Chitinases in Asthmatic Children Undergoing Tonsillectomy “This study is a collaboration between the YCAAD and CPO investigating the effect of adenotonsillectomy on inflammation and asthma control in pediatric patients.” Maria Koenigs Yale School of Medicine Class of 2014 Maria B. Koenigs, BA1, Jonathan C Levin, BS1, Lisa Gagnon, APRN2, Eric D Baum, MD2, David E Karas, MD2 and Geoffrey L Chupp, MD1 1Section of Pulmonary and Critical Care, Yale University School of Medicine, New Haven, CT, 2Section of Otolaryngology, Yale University School of Medicine, New Haven, CT

Chitin and Chitinases Chitin is the 2nd most abundant polysaccharide in nature Contributes to Th2 inflammation associated with asthma (Ober and Chupp, Curr Opin Allergy Clin Immunol 2009) Chitinases break down chitin and modulate host immune response Chitotriosidase (true chitinase), YKL-40 (chitinase-like) Seibold, J Allergy Clin Immunol 2008 Humans don’t have chitin but have conserved chitinases to deal with chitin in environment Chitotriosidase- primary human chitinase in the lung, found to be elevated in serum of asthmatics and decreased in BAL fluid, found to be mutant copy in Lung Chitin Chitinases Chitin Chitotriosidase YKL-40 Th2 Inflammation

Human chitinase and chitinase-like proteins are associated with asthma in children Chitotriosidase YKL-40 Primary chitinase of the lung Higher activity in the serum in asthmatics (Bargagli, Respiration 2010) Truncated form of protein found in asthmatic children (Vicencio, Pediatrics 2010) Elevated in serum of asthmatics (Chupp et al NEJM 2007) Elevated levels associated with increased hospitalization in asthmatic children (Cunningham, Annals of Aller, Asthma, and Immuno, 2010) YKL-40 Chitotriosidase

Chitotriosidase is overexpressed in hypertrophied adenoid tissue in children Chitotriosidase found to be over-expressed in adenoids 41 children with Chronic rhinosinusitis Otitis Media Allergic rhinitis The presence of Chitotriosidase in the upper airway suggests that there may be a relationship between inflammation in the upper and lower airways Heo, Otolaryngol Head Neck Surg, 2011

Adenotonsillectomy and Asthma 1986- 40-45% improved asthma control (Garabedian, Ann Otolaryngol Chir Cervicofac) Since then- small mostly single-armed studies 2010-2012 renewed interest Decreased ER visits and pediatrician visits Decreased medication usage Increased ACT score (Ulis et al Unpublished data, (Piessens, Int Journal of Pediatric Otolaryngology, Busino, Laryngoscope) Common surgical procedure in children, performed most commonly for symptoms of sleep disordered breathing or chronic/recurrent tonsillar infections 1986 - Observational study of 55 asthmatic patients, “a remarkable finding, 40-45% of cases it was markedly improved.” 1996 – Observational study 2011 – Retrospective chart review - 2.2% of asthmatics had tonsillectomy for chronic tonisllitis (versus 9.9%) of controls 2012 – Retrospective chart review However, whether or not AT has a biological effect on airway inflammation and asthma has not yet been studied Biological effect of adenotonsillectomy on asthmatic inflammation has not yet been studied

Hypothesis and Aim Hypothesis Adenotonsillectomy will improve asthma control and modulate inflammatory pathways associated with asthma Aim A prospective study to investigate the clinical and biological effect of adenotonsillectomy on asthma, chitotriosidase activity, and YKL-40 levels before and after surgery

(Physician Diagnosis) Study Design Baseline - Asthma Questionnaires - Genotypes - Chitinase Levels - PFTs (age ≥6) 6 month follow-up - Asthma Questionnaires - Chitinase Levels - PFTs (age ≥6) 184 pediatric patients (ages 2-18) undergoing adenotonsillectomy enrolled 107 Asthmatics (Physician Diagnosis) 77 Controls 137 enrolled, Physician diagnosis of asthma, 71 asthmatics vs 66 controls Baseline – Clinical (Qs, PFTs), Lab (Genotypes, Chitinase, IgE) 6-mo – Repeat Qs, PFTs, Labs Total 51 follow-ups (91 eligible for 6 month) Mean 6.8 months (range 5-12) until follow-up Asthmatics – 27 (19 labs) Controls – 24 (9 labs)

