but analyzing 89 double-blind RCTs with patients suffering from AA and receiving AIT. Patients improved in SS and MS, but not in CSMS (this benefit was clear for SCIT but questionable for SLIT). Patients undergoing SCIT also showed an improvement of asthma-related quality of life, as well as a decrease in allergen-specific airway hyperreactivity. On the contrary, AIT (both SCIT and SLIT) was associated with a moderate increased risk of developing adverse reactions 24.

Another systematic overview of systemic reviews (2017) of AIT for AA concluded that SCIT improves medication and SS significantly for asthmatic patients (SLIT did not show clear benefits), however there was no clear evidence of lung function improvement (for both SCIT or SLIT) [25]. Not only the efficacy of AIT for allergic diseases has been demonstrated by objective measures, but also by subjective measures like patients and physician’s perspective. In 2019, a cross-sectional study in Korea included 267 patients with AR, AA and/or dermatitis that were undergoing at least 1 year of SCIT. During this period, researchers asked patients (as well as their respective physicians) to answer a questionnaire regarding their satisfaction with the therapy. The majority of patients and doctors reported to be satisfied with the efficacy and safety but not with the cost. Clinical symptoms, severity rates and medication prescription rates decreased significantly for AR and AA. This change was noticed even higher in the pediatric group compared to the adult group (all except clinical symptoms) [26].

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9.6. Comparison of AIT effectiveness for AA and AR between subgroups according to age, gender, allergic disease diagnosis, duration of immunotherapy

It seems like AIT is equally effective (no significant differences) between male and females, children and adults, patients with AR with AA and patients with AR without AA. However, the data from the literature it’s a bit controversial due to the limited amount of studies performed to compare the effectiveness of AIT between different subgroups according to age, gender, allergic disease diagnosis, etc…

In 2014, a retrospective cohort study in Israel evaluated the effectiveness of aeroallergen and hymenoptera subcutaneous immunotherapy on 232 patients with allergic airway diseases [27]. As a result, the study confirmed that immunotherapy was effective on reducing asthma and rhinitis

medications use. On top of that, Rottem et al. found no correlation between treatment efficacy and age, gender or patients´ primary allergic condition (AR with or without AA).

Two years after, Lourenço et al. published a retrospective study in Brazil, analyzing the effectiveness of subcutaneous immunotherapy in 281 patients with AR and AA. The authors demonstrated the efficacy of subcutaneous immunotherapy in terms of decreasing symptoms of itching, rhinorrhea, sneezing and nasal congestion. Additionally, no significant differences were found between both genders [28].

A two year-prospective observational study in Germany (2017) evaluated the effectiveness of SLIT with grass pollen tablet on patients with grass pollen-caused ARC. Shah-Hosseini et al. concluded that there was a significant reduction in symptoms and in medication use. Furthermore, they compared the effectiveness between different subgroups finding these results: no significant differences on ARC SS between children, adolescents and adults; patients without AA had a significantly higher reduction on ARC SS compared to those patients without AA [29].

In 2019, another two-year observational study that supports the efficacy of SCIT was performed in China [30]. This study included 58 house dust mite- allergic patients that were under subcutaneous immunotherapy during 2 years. Tu et al. found that patients had a significant reduction in AR and AA SS at 6, 12 and 24 months compared to baseline, and this reduction was statistically higher at 24 months compared to 6 months. Similarly, the reduction of MS in AR and AA was significantly higher in 6 months to baseline, 12 to 6 months and 24 to 12 months.

In 2018, Huang et al. [31] compared the long-term efficacy of HDM SCIT in pediatric and adult patients with AR. In this study, 118 patients were given HDM-SCIT for 3 years and followed-up for 2 more years. As a consequence, both groups improved their SS, CSMS and health-related quality of life (HRQoL) significantly at the end of the third year and fifth year compared to the baseline, and this difference was much higher in the pediatric group.

