Novel laboratory markers for diagnosis and monitoring of ANCA associated vasculitis and idiopathic inflammatory myopathies interstitial lung disease

INTRODUCTION Interstitial lung disease (ILD) may complicate the course of systemic autoimmune rheumatic disease (SARD), particularly ANCA associated vasculitis (AAV) and idiopathic inflammatory myopathies (IIM). Here, we explored the landscape of serum biomarkers in idiopathic and SARD-ILD in four different setting. First, as a proof of concept, preliminary study, we evaluated IL-6, SMRP, KL-6 and ferritin in idiopathic and SARD-ILD in a large cohort of patients from the UK Biomarkers in Interstitial Lung Disease (UK-BILD) Study. Secondly, we aimed to assess whether serum ferritin could be considered a specific and sensitive biomarker for IIM-ILD, as well as to assess whether it correlates with clinical, radiological, functional and routine laboratory findings. As a corollary, some IIM patients from this cohort were furtherly studied in order to assess the prevalence of subclinical renal injury by elevated NGAL, KIM1, Activin A, CD163, and Cys-s. Finally, given the evidence coming from the previous studies for the abovementioned biomarkers, we prospectively studied a cohort of AAV, with and without ILD, in order to evaluate serum CD163 and KL6 levels and whether their levels correlate with disease severity and extent. Subsequently, a proof-of-concept, corollary study, ILD-AAV patients were treated with a protocol of Rituximab (RTX) and a short dosage of GCs in order to assess whether this scheme could be effective in stabilizing respiratory functional parameters and imaging findings. METHODS Patients included in the UK-BILD Study” were retrospectively enrolled from 39 UK recruitment centers. Inclusion criteria were a diagnosis of IPF, idiopathic NSIP and SARD-ILD. Serum samples were obtained from recruited patients, anonymized in an electronic database and marker concentrations were measured singly by KL-6, IL-6, FER and SMRP reagent assays. Patients were further stratified according to HRCT findings and comparative analysis of serum marker concentrations were performed within and between groups. For ferritin evaluation, all patients affected by IIM and followed at Siena (Italy), Palermo (Italy), Bari (Italy) and Lucknow, (India) IIM referral centers were retrospectively included. Primary endpoints were to assess whether abnormal ferritin serum levels are associated with the presence of ILD and to evaluate whether high ferritin serum levels can be evidenced only in anti-MDA5 DM or also in ASS ILD. In order to evaluate urinary biomarkers in IIM, clinical data, core set measures, sera and urine were prospectively collected from all patients enrolled in the MyoCite cohort from 2017 to 2021. Twenty healthy individuals (HCs) and 16 patients affected by acute kidney injury were included as a control group. For patients with IIMs, data from both baseline and follow-up visits within the observational period were included. Finally, for ILD-AAV study, patients with MPA or GPA with and without ILD were prospectively collected and treated with RTX and oral GCs according to PEXIVAS protocol, measuring serum CD163 and KL6 levels. RESULTS 1013 patients were selected: 520 (51.3%) had idiopathic ILD and 493 SARD-ILD. Idiopathic ILD patients displayed higher KL-6 values than SARD-ILD. Ferritin and SMRP, though within normal ranges, were significantly higher in idiopathic ILD. Logistic regression showed good sensitivity and specificity selecting the variables ferritin and KL-6 concentrations, age and gender-male correlated with a diagnosis of idiopathic ILD. A total of 139 IIM patients underwent ferritin measurement. When patients were stratified between ASS, DM and anti-MDA5 DM, a statistically significant difference was found only for ferritin, which was positively associated with the presence of anti-MDA5-DM with ILD. ROC curve analysis obtained a significant AUROC with the best cut-off value of 303.5 for distinguishing ASS and MDA5-DM with ILD. Analysis of 201 visits of 110 adult patients with IIMs indicated higher normalized biomarker levels compared to HCs, and comparable to patients with AKI, with the exception of NGAL, which was higher in the AKI group. Notably, 49% patients with IIMs had eGFR<90; the levels of the 5 biomarkers were comparable between active and inactive IIMs, and different subtypes of IIMs. Similarly, a poor correlation between urine biomarker levels and core set measures of activity and damage was found. Changes in biomarker levels on follow-up did not correlate with eGFR changes. Eight patients, 3 affected by GPA and 5 by MPA, were included in AAV-ILD study. All but one displayed positivity for anti-MPO and 3 of them did not have any extra-pulmonary sign of vasculitis. At baseline, LFT evidenced a restrictive pattern (mean FVC 83%) associated with a moderate impairment of diffusion capacity (mean DLCO 54%). After 6 and 12 months, a stabilization of LFT findings was evidenced, while no sign of progression of ILD was assessed at CT scan. No patient suffered from severe AE, except one who had bacterial pneumonia. All patients but three discontinued GCs. DISCUSSION Our study showed the excellent diagnostic value of KL-6 for detecting ILD, which irrespective of the final diagnosis and extent of disease, is always elevated and is a reliable biomarker of lung fibrosis in various diseases, ranging from idiopathic to autoimmune forms. Our study proposed an ILD differentiation model including clinical background. Moreover, in a large, multicentric, international cohort, we evidenced that serum, ferritin represents a reliable and specific biomarker for the diagnosis of patients with MDA5-ILD. Ferritin represents the sole clinical and serological feature able to address to a positivity of MDA5, as well as to distinguish between ASS and anti-MDA5-DM with ILD. At the same time, exploratory analysis of urinary biomarkers identified low eGFR and elevated biomarkers of CKD in nearly half of the patients with IIMs, comparable to patients with AKI and higher than HCs, indicative of potential renal damage in IIMs that may have a lead to complications in other systems. Finally, through the first prospective study specifically designed to evaluate the efficacy of RTX in AAV-ILD. Our findings have displayed that RTX, prescribed in association with a short course of GCs, is able to stabilize imaging features and respiratory functionality.