Virograms for prediction of predisposition to asthma exacerbation

Background: Clinical transcriptomics of peripheral blood mononuclear cells (PBMC) are coming into focus as a surrogate approach for prognosis, diagnosis, biomarker discovery and examination disease mechanisms. However, bioassays paired with transcriptomic analytic tools are yet to be developed and made available at point of care. Harnessing personal dynamic genomic responses to tailor patient asthma treatment or prevent disease exacerbations remain unmet medical needs. Method: We developed a rhinovirus-stimulated peripheral blood based-assay (virogram assay) coupled with single-subject analytics (N-of-1-patwhays) to capture dynamic genome-wide expression and dysregulated pathways to retrospectively predict childhood asthma exacerbation. We hypothesized that some genomic factors might predispose any given individual, healthy or asthmatic, to a set of similar transcriptional responses to rhinovirus stimulation. We first generated a classifier from paired sample microarrays, control and stimulated PBMC from healthy subjects and applied this classifier on the transcriptomic analysis of control and HRV-stimulated PBMC samples (virogram assay) from children with asthma. Results: The analysis of the different genomic responses of single-subject paired PBMC samples (HRV-stimulated and control) derived from healthy individuals (external dataset) enabled the discovery of dysregulated pathways related to acquired immunity, epigenetics and morphogenesis. The classifier built on these results and applied on the transcriptional analysis derived from the virogram assay predicted that the risk of asthma exacerbation among asthmatic subjects with an accuracy of 70%. Conclusion: We provide evidence that clinical prognosis can be predicted with a PBMC based-bioassay aligned with adequate single-subject analytics to assess dynamic transcriptomic response to specific disease-associated stimuli. Virogram assay of rhinovirus-stimulated PBMC samples and microarra Prepherial blood mononuclear cells (PBMCs) were isolated from blood samples collected from the 23 pediatric asthma subjects enrolled in the extension study following their participation in TREXA and BADGER clinical trials. Based on exacerbation status, the pediatric asthma subjects were classified in two groups: 11 subjects coded as no exacerbation (NE) for those that did not receive oral corticosteroids during the year of the study and (ii) 12 subjects coded as recurrent exacerbation (RE) for those that received two or more courses of oral corticosteroids during the year of the study. None of the 23 asthma patients received steroids during the month prior to the extension study when blood samples were collected for PMBCs. Each individual’s PBMCs are divided into two cultures, non-stimulated (PBMC control) and stimulated with human rhinovirus (PBMC stimulated) at 37°C in 5% CO2 for 24 hours. Paired PBMCs samples from each subject were harvested separately for RNA extraction. A total of 46 samples (23 pairs) were assessed for RNA quality, then amplified, tagged, and hybridized on Affymetrix Human Gene 1.0 ST microarrays according to standard operating procedures. Microarray datasets and gene expression processing Robust Multiple-array Average (RMA) normalization was applied on each patient data independently (2 paired samples at a time, to avoid bias in the single-patient experiments) using Affymetrix Power Tools (APT).