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17 9.7. Multiple allergen vs single-allergen immunotherapy in polysensitized and

monosensitized patient

Normally, monosensitized patients are treated by single-allergen immunotherapy due to ethical reasons and its proven efficacy [32], but there has been a debate whether polysensitized patients should be treated with single or multiple-allergen immunotherapy. In United States, multiple allergen immunotherapy tends to be the approach (due to convenient, epidemiologic and economic reasons), whereas in Europe single-allergen immunotherapy is preferred [33, 34]. The reasoning behind the European approach is that polysensitized patients are not necessarily polyallergic, thus it is only necessary treat the most relevant allergen. Also, if a patient experiences an adverse reaction, it is not possible to identify the causative allergen on a multiple-allergen immunotherapy [32, 35].

In terms of evidence, single-allergen immunotherapy seems to be equally effective for both monosensitized and polysensitized patients.

In 2016, a prospective, self-placebo controlled, parallel-group trial studied the effectiveness of HDM SCIT (using a single allergen) in monosensitized and polysensitized patients who had AR with or without AA. Soyyigit et al. [36] concluded that the efficacy of HDM SCIT was similar in both monosensitized and polysensitized patients, with differences in short-term immune changes.

Malling et al. [37] found similar results in their randomized double-blind, placebo-controlled trial which consisted of 628 patients receiving grass pollen sublingual tablets of different indexes of reactivity. As a result, no differences in terms of efficacy were found between the group of monosensitized patients and the group of polysensitized patients.

On the other hand, the effectiveness of multiple-allergen immunotherapy for polysensitized patients has been controversial and not proved yet due to the limited amount of studies performed [32, 35, 38]. One study supporting the efficacy of multiple-allergen immunotherapy was published in 2017. In this prospective randomized study, Wang et al. [39] compared the efficacy of single-allergen (HDM) SLIT vs multiple-allergen SCIT in polysensitized children with AR. The authors concluded that no significant differences were found between the efficacy of both immunotherapies.

Another retrospective study in 2017, found a higher remission rate in polysensitized patients (to HDM and any pollen) who were under SCIT with HDM only compared to polysensitized patients that underwent immunotherapy with multiple allergens. This difference was not statistically significant [40].

Furthermore, multiple-allergen immunotherapy has been associated with problems such as incompatibilities between the extracts (due the presence of proteolytic enzymes that decreases the potency of the main allergen) and dilution of the main extract to lower doses [41, 42].

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9.8. Effectiveness evaluation

There are many clinical parameters to evaluate the effectiveness of AIT for allergic diseases, some examples can be:

 Symptom score  Medication score

 Combined symptom-medication score  Health-related quality of life

 Patient and physician-rated satisfaction scales,  Visual analogue scales

 Allergen provocation tests  Functional measures

Due to this wide variety of clinical parameters, the lack of a validated method for measurement of these parameters and the lack of consensus, it is difficult to compare results from clinical trials. To solve this problem various organizations have announced some recommendations for future clinical trials.

One of them being the World Allergy Organization (WAO) taskforce whose recommendations (2007) were to use CSMS as primary outcome [43].

One year later, the European Medicine Agency (EMA) released a guideline on the clinical development of products for AIT where they stated that a primary endpoint should reflect both symptom severity as well as medication usage, but not specifying whether they should be combined or not.

Lastly, the European Academy of Allergy and Clinical Immunology (EAACI) published a literature review (2014) comparing different methods of efficacy evaluation of AIT for patients with ARC, with the aim of establishing an optimal outcome measure to be used. As a result, the EAACI taskforce not only recommended to use CSMS as primary endpoint for future RCTs for ARC, but also proposed a simple standardized method that measures SS and MS simultaneously in an equally weighted manner [44].

