Theerapan Songnuy, M.D.

Asthma Biomarkers

Establishing standard definitions and data collection methodologies for validated outcomes measures in asthma clinical research

Identifying promising outcomes measures for asthma clinical research that require further development

Szefler SJ et al. Asthma outcomes: Biomarkers. JACI. 2012; 129( 3Suppl):S9-23.

Objectives of Outcome Standardization

Core outcome - A set of asthma outcomes considered by NIH and other agencies as requirement

for funding support : Multi-allergen screening to define atopy

Szefler SJ et al. Asthma outcomes: Biomarkers. JACI. 2012; 129( 3Suppl): S9-23.

Three Categories of Asthma Biomarkers

Standard definitions have been developed, method for measurement can be specified, validity has been proved but whose inclusion in

funded clinical asthma research will be optional

- CBC to measure total eosinophils - Fractional exhaled nitric oxide ( Feno) - Sputum eosinophils - Urinary leukotrienes - Total and allergen-specific IgESzefler SJ et al. Asthma outcomes: Biomarkers. JACI. 2012;

129( 3Suppl): S9-23.

Supplemental Outcomes

Have potential to : expand/ improve current aspects of disease monitoring and improve

translation of basic and animal model-based asthma research into clinical research. They are not yet standardized and need more development and validation

- Sputum PMN leukocytes - Cortisol measures - Airway imaging - Breath markers - System-wide studies ( genomics, proteomics)Szefler SJ et al. Asthma outcomes: Biomarkers. JACI. 2012;

129(3Suppl): S9-23.

Emerging Outcomes

Nitric Oxide ( NO) is an pollutant emitting from vehicle and cigarette smoke

A biological mediator in animal and human Produced by human lungsRoles of NO: - Vasodilator - Bronchodilator - Neurotransmitter - Inflammatory mediatorPalmer RM, Ashton DS, Moncada S. Vascular endothelial cells synthesize

nitric oxide from L-arginine. Nature 1988; 333: 664-666.

Nathan C, Xie QW. Nitric oxide synthases: roles, tolls, and controls. Cell 1994; 78:

915-918.

Fractional Exhaled Nitric Oxide ( FENO) : A biomarker of asthma

Highly reactive molecule/ free radical and has oxidant property

Bactericidal and cytotoxicity for tumor cellPathophysiology of NO in airway and lung: - A pro-inflammatory mediator lead to airway hyper-responsiveness

Reid DW, Johns DP, Feltis B, Ward C, Walters EH. Exhaled nitric oxide continues to reflect airway hyperresponsiveness and disease activity in inhaled corticosteroid- treated adult asthmatic patients. Respirology. 2003; 8: 479-486.

Fractional Exhaled Nitric Oxide ( FENO) : A biomarker of asthma

FENO used as quantitative method of airway nitric oxide ( NO), produced by NO synthasesNoninvasive, simple, and safe methodAn indirect marker for eosinophilic airway inflammation Support diagnosis of eosinophilic asthmaDetermining corticosteroid responsiveness ( more consistently than other methods)Evaluation of adherence to anti-inflammatory drugs

Dweik RA et al. An official ATS clinical practice guideline: Interpretation of exhaled nitric oxide levels ( (FENO) for clinical applications. Am J Respir Crit Care Med.

2011; 184: 602-615.

Fractional Exhaled Nitric Oxide ( FENO) : A biomarker of asthma

Low FENO ( < 25 ppb in adult, < 20 ppb in kids)

- Less likely eosinophilic inflammation & responsiveness to corticosteroidHigh FENO ( > 50 ppb in adult, > 35 ppb in

kids) - More likely for eosinophilic inflammation

& in symptomatic patient, more response to corticosteroid

Clinically Significant Cut Points of FENO

Significant increase in FENO - Greater than 20% ( if value > 50 ppb) - More than 10 ppb ( if value < 50 ppb)Significant decrease in FENO - Greater than 20% (if value > 50 ppb) - More than 10 ppb ( if value < 50 ppb)

Clinically Significant Cut Points of FENO

Not associated with neutrophilic airway InflammationChildren younger than 4 yearsFalse negative in patient already treated with

ICS

Limitation of FENO for Diagnosis Asthma

Age , FENO increase 5% per year ( important in children)

HeightAtopic symptomsSmokingAnti-inflammatory drugsMeasurement techniqueExhalation flow rateNasal NO contaminationNO analyzer used

Borrill Z, Clough D, Truman N, Morris J, Langley S, Singh D, A comparison of exhaled nitric oxide measurements

performed using three different analyzers. Respir Med 2006; 100: 1392-1396.

