Interferon-alpha2 Treatment Reduces Circulating Neutrophil Extracellular Trap Levels in Myeloproliferative Neoplasms

Background and aim Arterial and venous thrombosis are major life-threatening events for patients with Philadelphia-negative chronic myeloproliferative neoplasms (MPNs). Neutrophil activation and, in particular, the formation of neutrophil extracellular traps (NETs) have been implicated in the pathogenesis of MPNs and thrombosis, particularly in patients with the most common MPN phenotypic driver mutation, JAK2V617F. Moreover, ruxolitinib treatment reduces ex vivo NET formation in JAK2V617F-positive patients with MPNs. In this study, we aimed to clarify the effect of interferon alpha (IFNα) and hydroxyurea (HU) treatment on the levels of circulating NETs in patients with MPNs.

Methods Serum NET (myeloperoxidase-DNA complexes) levels were measured using an in-house ELISA in pre-treatment samples collected from 128 patients with MPNs enrolled in the DALIAH trial (NCT01387763), a randomized controlled phase 3 clinical study. NETs were likewise quantified in 85 paired samples collected after 12 months of therapy with HU or either of two different formulations of pegylated IFNα (PEG-IFNα2a or PEG-IFNα2b).

Results At baseline, NET levels did not vary significantly between patients with different MPN sub-diagnoses (ET: 48, PV: 61, Pre-MF: 10, PMF: 9), phenotypic driver mutations (JAK2V617F, CALR, MPL, or triple negative), or between those with or without a history of major thrombosis. However, patients with PV and a JAK2V617F allele burden ≥ 50% at baseline had significantly higher baseline NET levels than those with an allele burden < 50% (p=0.006). NET levels at baseline correlated significantly with neutrophil count (r=0.29, p=0.001), neutrophil-to-lymphocyte ratio (NLR)(r=0.26, p=0.004) and JAK2V617F allele burden (r=0.22, p=0.03), particularly in patients with PV and allele burden ≥ 50% (r=0.50, p=0.01, r=0.56, p=0.007 and r=0.45, p=0.03 respectively), while the same was not observed at follow-up. Furthermore, after 12 months of treatment, NET levels decreased in 70% of patients, with an average decrease of 48%, compared to baseline levels. The effect of treatment was particularly pronounced in patients with PV with an allele burden ≥ 50%, of whom 76% showed decreased NET levels, with an average decrease of 60%, while 67% of patients with allele burden < 50% showed an average reduction in NET levels of only 36% (Figure 1). While 77% and 73% of patients treated with PEG-IFNα-2a or PEG-IFNα-2b, respectively, had decreased NET levels, the same applied to only 53% of patients treated with HU (Figure 2).

Discussion and conclusion The increased NET levels in patients with PV with a JAK2V617F allele burden ≥ 50% indicate that the JAK/STAT pathway is involved in NET formation and/or clearance. In keeping with this, the highest proportion of patients with decreased NET levels after treatment and the highest average decrease were seen in patients with PV and a baseline JAK2V617F allele burden ≥ 50%. NET levels at baseline correlated with neutrophil count and NLR, however normalization of blood counts alone did not account for the observed decrease in NET levels. Accordingly, treatment with IFNα, which has previously been shown to target the JAK2V617F clone preferentially, effectively reduced circulating NET levels, and to a greater extent than HU treatment. This may relate to our previous finding that three months' IFNα2 treatment downregulates the expression of PAD4, which is implicated in NET formation. Although, we found no difference in NET levels in patients with and without a history of major thrombotic events, these data suggest that IFNα2 treatment, by reducing levels of potentially prothrombotic NET, may contribute to reduced risk of this complication in patients with MPNs, and that it is an advantageous treatment compared to HU in this respect.

Hasselbalch:Novartis: Consultancy, Other: Grants; AOP Orphan Pharmaceuticals: Consultancy, Other: Data monitoring board.

Pegylated interferon-alpha-2a (Pegasys) Pegylated interferon-alpha-2b (PegIntron)