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10. RESEARCH METHODOLOGY AND METHODS

10.1. Study design and object of study

This retrospective observational study was performed at the Department of Immunology and Allergology of the Hospital of Lithuanian University of Health Sciences, Kaunas Clinics. A total of 40 patients with airway allergic diseases (AR, AA or both) that underwent SCIT using chemically modified allergens of HDM in Kaunas Clinics were included in this study. During the period in which patients were undergoing SCIT, a time point in autumn 2019 (between 01-10-2019 and 05-12-2019) was utilized to collect patients´ medical data by using their medical record to evaluate the effectiveness of SCIT. Autumn was chosen to avoid changes in MS that could happen in seasons where plants blossoming might impact polysensitized patients from the study. The study design was approved by the Ethics Committee of Lithuanian University of Health Sciences. Patients´ consent was obtained orally.

10.2. Participant selection

All patients diagnosed with a symptomatic allergic airway disease (AR and/or AA) that were undergoing SCIT at Kaunas Clinics were selected for this study. From those patients, only patients receiving SCIT with HDM (50% D. pteronyssimus and 50 % of D. farinae) or HDM mixture with other chemically modified allergens were included. A total of 40 patients met the inclusion criteria. Patients that were under HDM SCIT for less than 1 month were excluded from the study (4 patients met the exclusion criteria)

10.3. Research methods

All relevant clinical data was obtained anonymously through case histories from the Immunology and Allergology Department of Kaunas Clinics. Collected data for this study included: Patient’s gender, age, diagnosed allergic disease(s), allergen sensitization, used chemically modified allergen for immunotherapy and used medications for symptom control before and during SCIT. For each patient, used medications were recorded on a scale with different scores based on the type of drug. Clinical efficacy was evaluated by using these MS. Despite EAACI recommendations on using CMSS, symptom score was not used due to limited availability of information.

Used asthma medication score for this study was created based on GINA recommendations in 2018 for asthma treatment. This scoring system consisted of 5 total points: 1 point for patients not using any

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controller medication; 2 points for patients using daily low dose inhaled corticosteroids (ICS) or leukotriene receptor antagonist (LTRA); 3 points for patients taking low dose ICS and long-acting beta2-agonist (LABA); 4 points for patients using medium or high dose ICS and LABA; and 5 points for patients taking high dose ICS and LABA with a possible adjuvant therapy such as tiotropium or anti IgE. As reliever medication all patients were using short-acting beta2 agonist as they needed. This is explained thoroughly on Table 1.

Table 1. Asthma medication score

Score Controller Medication Alternative medication Reliever medication

1 SABA as needed

2 Low dose ICS LTRA SABA as needed

3 Low dose ICS+ LABA Medium dose ICS or low dose ICS+LTRA

SABA as needed 4 Medium or high dose

ICS+LABA Add Tiotropium or LTRA SABA as needed 5 High dose ICS+LABA±tiotropium or anti-IGE or anti-IL5 High dose

ICS+LABA+ low dose OCS

SABA as needed

Abbreviations: ICS inhaled corticosteroids; LTRA leukotriene receptor inhibitors; SABA short-acting beta2-agonist; LABA long-acting

beta2-agonist; OCS oral corticosteroids.

Allergic rhinitis medication score was created using ARIA guidelines for AR treatment and modified according to the clinical practice and treatment patterns used in Immunology and Allergology Department of the Hospital of Kaunas Clinics. AR MS consisted on a scale from 0 to 4 points: patients not using any medication were given 0 points; patients using oral antihistamines as needed were given 1 point; patients using peroral antihistamines daily were given 2 points; patients taking intranasal corticosteroids were given 3 points and patients taking intranasal corticosteroids and oral antihistamines or any other medication were given 4 points. This is summarized on Table 2.

Table 2. Allergic rhinitis medication score

Score Medication

0 No need of medications

1 Oral antihistamines as needed or intranasal H1-antihistamines and/or decongestant or LTRA 2 Oral antihistamines daily

3 Intranasal corticosteroids

4 Intranasal corticosteroids + oral antihistamines or any other medication

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21 SCIT with HMD only consisted exclusively of HDM chemically modified allergens (50 % D. farinae and 50% of D. pteronyssimus). On the contrary, SCIT with mixture of HDM and other chemically modified allergens was composed of HMD in different concentrations (varying from 25% to 70%) and other allergens (such as mugwort, birch pollen and cat dander).