Buchvald F et al. Measurements of exhaled nitric oxide in healthy subjects age 4 to 17 years. JACI 2005;115: 1130-1136.

Confounders Affecting FENO Measurement

www.ergonomidesign.se

Pediatr Allergy Immunol 2012; 23: 59-64.

To study association between exhaled nitric oxide, asthma and atopy in a variety clinically relevant phenotypes in a cohort of 6-year-old children

Aim

The New Zealand Asthma and Allergy Cohort StudyAt Wellington and ChristchurchFrom 1997-2001Participants: - Mothers were recruited randomly by midwives ( previous study) - Questionnaires were administered by nurses at start, 3, 15, 24, 36, 48 & 60 mo of age - Home visit at 3 & 15 months & others by telephone - Outcomes were measured at 15, 36, 48 & 60 months

Epton M, Town G, Ingham T, et al. The New Zealand infant cohort study : assembly, demographics and investigations. BMC Public Health 2007; 7: 26.Pediatr Allergy Immunol 2012; 23: 59-64.

Methods

Participants: - Between the ages of 6-7 years - Questionnaires & clinical assessments : - FENO measurement - SPT to food & environmental allergens - Blood for total & specific IgE

Measurement of exhaled nitric oxide : - Using Aerocrine NIOX chemiluminescence analyser at flow rate 50

ml/s - Range of detection from 2- 200 p.p.b. - FENO was measured according to American Thoracic Society / European Respiratory Society guidelines - Before NO measurement, mothers were asked whether the child had a

cold, respiratory infection or had exercised a last hour before

Pediatr Allergy Immunol 2012; 23: 59-64.

Methods

SPT: - Allergens: Der p, cat, dog, horse, cockroach mix, rye grass, olive tree, Aspergillus fumigatus, peanus, Alternaria tenuis, egg white, & cow’s milk ( Dome/ Hollister-Stier, Spokane, WA, USA)

- Mean wheal diameter was measured - A positive reaction defined as a mean wheal diameter of 3 mm or greater

- Atopy defined as positive one or more reaction to SPT Pediatr Allergy Immunol 2012; 23: 59-64.

Methods

Wheezing, asthma & family history definitions

- Mother/caregiver was asked for child’s wheezing at each visit

- Wheezing classified as: - Early transient ( occur from birth to 3 y ) - Late onset ( from 3-6 y) - Persistent wheeze ( from birth-6 y)

- Current asthma ( previous doctor’s diagnosis of asthma at any time & current wheeze, inhaler use - A family history of allergic disease ( a parental report of either

parent having a history of asthma, allergic rhinitis or eczema ) Pediatr Allergy Immunol 2012; 23: 59-64.

Methods

IgE measurement - Total IgE - Specific IgE - These were measured by IMMULITE 2000 Siemens Medical, Deerfield, IL, USA - Atopy defined as any sIgE 0.35 kU/l or more

Pediatr Allergy Immunol 2012; 23: 59-64.

Methods

At birth 3-mo. 15-mo. 6 yr. 6 yr.

Attend Lab complete

FENO test

Results

1105

1064(96.3

%)

1011(91.4

%)846

681(62%

)

Pediatr Allergy Immunol 2012; 23: 59-64.

Pediatr Allergy Immunol 2012; 23: 59-64.

Pediatr Allergy Immunol 2012; 23: 59-64.

Pediatr Allergy Immunol 2012; 23: 59-64

Pediatr Allergy Immunol 2012; 23: 59-64

Pediatr Allergy Immunol 2012; 23: 59-64

FENO is closely related to specific IgE to aeroallergens among allergic children

The main factor of airway inflammation is sIgE

to aeroallergen & to Der p that children have continually exposed at a high level

FENO is not elevated in non-allergic asthma Children

Conclusion

JACI. 2011; 127 ( 5) : 1165-72.e5.