YCAAD Asthma Questionnaire (Pediatric Version) Study Methods YCAAD Asthma Questionnaire (Pediatric Version) Asthma Symptoms: Symptoms history, Triggers, Medication usage, ED visits Comorbidities: sinusitis, atopy (allergies and eczema), GERD Environmental, home, and product exposures Personal and family med history Socioeconomic data, school and work days missed Asthma Control Test (ACT) Objective Data Chitinase Genotype Chitinase Activity YKL-40 Level IgE Levels Pulmonary Function Testing (age ≥ 6, and otherwise able) Asthma Questionnaire surveys symptoms, comorbidities, exposures, personal and family med hx, SES data, school & work days missed, and ACT Baseline ACT collected retrospectively on initial group of patients, however, data was consistent with ACTs collected prospectively Objective data Asthma Symptoms: Symptoms history, Triggers, Medication usage, ED visits Comorbidities: sinusitis, atopy (allergies and eczema), GERD Environmental, home, and product exposures Personal and family med history Socioeconomic data, school and work days missed Asthma Control Test Genotype CHIT1 exon 10 duplication (rs3831317) CHI3L1 Promoter SNP (rs4950928) Chitinase Activity – measure of Chitotriosidase (Enzyme Assay) YKL-40 Level (ELISA) IgE Levels Pulmonary Function Testing (age ≥ 6, and otherwise able)

Study Population at 130 patients Mean Age: 6.2 (2-18 range) Follow up rate: 80.7% Primary Indication for tonsillectomy 75% for tonsillar hypertrophy and OSA 23% recurrent infection Differences in asthmatics versus controls Hispanic ethnicity Comorbities: Sinusitis, hx of GERD, Atopy Decreased FEV1 No sig diff in age, gender, race, second hand smoke, T&A primary indication Sig: Hispanics/Latinos in asthmatics, Atopy, Hx GERD, Sinusitis, FEV1% (consistent) IgE higher in asthmatics, not sig

Clinical Response to T&A in Asthmatics In asthmatics – sig decreased chitinase activity Follow-up data for asthmatics showed… Sig increases ACT score; fewer ED visits, steroid courses, ear infections, other antibiotic use, missed school and parental work days Levin et al Unpublished Data

Chitinase Activity is modified by T&A Correspondingly, Chitinase activity significantly lower in Asthmatics, YKL-40 levels NS Levin et al Unpublished Data

Subpopulation Analysis Poorly controlled asthmatics Poor asthma control is defined as ACT ≤ 19 More than 2 ED/Urgent care visits per year More than 2 oral steroid courses per year More than 2x/week rescue SABA use Poorly controlled asthma- ED visits and ACT(N Engl J Med. 2009 April 9; 360(15): 1487–1499.) Steroid use in > 2 Ann Allergy Asthma Immunol 2004; 92:312-9 Frequent exacerbations: Aidan Long Mass general, hospital

Composite Variable Asthma improvement in uncontrolled asthmatics at baseline Asthma Improved in 36/48 (75%) SABA use decreased ACT Score Increase > 3 Number Oral Steroid Courses Decreased ED/Urgent Care Visits Decreased 18/28 (64%) 16/20 (80%) We defined improvement in asthmatic symptoms by any of the following:ED visits decreased, steroid courses decreased, ACT +3, Albuterol use in last month decreased, controller stopped 24/35 (68%) Levin et al Unpublished Data 19/22 (86%)

Baseline chitinase activity is elevated in poorly controlled asthmatics who improve with T&A Levin et al Unpublished Data

Hypertrophy/ Inflammation Concept: Tonsillectomy induces changes in systemic inflammation associated with asthma Tonsil Hypertrophy/ Inflammation Before Tonsillectomy   Serum Chitinases Chitin  Systemic Th2 Response Asthma Severity After Tonsillectomy  Serum Chitinases Our primary study results suggest that patients who have a higher chitinase level (animate) at baseline with tonsilar hypertrophy/inflammation & asthma are more likely to see improvement after tonsillectomy Animate Given high serum levels, indicates a systemic component to inflammation at baseline After tonsillectomy, serum chitinase levels fall Suggesting a decreased systemic inflammatory response along with decreased asthma severity Chitin  Systemic Th2 Response Asthma Severity