In this study, patients that were sensitized to HDM (D. farinae and/or D. pteronyssimus) were

considered monosensitized, while patients that were sensitized to HDM and any other allergen(s) were considered to be polysensitized.

10.4. Methods of data analysis

Statistical analysis was performed using XLSTAT software. Studied variable for HDM SCIT effectiveness evaluation was categoric and ordinal (medication score), and it was analyzed and described by mean and standard deviation. Non-parametric tests were used over parametric tests because the data was not normally distributed.

Wilcoxon Signed-Rank test (WSRT) was used to evaluate intraindividual differences in medication score changes before and during/after immunotherapy. This test was chosen due to the presence of a dependent, categorical, ordinal variable (medication score) and an independent categorical variable which consists of matched pairs (before and during HDM SCIT). Differences between subgroups were calculated by using Mann-Whitney U test (MWUT). Differences were considered to be significant when p <0.05.

All patients were grouped according to:

• diagnosis (patients with allergic rhinitis without allergic asthma and patients with allergic rhinitis and allergic asthma)

• diseases (allergic rhinitis cases and asthma cases) • age (adults ≥18 years old, children<18 years old) • gender (male, female)

• duration of HDM SCIT (1-6 months, 7-12 months, 13-18 months, 19-24 months, >24 months) • sensitization (monosensitized, polysensitized)

• number of allergen extracts used for SCIT (only HDM or mixture of HDM and other chemically modified allergens)

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11. RESULTS

A total of forty patients underwent SCIT with house dust mites. All 40 patients were diagnosed with allergic rhinitis and 19 of them were diagnosed with allergic asthma as well. From those forty patients: thirty-six (90%) were adults and four were children (10%); eighteen (45%) were males and twenty-two (55%) were females; 6-12 months was the most common duration of SCIT (28%); fourteen were monosensitized (35%) and thirty-six were polysensitized (65%); the majority received SCIT with HDM allergoid (78%), and the rest got SCIT with a mixture of HDM and other allergoids (22%); twenty-one (52%) only had allergic rhinitis, while the rest (48%) had allergic rhinitis associated with allergic asthma. Further information is showed in Table 3.

Table 3. Patients´ demographic and baseline characteristics

Parameter Number of patients (%) Age Children (<18y) Adults (≥18y) 4 (10%) 36 (90%) Gender Male Female 18 (45%) 22 (55%) AIT duration 1-6 months 7-12 months 13-18 months 19-24 months >24 months 10 (25%) 11 (28%) 7 (18%) 4 (10%) 6 (15%) Sensitization Monosensitized Polysensitized 14 (35%) 26 (65%) Amount of allergen extracts

HDM only

Mixture of HDM and other chemically modified allergens 31 (78%) 9 (12%) Diagnosis Allergic rhinitis

Allergic rhinitis and asthma

21 (52%) 19 (48%)

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23 11.1. Clinical effectiveness assessment

Overall, there was a significant reduction (p<0.001) in medication score for allergic rhinitis in all patients before and during/after HDM SCIT, decreasing from a mean of 3.5±0.877 up to 2.375±1.580. Conversely, medication score for asthma did not show a significant difference (p>0.05) before and during/after the treatment. Specific details are represented on Table 4.

Table 4. Changes on medication score of both allergic airway diseases

Disease n MS μ±SD before HMD SCIT MS μ±SD after HDM SCIT MS mean improvement, μ±SD p value (WSRT) AR 40 3.5±0.877 2.375±1.580 1.125±1.488 0.0002 AA 19 2.278±0.826 2.167±0.857 0.111±0.471 0.317

Abbreviations: AR allergic rhinitis; AA allergic asthma; MS medication score; μ mean; SD standard deviation; HDM house dust mite; SCIT subcutaneous allergen specific immunotherapy; n number of patients; WSRT Wilcoxon Signed-Rank test.

