biology Article KL-6 in ANCA-Associated Vasculitis Patients with and without ILD: A Machine Learning Approach Edoardo Conticini 1,† , Miriana d’Alessandro 2, *,† , Laura Bergantini 2 , Diego Castillo 3 , Paolo Cameli 2 , Bruno Frediani 1 , Luca Cantarini 1 and Elena Bargagli 2 1 Rheumatology Unit, Department of Medicine, Surgery & Neurosciences, University of Siena, 53100 Siena, Italy; conticini.edoardo@gmail.com (E.C.); fredianibruno60@gmail.com (B.F.); cantarini@unisi.it (L.C.) 2 Respiratory Diseases and Lung Transplantation Unit, Department of Medical and Surgical Sciences & Neurosciences, University of Siena, 53100 Siena, Italy; laurabergantini@gmail.com (L.B.); paolocameli88@gmail.com (P.C.); bargagli2@gmail.com (E.B.) 3 Respiratory Department, Hospital de la Santa Creu i Sant Pau, Sant Pau Biomedical Research Institute (IIB-Sant Pau), 08041 Barcelona, Spain; dcastillo@santpau.cat * Correspondence: dalessandro.miriana@gmail.com; Tel.: +39-057-758-6713; Fax: +39-057-728-0744 † These authors equally contributed to the study. Simple Summary: Krebs von den Lungen-6 (KL-6) is a mucin involved in several cellular pro- cesses, and its expression increases following injured or regenerating type II pneumocyte. KL-6 was suggested to identify patients affected by fibrotic interstitial lung diseases (ILD) associated with rheumatologic disorders. This is the first study that has assessed whether serum KL-6 can distinguish ANCA-associated vasculitis (AAV) patients with ILD from those without ILD, and whether this biomarker and its changes over time are correlated with disease activity, vasculitic damage, and  response to treatment. Thirteen AAV patients were enrolled, four of them with ILD. Higher serum  KL-6 was found in AAV-ILD patients in comparison with those without ILD. The best KL-6 cutoff Citation: Conticini, E.; d’Alessandro, values of 368 U/mL and 301 U/mL at baseline and during follow-up, respectively, were suggested to M.; Bergantini, L.; Castillo, D.; distinguish the two groups. A direct correlation was found between serum KL-6 and disease activity. Cameli, P.; Frediani, B.; Cantarini, L.; Our multicenter study demonstrated KL-6 as a reliable, non-invasive, and easy-to-perform marker of Bargagli, E. KL-6 in ANCA-Associated Vasculitis Patients with and without ILD in AAV patients, being helpful for disease activity assessment. Changes in serum concentrations ILD: A Machine Learning Approach. of KL-6 over time could be useful for monitoring AAV patients. Further study of KL-6 as a marker of Biology 2022, 11, 94. https://doi.org/ response to therapy during long-term follow-up would also be worthwhile. 10.3390/biology11010094 Abstract: Background: ANCA-associated vasculitis (AAV) are small vessel vasculitis distinguished Academic Editor: Frédéric Becq between microscopic polyangiitis (MPA) and granulomatosis with polyangiitis (GPA). The former Received: 9 December 2021 may have interstitial lung disease (ILD) associated with high morbidity and mortality. Here, Krebs Accepted: 6 January 2022 von den Lungen-6 (KL-6), a marker of fibrotic ILD, was assessed for distinguishing AAV patients Published: 8 January 2022 with ILD from those without ILD, and whether its changes over time are correlated with disease Publisher’s Note: MDPI stays neutral activity. Materials and Methods: Thirteen AAV patients (eight females, mean age 61 ± 14.8 years) with regard to jurisdictional claims in were enrolled: six MPA and six GPA. Serum samples were assayed for KL-6 concentrations (Fujirebio published maps and institutional affil- Europe, Belgium). To investigate potential binary classifiers for diagnosis of AAV-ILD, we constructed iations. a regression decision tree model. Results: Higher serum KL-6 were in AAV-ILD compared with those without ILD (972.8 ± 398.5 vs 305.4 ± 93.9, p = 0.0040). Area under the receiver operating characteristics curve showed 100% of the diagnostic performance of KL-6 for identifying the ILD involvement (accuracy 91.7%) and the best cutoff value of 368 U/mL (sensitivity 100% and specificity Copyright: © 2022 by the authors. 