POS1180 CELL FREE MITOCHONDRIAL DNA AS A BIOMARKER IN PATIENTS WITH EOSINOPHILIC GRANULOMATOSIS WITH POLYANGIITIS
Authors: N. Venhoff, S. Giaglis, N. Frede, M. Rizzi, J. Thiel, D. Kyburz, U. Walker
Journal: Annals of the Rheumatic Diseases (2024)

<h3>Background:</h3> The activation of neutrophils via anti-neutrophil cytoplasmic antibodies (ANCA), degranulation and formation of extracellular traps (NETs) are central processes in the pathogenesis of ANCA-associated vasculitis. As a result of increased NETosis, increased cell-free DNA, in particular mitochondrial DNA, was observed in the plasma of these patients (1). In contrast to granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA), eosinophilic granulomatosis with polyangiitis (EGPA) is driven by eosinophils and almost 70% of patients are ANCA negative. <h3>Objectives:</h3> The aim of the study was to investigate circulating cell-free DNA as a potential biomarker in patients with EGPA compared to GPA and MPA patients and healthy controls. <h3>Methods:</h3> Total DNA was isolated from platelet-free plasma samples from patients with EGPA, GPA or MPA and healthy donors (HD). Copy numbers were quantified by qPCR for mitochondrial (mt) DNA (ATP-6 gene) and nuclear (n) DNA (GAPDH gene). <h3>Results:</h3> 30 EGPA patients (mean age 56.8 ± 13.3 years, mean BVAS 1.73 ± 5.13, 13.33% ANCA positive (4/30)), 80 GPA patients (mean age 62.4 ± 15.1 years, mean BVAS 1.79 ± 4.86), 21 MPA patients (66.0 ± 9.72 years, mean BVAS 2.14 ± 5.14) and 128 HD (48.4 ± 15.3) were analyzed. Compared to HD, median total cell free DNA was 1.5-fold higher in EGPA (p=0.0291, Mann-Whitney test) and was comparable to values in GPA and MPA patients (Figure 1A). Especially mtDNA levels were significantly increased in EGPA (1.36x10<sup>8</sup> copies/ml plasma, 95% CI: 6.95x10<sup>7</sup> to 2.02x10<sup>8</sup>) compared to HD (7.8x10<sup>6</sup> copies/ml plasma, 95% CI: 6.70x10<sup>6</sup> to 8.94x10<sup>6</sup>, p&lt;0.0001) (Figure 1C). The amount of circulating mtDNA in EGPA was similar to that in patients with GPA or MPA. The nDNA concentrations in plasma did not differ between EGPA (6.61x10<sup>6</sup> copies/ml plasma, 95% CI: 4.08x10<sup>6</sup> to 9.15x10<sup>6</sup>), GPA (8.38x10<sup>6</sup> copies/ml plasma, 95% CI: 5.1x10<sup>6</sup> to 11.65x10<sup>6</sup>), MPA (2.28x10<sup>7</sup> copies/ml plasma, 95% CI: -9.73x10<sup>6</sup> to 5.53x10<sup>7</sup>) and HD (6.55x106 copies/ml plasma, 95% CI: 2.66x10<sup>6</sup> to 10.44x10<sup>6</sup>). In a linear regression model, pulmonary involvement, BVAS score and cell-free DNA levels significantly predicted mtDNA levels in EGPA/GPA/MPA patients (p&lt;0.001, respectively p=0.031 and p=0.037). Receiver operating characteristic curve (ROC) analysis showed that a cut-off value of 1.50x10<sup>7</sup> mtDNA copy numbers differentiated between EGPA and HD with 86.67% sensitivity, 86.72% specificity and an AUC of 0.95 (Figure 2 C). In this cohort, only 4 patients had active EGPA with elevated CRP, eosinophils and BVAS at the time of analysis. MtDNA levels were increased but not significantly higher than in patients with inactive disease. There were no significant differences between ANCA positive (4/30) vs. ANCA negative EGPA patients regarding cell-free DNA levels. <h3>Conclusion:</h3> The quantification of cell free mtDNA, but not nDNA copy numbers allows a differentiation between healthy donors and patients with EGPA. There were no significant differences to the patients with GPA or MPA, although the latter were much more frequently ANCA positive and were presumably more strongly driven by neutrophils than EGPA. Future studies should include more patients with active disease and follow-up visits. <h3>REFERENCES:</h3> [1] Giaglis S, et al. Mitochondrial DNA: a novel indicator of active inflammation in ANCA-associated vasculitides. Rheumatology (Oxford). 2023 Aug 1;62(8):2930-2937. doi:10.1093/rheumatology/kead015. PMID: 36645235; PMCID: PMC10393440. [2] Hashimoto T, et al. Increased Circulating Cell-Free DNA in Eosinophilic Granulomatosis With Polyangiitis: Implications for Eosinophil Extracellular Traps and Immunothrombosis. Front Immunol. 2022 Jan 12;12:801897. doi: 10.3389/fimmu.2021.801897. PMID: 35095884; PMCID: PMC8790570. <h3></h3> <h3>Acknowledgements:</h3> <b>NIL.</b> <h3>Disclosure of Interests:</h3> <b>UW is coinventor of patents owned by Freiburg University; NV is coinventor of patents owned by Freiburg University. Some parts of this work have already been published (1).</b>