Whether FENO was increased in children with allergic sensitization or asthma

Whether specific allergen exposure increased FENO level in sensitized , but not in un-sensitized children

Whether sedentary behavior increased FENO, independent of allergen exposures

JACI. 2011; 127 ( 5) : 1165-72.e5

Objectives

Children whose mothers reside in Boston Metropolitan area Between Sep 1994-Aug 1996 ( cohort study)*Children whose mothers: - Age at least 18 y - History of hay fever, asthma, or allergy in at least one of the child’s parents - Families were not screened if NB was admitted in NICU, maternal gestational age < 36 wk or he/she had a congenital anomaly

*Gold DR, Burge HA, Carey V, Milton DK, Platts-Mills T, Weiss ST. Predictors of repeated

wheeze in the first year of life : The relative roles of cockroach, birth weight, acute lower

respiratory illness , and matrnal smoking. Am J Respir Crit Care Med. 1999; 160(1): 227-36.

JACI. 2011; 127 ( 5) : 1165-72.e5.

Methods

A series of home visit: - At age 2-3 months, 7 y, 12 yQuestionnaires : by trained research assistants ( home visit) - Demographics - Home characteristics - Environmental exposures - Tobacco use - Health outcomesQuestionnaires : by telephone every 6 months

JACI. 2011; 127 ( 5) : 1165-72.e5.

Methods

Participants :

Enrolled F/U until age 12 y FENO

measurement

JACI. 2011; 127 ( 5) : 1165-72.e5.

Methods

505430

(85%)277

( 64%)

Home visit & dust sample collection - Measurement of bed dust mite ( by Eureka Mighty-

Mite vacuum cleaner : Model 3621; Eureka Co. , Bloomington IN) modified to hold 19*90 mm cellulose extraction thimbles

- All layers of the bedding were vacuumed for 10 min - Allergen concentrations (ug/g dust) for Der p1,Der f1, Fel d1, Bla g2 quantified by ELISA - Cut off point : for Der p 1, Der f1 greater than 10 ug/g for Cat allergen greater than 8 ug/g for Cockroach above detectable levels JACI. 2011; 127 ( 5) : 1165-72.e5

Methods

Assessment of allergic sensitization

- Specific IgE to common ( outdoor & indoor) allergens were done using the UniCap 250

system in 189 children - Positive test was greater than 0.35 IU/ml - Specific IgE + SPT + FENO in 208 children JACI. 2011; 127 ( 5) : 1165-72.e5

Methods

Assessment of TV watching /video game playing - Collecting data every 6 months by telephone - Using the closest time to FENO measurement - Assess hour of weekday & weekend separately - Categories: ( none, 1-5 hr, 6-10 hr, 11-15 hr, or 16-20 hr) - Also ask for “ physical activity” day/weekDefinition of respiratory symptom outcomes - Collecting every 6 months by telephone - Outcomes: current asthma; any wheeze; dry cough at night; current rhinitis

JACI. 2011; 127 ( 5) : 1165-72.e5

Methods

Spirometry: - Albuterol was administered ( 180 ug; 2 puffs with spacer) & wait 10 min - Apply spirometry before & after medication - Positive bronchodilator response : 12% increase in FEV1

JACI. 2011; 127 ( 5) : 1165-72.e5

Methods

Measurement of FENO : - FENO level by using a portable electrochemical device ( NIOX MINO); Aerocrine AB - Validated by chemiluminescene technology; +-5 ppb - Subject breathed in through an NO scrubbing filter &

exhaled out into the room air twice - Then inhaled a third time through the filter & exhaled into the FENO analyzer - Flow rate of 50 ml/s without a nose clip - The last 3 seconds of exhalation was assessed - This procedure was done 3 times, the median value was

used

Methods

JACI. 2011; 127 ( 5) : 1165-72.e5

PICT

Results

JACI. 2011; 127 ( 5) : 1165-72.e5

JACI. 2011; 127 ( 5) : 1165-72.e5

JACI. 2011; 127 ( 5) : 1165-72.e5

JACI. 2011; 127 ( 5) : 1165-72.e5

JACI. 2011; 127 ( 5) : 1165-72.e5

JACI. 2011; 127 ( 5) : 1165-72.e5

The first study to investigate the independent impact of home allergens and sedentary home behavior on FENO in high risk children for allergies & asthma

- Sensitization : predictor of airway inflammation - Allergic sensitization : key factor in NO production - Sedentary behavior has positive association with

FENO

In Conclusion

FENO : used as a biomarker in assessment and management of airway inflammatory disease

FENO is a noninvasive, ease of repeat measurement, & easy use in patients

Take Home Messages

THANK YOU VERY MUCH