Asthma Biomarkers; FENO Presented by Theerapan Songnuy, MD. March1, 2013

1. Theerapan Songnuy, M.D.

2.  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.

3.  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.

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

5.  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.

6.  Nitric Oxide ( NO) is an pollutant emitting from
vehicle and cigarette smoke
 A biological mediator in animal and human
 Produced by human lungs
 Roles of NO:
- Vasodilator
- Bronchodilator
- Neurotransmitter
- Inflammatory mediator
Palmer 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.

7.  Highly reactive molecule/ free radical and has oxidant
property
 Bactericidal and cytotoxicity for tumor cell
 Pathophysiology 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.

8.  FENO used as quantitative method of airway nitric oxide
( NO), produced by NO synthases
 Noninvasive, simple, and safe method
 An indirect marker for eosinophilic airway inflammation
 Support diagnosis of eosinophilic asthma
 Determining 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.

12.  Low FENO ( < 25 ppb in adult, < 20 ppb in kids)
- Less likely eosinophilic inflammation &
responsiveness to corticosteroid
 High FENO ( > 50 ppb in adult, > 35 ppb in kids)
- More likely for eosinophilic inflammation &
in symptomatic patient, more response to
corticosteroid

13.  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)

15.  Not associated with neutrophilic airway
Inflammation
 Children younger than 4 years
 False negative in patient already treated with ICS

16.  Age , FENO increase 5% per year ( important in children)
 Height
 Atopic symptoms
 Smoking
 Anti-inflammatory drugs
 Measurement technique
 Exhalation flow rate
 Nasal NO contamination
 NO 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.

17. www.ergonomidesign.se

18. Pediatr Allergy Immunol 2012; 23: 59-64.

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

20.  The New Zealand Asthma and Allergy Cohort Study
 At Wellington and Christchurch
 From 1997-2001
 Participants:
- 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.

21. 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.

22.  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.

23.  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.

24.  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.

25. At birth 3-mo. 15-mo. 6 yr. 6 yr.
1064 1011 681
1105 (96.3%) (91.4%)
846 (62%)
Attend Lab complete
FENO test
Pediatr Allergy Immunol 2012; 23: 59-64.

26. Pediatr Allergy Immunol 2012; 23: 59-64.

27. Pediatr Allergy Immunol 2012; 23: 59-64.

28. Pediatr Allergy Immunol 2012; 23: 59-64

29. Pediatr Allergy Immunol 2012; 23: 59-64

30. Pediatr Allergy Immunol 2012; 23: 59-64

31.  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

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

33.  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

34.  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.

35. A series of home visit:
- At age 2-3 months, 7 y, 12 y
Questionnaires : by trained research assistants ( home visit)
- Demographics
- Home characteristics
- Environmental exposures
- Tobacco use
- Health outcomes
Questionnaires : by telephone every 6 months
JACI. 2011; 127 ( 5) : 1165-72.e5.

36. Participants :
Enrolled F/U until age 12 y FENO
measurement
430 277
505 ( 64%)
(85%)
JACI. 2011; 127 ( 5) : 1165-72.e5.

37.  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

38.  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

39. 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/week
Definition 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

40. 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

41. 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
JACI. 2011; 127 ( 5) : 1165-72.e5

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

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

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

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

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

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

49.  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

50.  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


51. THANK YOU VERY MUCH