11.2. Subgroup analysis by patients’ allergic disease diagnosis

As mentioned previously, allergic rhinitis seems to be strongly associated to asthma. From this study, 48% of patients had allergic rhinitis associated with asthma. Both subgroups showed a significant improvement in medication score for allergic rhinitis (p<0.05) as it is exposed on Table 5. No significant differences were found between patients with allergic rhinitis and patients with allergic rhinitis and asthma (p>0.05).

Table 5. Changes in medication score according to diagnosed allergic airway diseases

Diagnosis n AR MS μ±SD before HDM SCIT AR MS μ±SD after HDM SCIT MS mean improvement, μ±SD p value (WSRT) p value (MWUT) AR without AA 21 3.524±0.814 2.095±1.546 1.350±1.461 0.001 0.323 AR with AA 19 3.474±0.964 2.632±1.571 0.857 ±1.406 0.041

Abbreviations: AR allergic rhinitis; AA allergic asthma; MS medication score; μ mean; SD standard deviation; HDM house dust mite; SCIT subcutaneous allergen specific immunotherapy; n number of patients; WSRT Wilcoxon Signed-Rank test; MWUT Mann-Whitney U test. Intraindividual differences were calculated by Wilcoxon Signed-Rank test. Differences between subgroups were calculated by using Mann-Whitney U test.

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11.3. Subgroup analysis by age

Patients’ overall age was 29±9 years. There were 36 adults and only 4 children. Adults improved their medication score significantly on allergic rhinitis (p<0.05), while children did not (p>0.05). Both subgroups did not express significant changes in their medication score for asthma (p>0.05). No significant difference was observed between adults and children in both diseases (p>0.05). Details of changes in medication score according to age are presented on Table 6 and Table

7.

Table 6. Changes in AR medication score according to age

Age n AR MS μ±SD before HMD SCIT AR MS μ±SD after HDM SCIT MS mean improvement, μ±SD p value (WSRT) p value (MWUT) Adults 36 3.5±0.878 2.333±1.604 1.167±1.5 0.0004 0.970 Children 4 3.5±1 2.5±1.291 1±1.414 0.5

Abbreviations: AR allergic rhinitis; MS medication score; μ mean; SD standard deviation; HDM house dust mite; SCIT subcutaneous

allergen specific immunotherapy; n number of patients;WSRT Wilcoxon Signed-Rank test; MWUT Mann-Whitney U test. Intraindividual

differences were calculated by Wilcoxon Signed-Rank test. Differences between subgroups were calculated by using Mann-Whitney U test.

Table 7. Changes in asthma medication score according to age

Age n AA MS μ±SD before HMD SCIT AA MS μ±SD after HDM SCIT MS mean improvement, μ±SD p value (WSRT) p value (MWUT) Adults 16 2.250 ±0.856 2.188±0.911 0.063±0.443 1 0.217 Children 3 2.667±0.577 2.333±0.577 0.333±0.577 1

Abbreviations:AA allergic asthma; MS medication score; μ mean; SD standard deviation; HDM house dust mite; SCIT subcutaneous

11.4. Subgroup analysis by gender

Both gender subgroups showed a significant improvement on allergic rhinitis medication score after undergoing HDM SCIT (p<0.05), with a significant larger reduction in medication scores on male patients over female patients (p<0.05).