87.5%). The decision tree model showed a 33% improvement in class purity using a cut-off value Licensee MDPI, Basel, Switzerland. of 513 U/mL to distinguish AAV patients with and without ILD. Stratifying AAV patients as MPA This article is an open access article distributed under the terms and and GPA with and without ILD considering T0 and T1 KL-6, the model obtained an improvement of conditions of the Creative Commons 40% for classifying GPA non-ILD with a T0 serum KL-6 cut-off value of 513 U/mL and a T1 KL-6 Attribution (CC BY) license (https:// cut-off of 301 U/mL. A direct correlation was found between serum T0 KL-6 and T0 BVAS (r = 0.578, creativecommons.org/licenses/by/ p = 0.044). Conclusion: Our multicenter study demonstrated KL-6 as a reliable, non-invasive, and 4.0/). easy-to-perform marker of ILD in AAV patients and its helpfulness for disease activity assessment. Biology 2022, 11, 94. https://doi.org/10.3390/biology11010094 https://www.mdpi.com/journal/biology Biology 2022, 11, 94 2 of 10 Changes in serum concentrations of KL-6 over time could be useful for monitoring AAV patients. Further study of KL-6 as a marker of response to therapy during long-term follow-up would also be worthwhile. Keywords: KL-6; ANCA-associated vasculitis; interstitial lung disease 1. Introduction ANCA-associated vasculitis (AAV) are small vessel vasculitis affecting the kidneys, upper and lower airways, skin, and central and peripheral nervous systems [1]. They are distinguished between microscopic polyangiitis (MPA), granulomatosis with polyangiitis (GPA), or eosinophilic granulomatosis with polyangiitis (EGPA), which have different signs, symptoms, and autoimmune profiles. MPA is usually associated with ANCA-MPO positivity and GPA with ANCA-PR3 positivity, whereas up to 50% of patients with EGPA are ANCA-negative [2,3]. Patients with MPA, especially those who are ANCA-MPO-positive, may have intersti- tial lung disease (ILD) [4,5], which is associated with high morbidity and mortality as it is often underdiagnosed and responds poorly to conventional treatments [6]. Interstitial lung disease associated with AAV does not have specific imaging or bronchoalveolar lavage (BAL) findings. Moreover, due to lack of validated diagnostic criteria [7], the diagnosis of AAV relies on clinical expertise and often requires invasive biopsy procedures. In the lung, Krebs von den Lungen-6 (KL-6) is a key transmembrane mucin implicated in the process of cell proliferation, growth, and apoptosis; its expression increases in injured or regenerating type II pneumocytes [8–11]. KL-6 was recently proposed as a diagnostic biomarker for ILD and for predicting response to antifibrotic therapies [12,13]. Different reports have demonstrated elevated serum KL-6 concentrations in various ILDs, including connective tissue disorders associated with ILD [14–17], suggesting that this marker is not only useful for diagnosis but also for prognosis and for monitoring response to therapy. Some authors have reported higher serum concentrations of KL-6 in AAV patients with ILD than in those without lung involvement, suggesting that this mucin could be a useful prognostic marker [18–20]. However, these few studies were conducted in small cohorts and did not consider disease activity or extrapulmonary manifestations. Our aim here was therefore to assess whether serum KL-6 can distinguish AAV patients with ILD from those without ILD, and whether this biomarker and its changes over time are correlated with disease activity, vasculitic damage, and response to treatment. 