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Other-Diseases Comparison Group and Control Group: For the other-diseases group, comparison cases were selected to achieve a balanced sample size age matched with the NEHI cases and to include other pulmonary disorders associated with NEC hyperplasia (bronchiolitis, BPD, pulmonary hypertension). As a control group, uninvolved lung from lobectomy cases for congenital cystic adenomatoid malformations served as an additional comparison (n = 6).
Infant Pulmonary Function Tests
Infant pulmonary function tests (PFTs) were performed as part of clinical care for some of the subjects in these cohorts. Testing was performed with oral chloral hydrate sedation (75-100 mg/kg) using the nSpire Infant Pulmonary Laboratory system and its updated integrated software (vCM55Ra; nSpire; Longmont, Colorado). After static measures, plethysmography was performed as described by Castile and then flow-volume curves were obtained using the raised-volume rapid thoracoabdominal compression technique described by Jones Studies were performed and interpreted according to American Thoracic Society/ European Respiratory Society guidelines.
Immunohistochemistry
To delineate NECs, immunohistochemistry for bombesin (Immu-noStar, Inc; Hudson, Wisconsin) was performed on formalin-fixed 5-pm paraffin sections as previously described2 Dual immunofluorescence for bombesin with Ki67 (1:100) (Dako M7249; Dako North America; Carpinteria, California) was carried out on paraffin sections following antigen retrieval. Bombesin was developed with antirabbit fluorescein and Ki67 with biotinylated antimouse IgG (both from Vector Laboratories Inc; Burlingame, California) followed by Cy3-streptavidin (Jackson ImmunoResearch; West Grove, Pennsylvania).
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Morphometry was performed on sections immunostained for bombesin similar to that previously described, using images captured by a SPOT RT Slider digital camera (Diagnostics Instruments, Inc; Sterling Heights, Michigan) mounted on a Nikon E400 microscope (Nikon Instruments Inc; Melville, New York).