On the contrary, none improved significantly on asthma medication score (p>0.05), without significant differences between both subgroups (p>0.05). Changes in medication score according to gender can be seen on detail on

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Table 8. Changes in AR medication score according to gender

Gender n AR MS μ±SD before HMD SCIT AR MS μ±SD after HDM SCIT MS mean improvement, μ±SD p value (WSRT) p value (MWUT) Male 18 3.444±0.922 1.611±1.501 1.833±1.618 0.003 0.016 Female 22 3.545±0.858 2.955±1.362 0.590±1.098 0.016

Table 9. Changes in asthma medication score according to gender

Gender n AA MS μ±SD before HMD SCIT AA MS μ±SD after HDM SCIT MS mean improvement, μ±SD p value (WSRT) p value (MWUT) Male 8 2.375±0.744 2.125±0.835 0.25±0.463 0.5 0.170 Female 11 2.273±0.905 2.273±0.905 0±0.447 1

Abbreviations: AA allergic asthma; MS medication score; μ mean; SD standard deviation; HDM house dust mite; SCIT subcutaneous

11.5. Subgroup analysis by AIT duration

The duration of HDM SCIT in general was very heterogenous, ranging from one month (shortest) to thirty-six months (longest), with a mean around 13±9 months. Patients´ improvements on allergic rhinitis medication score appeared to be directly proportional to the duration of HDM SCIT (except for the subgroup of 19-24 months which might be due to the small number of patients belonging to this subgroup). This improvement was very small within 1 to 6 months of SCIT

(p=0.317) and started to be statistically significant from 7 to 12 months (p=0.041), peaking in patients over more than 24 months of HDM SCIT (p=0.026). Overall, patients that received more than a year of immunotherapy had a significant improvement over patients having undergone 1-6 months of immunotherapy.

On the other hand, asthma medication score did not improve significantly in any of the subgroups (p>0.05). Changes in medication score according to immunotherapy duration are further explained on

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Table 10. Changes in AR medication score according to immunotherapy duration AIT duration n AR MS μ±SD before HDM SCIT AR MS μ±SD after HDM SCIT MS mean improvement, μ±SD p value (WSRT) 1-6 months 10 3.4±0.966 3.1±1.197 0.3±0.949 0.317 7-12 months 11 3.467±0.834 2.733±1.335 0.733±1.163 0.041 13-18 months 7 3.5±0.837 1.833±1.722 1.667±1.366 0.041 19-24 months 4 4±0 3±1.732 1±1.732 0.317 >24 months 6 3.5±1.225 0.333±0.516 3.167±1.169 0.026

allergen specific immunotherapy; n number of patients; WSRT Wilcoxon Signed-Rank test.

Table 11 Changes on asthma medication score according to immunotherapy duration

AIT duration n Asthma MS μ±SD before HDM SCIT Asthma MS μ±SD after HDM SCIT MS mean improvement, μ±SD p value (WSRT) 1-6 months 6 2.333±1.033 2.333±1.033 0±0 1 7-12 months 7 2.429±0.787 2.429±0.535 0±0.577 1 13-18 months 2 1.5±0.707 1.5±0.707 0±0 1 19-24 months 2 2.5±0.707 2±1.414 0.5±0.707 0.317 >24 months 2 2.5±0.707 2±1.414 0.5±0.707 0.317

Abbreviations: MS medication score; μ mean; SD standard deviation; HDM house dust mite; SCIT subcutaneous allergen specific

immunotherapy; n number of patients; WSRT Wilcoxon Signed-Rank test.

11.6. Subgroup analysis by amount of allergen extracts

There were 31 patients receiving SCIT with HDM only and 9 patients undergoing SCIT with mixture of HDM and other allergoids. Significant improvement on allergic rhinitis medication score was reached by the subgroup receiving HDM only (p<0.05). On the contrary, patients receiving

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27 significantly (p>0.05). Asthma medication score did not change significantly for neither of the

subgroups (p>0.05). Differences between both subgroups for both diseases were not significant (p>0.05). Specific changes in medication score of these subgroups are exposed on Table 12 and Table

13.