Summary and Conclusions Tonsillectomy improves asthmatic control in a majority of pediatric patients Chitinase activity is decreased in asthmatics following adenotonsillectomy Higher baseline chitinase activity may predict which poorly controlled asthmatics will have improved asthma control after adenotonsillectomy In summary, it was found that…

Limitations Seasonal variability Batch effect – sample collection and processing Limited PFT data in young population Small loss to follow-up

Future Directions Currently: Genome-Wide Expression Analyses on Tonsillar Tissue, Nasal Epithelial Tissue, and Blood Prospective study to explore the use baseline chitinase activity to predict response to adenotonsillectomy Comparison with matched non-surgical control group Personalized Medicine T&A on systemic inflammation

Acknowledgements Jon Levin, Yale School of Medicine ‘13 Lisa Gagnon, APRN Dr. Geoffrey Chupp and Dr. David Karas Dr. Susan He Connecticut Pediatric Otolaryngology Yale Center for Asthma and Airway Diseases Yale-New Haven Hospital Pediatric Surgery Center North Haven Surgical Center

Questions?

Adenotonsillectomy and Asthma Mostly small, single-arm studies 1986: 40-45% showed improvement (Garabedian, Ann Otolaryngol Chir Cervicofac) 2010: Prospective study of asthmatics and controls Fewer ER/pediatrician visits, steroid courses, decreased asthma severity medication use post-op (Ulis et al Unpublished data) 2011: Retrospective study of asthmatics and controls Fewer hospital visits, steroid courses, medication use Improved ACT scores (Busino, Laryngoscope) 2012: Decreased respiratory meds after surgery (Piessens, Int Journal of Pediatric Otolaryngology) Common surgical procedure in children, performed most commonly for symptoms of sleep disordered breathing or chronic/recurrent tonsillar infections 1986 - Observational study of 55 asthmatic patients, “a remarkable finding, 40-45% of cases it was markedly improved.” 1996 – Observational study 2011 – Retrospective chart review - 2.2% of asthmatics had tonsillectomy for chronic tonisllitis (versus 9.9%) of controls 2012 – Retrospective chart review However, whether or not AT has a biological effect on airway inflammation and asthma has not yet been studied Biological effect of adenotonsillectomy on asthmatic inflammation has not yet been studied

Chitotriosidase (CHIT1) in Asthmatics Primary active chitinase in the human lung (Seibold, J Allergy Clin Immunol 2008) CHIT1 24-bp exon 10 duplication (rs3831317): nonfunctional protein  rate of mutant allele in 6 asthmatic children with fungal sensitization (Vicencio, Pediatrics 2010) Higher activity in serum of asthmatics (Bargagli, Respiration 2010) Lower activity in BAL of asthmatics (Siebold, J All Clin Immuno 2008) Hypertrophied Adenoids: chitotriosidase overexpression in 41 children with chronic rhinosinusitis, otitis media, and allergic rhinitis (Heo, Otolaryngol Head Neck Surg, 2011) genotype for CHIT1 and asthma, hospitalizations, or ED visits EXON 9 EXON 10 EXON 11 Wild Type Mutant 24 bp duplication CHIT1 encodes, duplication exists that makes nonfunctional protein 422 patients showed no association between genotype for CHIT1 and asthma, hospitalizations, or ED visits (Wu A, J All Clin Immunol 2010) Additionally, Hypertrophied adenoids…in chronic upper airway disease

YKL-40 (CHI3L1) in Asthmatics Elevated in the serum of asthmatics (Chupp et al., NEJM 2007) CHI3L1 Promoter SNP (rs4950928, -131CG) Associated with elevated YKL-40 levels, severity of asthma, bronchial hyperresponsiveness, and reduced pulmonary function; minor allele is protective (Ober et al., NEJM 2008) Associated with asthma hospitalizations in children (Cunningham, Annals of Aller, Asthma, and Immuno, 2010) Chitinase-like protein In adults, Serum levels Correlated with severity, bronchial hyperresponsiveness, subepitithelial basement membrane thickening, and poor pulmonary function Genotype – SNP (C allele)  elevated; G allele protective