Session Title: Vasculitis II
Session Type: Abstract Submissions (ACR)
Background/Purpose: To discover new pathways involved in the pathophysiology of ANCA-associated vasculitis (AAV) and identify potential clinical biomarkers through use of whole-genome gene expression profiling. Given the recent discovery of a pathogenic link between AAV and neutrophil extracellular trap (NET) formation, the study also sought to determine if patients with AAV have low-density granulocytes (LDGs) in peripheral blood. LDGs are a distinct subset of neutrophils described in systemic lupus erythematosus (SLE) that separate with PBMC in density gradient preparations and are prone to form NETs.
Methods: The source of clinical data and link biospecimens was a randomized controlled treatment trial in AAV. RNA-sequencing of whole blood from 112 subjects with AAV was performed during active disease at the baseline study visit (BL) and during remission 6 months later (6M). Gene expression in subjects who met the primary trial outcome of clinical remission at 6M was compared to patients who did not enter remission at 6M (responders vs. nonresponders) using the generalized linear model likelihood ratio test. A multi-gene composite score was created by calculating z-scores on a per gene per sample basis. Enrichment of relevant gene set signatures was tested using Gene Set Enrichment Analysis (GSEA). Measurement of neutrophil-related gene expression in PBMC collected concomitantly to whole blood samples was performed by qPCR and compared by ANOVA. LDGs were directly isolated from PBMC fraction by negative selection in 5 additional patients with AAV not enrolled within the clinical trial.
Results: There were no baseline differences in disease activity, clinical features, treatment status, and neutrophil counts between responders (n=77) and nonresponders (n=35). After filtering transcripts expressed in <50% of subjects, there were 44,532 total aligned reads. Differential expression between responders and nonresponders was seen in 2,346 transcripts at BL visit (p<0.05). Unsupervised hierarchical clustering demonstrated a distinct cluster of granulocyte-related genes that included genes coding for myeloperoxidase (MPO) and proteinase 3 (PR3), the major autoantigens in AAV. A granulocyte gene signature composite score was 10-fold higher in nonresponders versus responders (p<0.001) and 4-fold higher in subjects during active disease (BL) than remission (6M) (p<0.001). The signature in AAV strongly overlapped an LDG signature seen in lupus (FDRGSEA<0.001). Increased transcription of PR3, but not MPO, measured in PBMC collected in a subset of subjects was associated with active disease (p<0.01) and failure to meet the primary trial outcome of remission at 6M (p<0.001), validating the findings from whole-blood profiling and localizing the source of granulocyte gene expression to LDGs. In isolation studies, LDGs were found in every patient with AAV and, similar to SLE, were noted to readily form NETs in the absence of stimulation.
Conclusion: In patients with AAV increased expression of a granulocyte gene signature is associated with disease activity and response to treatment. The source of this signature is likely LDGs, which may be a key pathogenic cell in AAV.
P. C. Grayson,
D. J. Phippard,
M. J. Kaplan,
P. A. Merkel,
Genentech and Biogen IDEC Inc.,
Actelion Pharmaceuticals US,
Actelion Pharmaceuticals US,
P. A. Monach,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-association-of-low-density-granulocytes-with-disease-activity-and-response-to-treatment-in-anca-associated-vasculitis/