Table 12. Changes in AR medication score according to amount of allergen extracts

Allergen n AR MS μ±SD before HDM SCIT AR MS μ±SD after HDM SCIT MS mean improvement, μ±SD p value (WSRT) p value (MWUT) HMD only 31 3.581±0.848 2.194±1.642 1.387±1.564 0.0004 0.073 Mixture of HDM and other chemically modified allergens 9 3.222±0.972 2.889±1.167 0.333±0.707 0.18

Table 13. Changes in asthma medication score according to amount of allergen extracts

Allergen n AA MS μ±SD before HDM SCIT AA MS μ±SD after HDM SCIT MS mean improvement, μ±SD p value (WSRT) p value (MWUT) HMD only 31 2.267±0.799 2.133±0.834 0.143±0.535 0.317 0.892 Mixture of HDM

and other chemically modified allergens

9 2.5±1 2.5±1 0±0 1

Abbreviations:AA allergic asthma; MS medication score; μ mean; SD standard deviation; HDM house dust mite; SCIT subcutaneous

11.7. Subgroup analysis by amount of allergen extracts and patient sensitization

There were 14 patients only sensitized to D. pteronyssinus or D. farinae (undergoing SCIT with HDM only) and 26 patients were sensitized to HDM and other chemically modified allergens. From these 26 patients, 17 underwent SCIT with HDM only and 9 received SCIT with a mixture of HDM and other chemically modified allergens. Monosensitized and polysensitized patients that

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received SCIT with only HDM showed a significant improvement in allergic rhinitis medication score (p<0.05) without significant differences between both subgroups (p>0.05). Additionally, none of these patients improved significantly in asthma medication score (p>0.05). On the contrary, polysensitized patients undergoing SCIT with HDM mixture and other chemically modified allergens did not improve their medication score on allergic rhinitis nor asthma (p>0.05). Furthermore, differences between polysensitized patients undergoing SCIT with only HDM and polysensitized patients undergoing SCIT with HDM mixture and other chemically modified allergens were statistically non-significant (p>0.05). All details are presented on Table 14.

Table 14 Changes on medication score according to sensitization and allergen number of AIT

Parameters Patient allergen sensitization

Monosensitized Polysensitized

Amount of allergen extracts HDM only HDM only HDM mixture with other chemically modified

allergens

Number of patients with AR (n) 14 17 9

AR MS μ±SD before HDM SCIT 3.286±1.069 3.824±0.529 3.222±0.972 AR MS μ±SD after HDM SCIT 1.714±1.729 2.647±1.498 2.889±1.167 MS mean improvement, μ±SD 1.571±1.604 1.176±1.629 0.333±0.707 AR MS p value (WSRT) 0.011 0.014 0.180 AR MS p value (MWUT) 0.631 - AR MS p value (MWUT) - 0.308

Number of patients with asthma (n) 6 9 4

AAMS μ±SD before HDM SCIT 2.333±0.516 2.111±0.928 2.5±1

AA MS μ±SD after HDM SCIT 2±0.894 2.222±0.833 2.5±1

MS mean improvement, μ±SD 0.333±0.516 -0.111±0.601 0±0

AA MS p value (WSRT) 0.157 0.564 1

AA MS p value (MWUT) 0.084 -

AA MS p value (MWUT) - 0.825

Abbreviations: AR allergic rhinitis; AA allergic asthma; MS medication score; μ mean; SD standard deviation; HDM house dust mite;

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12. DISCUSSION OF THE RESULTS

The results of this study are generally in agreement with the literature data regarding HDM SCIT utility in allergic airway diseases (as mentioned in the literature review section), supporting its effectiveness in the treatment of allergic rhinitis, especially reducing the need of medication usage to control symptoms from the disease.

This reduction was also seen in various subgroups. In both children and adults there was a reduction of mean medication score, but only being statistically significant for adults. This result does not concur with most of the recent studies, which support the effectiveness of HMD SCIT for allergic rhinitis in both children and adults [30, 45]. This might be due to the limited number of children patients included in this study.

In both male and female patients, there was a significant improvement in medication score, being statistically superior in male patients (which doesn’t match the results from the studies mentioned in the literature review stating that the effectiveness of AIT for allergic airway diseases between both genders is equal [27, 28]).

In terms of duration of HDM SCIT, according to the results of this study, significant medication score improvement is generally reached from the 7 to 12 months, and it is even more prominent at 24 months or more of HMD SCIT. This finding is in agreement with many of recent studies claiming to see HDM SCIT benefits on medication scores at 12 and 24 months of HDM SCIT [29, 46, 47].

Patients undergoing SCIT with HDM only improved their AR MS significantly and oppositely, patients that underwent SCIT with HDM mixture with other chemically modified allergens, did not improved their AR MS significantly. However, the difference between both of these subgroups was statistically not significant. As Soyyigit et al. concluded, single-allergen HDM SCIT seems to be effective in reducing the need of medication usage for allergic rhinitis in both monosensitized and polysensitized patients, without significant differences between both [36]. In this study, patients undergoing SCIT with only HDM had significant reductions on allergic rhinitis medication score regardless of being monosensitized to HDM or polysensitized (no statistical differences were found), while polysensitized patients undergoing SCIT with HDM mixture and other chemically modified allergens did not improve on their allergic rhinitis medication score. In addition, as Wang et al. [39] and Lee et al. [40] found on their respective studies, the difference between polysensitized patients undergoing SCIT with only HDM and SCIT with HDM mixture and other chemically modified allergens in this study were not statistically significant.

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The presence of other concomitant allergic disease like asthma did not affect significantly the

effectiveness of HDM SCIT on allergic rhinitis treatment, since the majority of patients with allergic rhinitis did experience a significant improvement on medication score being asthmatic or not.

On the contrary, the results did not show a significant improvement in asthmatic patients in terms of asthma medication necessity for symptom control. This finding is unmatched by the scientific literature, where many recent studies support the effectiveness of HDM SCIT in reducing asthma medication score and symptom score [30, 48]. This might be due to the fact that stepping down in asthma therapy requires a well-controlled, free symptom state of the patient during several months which makes it much harder than stepping down in allergic rhinitis drug therapy. No significant differences on asthma medication score change between subgroups were found.

There are some limitations that might have biased the results of this study. First of all, all data was collected from medical reports, not directly from the patient, so some data might not be updated. Secondly, the effectiveness of HDM SCIT was only evaluated by medication scores, not by combined symptom medication scores (as the EAACI and WAO recommend). Symptom scores could not be collected due to limited availability of information. And finally, collected data belonged to patients that were at different points of their SCIT. Additionally, there was a language barrier that could have affected the process of information collection.

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13. CONCLUSIONS

1) SCIT with HDM is an effective measure to treat allergic rhinitis on patients with HDM allergy. It provides significant reductions on allergic rhinitis medication needs, but not on asthma. Additionally, the effectiveness of SCIT with HDM for allergic rhinitis treatment is superior on male patients over female patients.

2) There are no significant differences in terms of HDM SCIT effectiveness for allergic rhinitis treatment between patients with allergic rhinitis only and patients with co-existing asthma. 3) HDM SCIT effectiveness on allergic rhinitis medication score usually starts to be significant at

7 to 12 months of undergoing immunotherapy.

4) SCIT with only HDM is equally effective in both monosensitized and polysensitized patients for the treatment of allergic rhinitis caused by HDM.

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14. PRACTICAL RECOMMENDATIONS

1) SCIT should be used for the management of patients with allergic rhinitis caused by HDM, since it has shown to effectively reduce the need of medications.

2) Patients should be encouraged to continue with their AIT despite not feeling improvements during the first few months, since it has shown to provide benefits in the long run.

3) When analyzing the effectiveness of AIT, patients should be evaluated at similar time points of their immunotherapy to get a more accurate view of the effect of AIT.

4) Medical doctors should use a standardized scoring system to better evaluate the effectiveness of AIT for allergic airway diseases.

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EVALUATION OF EFFECTIVENESS OF ALLERGEN-SPECIFIC SUBCUTANEOUS IMMUNOTHERAPY WITH HOUSE DUST MITES FOR PATIENTS WITH ALLERGIC AIRWAY DISEASES