2. Materials and Methods 2.1. Study Population We enrolled all consecutive patients assessed at the Vasculitis Clinic of the Rheumatol- ogy and Respiratory Disease Units of Siena University Hospital in the period December 2020 to November 2021 and ILD clinic of Hospital de la Santa Creu I Sant Pau (Barcelona) in the period 2017 to 2018. Inclusion criteria were granulomatosis with polyangiitis (GPA) or microscopic polyangiitis (MPA) diagnosed by a clinician with experience in vasculitis, active disease, and eligibility for rituximab treatment according to current recommendations [21]. Exclusion criteria were a diagnosis of EGPA and remitting disease. As part of our clinical practice, all patients underwent concomitant rheumatological and pneumological evaluation, lung function tests, routine blood tests, autoimmunity evaluation, and KL-6 assay. Current and previous treatments, Birmingham vasculitis score (BVAS), and vasculitis damage index (VDI) were also recorded. High-resolution computed tomography (HRCT) and BAL were performed if lung involvement was suspected. The first evaluation in the observation period at our vasculitis clinic (Siena) and ILD clinic (Barcelona) was defined as baseline (T0); the medical examination after treatment was defined as T1. Biology 2022, 11, 94 3 of 10 Serum samples from all patients were assayed for KL-6 concentrations by KL-6 reagent assay (Fujirebio Europe, United Kingdom), as previously reported [14]. The principle of the assay is agglutination of sialylated carbohydrate antigen in samples with KL-6 mAb by antigen–antibody reaction. The change in absorbance was measured to determine KL-6 levels. Serum concentrations of KL-6 were expressed in U/mL. A serum KL-6 cut-off value of 465 U/mL was considered to distinguish fibrotic-ILD patients from non-fibrotic patients and healthy subjects, as previously reported [22]. All patients gave their written informed consent to participate in the study, which was approved by our local ethics committee (Siena, Markerlung 17431 and Barcelona, IIBSP-KLS-2016-39). 2.2. Statistical Analysis The data are presented as mean ± standard deviation. Non-parametric one-way ANOVA (Kruskal–Wallis test) and Dunn’s test were performed for multiple comparisons. Wilcoxon’s rank sum test was used to compare data before and after treatment (T0–T1). Receiver operating characteristics (ROC) curve was employed to analyze the diagnostic performance of KL-6 for identifying the ILD involvement and to select the best cutoff threshold with high sensitivity and specificity. In order to investigate potential binary classifiers for diagnosis of AAV-ILD, we formed a group of patients with AAV but not ILD. Then, we constructed a regression decision tree for AAV with versus without lung involvement, MPA-ILD versus GPA non-ILD and MPA non-ILD, and previous versus non-previous treatment. We created a series of test/training partitions to evaluate the accuracy of potential binary classifiers by means of a confusion matrix. The best thresholds, in terms of specificity and sensitivity, of each binary classifier were chosen by Youden’s J method. The Spearman test was used to look for correlations. A p-value less than 0.05 was considered statistically significant. Statistical analysis was performed by GraphPad Prism 9.2 and XLSTAT 2021 software. 3. Results Demographic, immunological, and clinical data are shown in Table 1, together with BVAS, VDI, and organ involvement. A total of 13 patients (8 females, 5 males, median age 61 ± 14.8 years) were enrolled. One was excluded due to a concomitant diagnosis of lung cancer. Six had MPA and the other six had GPA; median duration of disease was 4.8 ± 6.5 years. Serum concentrations of KL-6 in AAV patients with ILD were compared with those of AAV patients without ILD (972.8 ± 398.5 vs 305.4 ± 93.9, p = 0.0040). Biology 2022, 11, 94 4 of 10 Table 1. Clinical, immunological, and demographic data of AAV patients. Abbreviations: AZA: azathioprine; BVAS: Birmingham vasculitis activity scale; F: female; GCs: glucocorticoids; GPA: granulomatosis with polyangiitis; KL6: Krebs von den Lungen; M: male; MMF: mycophenolate mofetil; MPA: microscopic polyangiitis; MTX: methotrexate; PNS: peripheral nervous system; RTX: rituximab; VDI: vasculitis damage index; * at T0. Length of Previous Organs Type of Lung T0 Treat- T0 GCs T0 KL6 T1 Treat- T1 GCs T1 KL6 T1 Sex/Age Diagnosis Disease * Treat- T0 BVAS T0 VDI T1 VDI Involved Involvement ment Dosage Levels ment Dosage Levels BVAS (months) ments Lung, ILD, alveolar F/83 MPA 28 kidney, RTX, AZA GCs 10 625 8 7 RTX 5 325 0 7 hemorrhage PNS Lung, ILD, alveolar M/77 MPA 8 kidney, - GCs, MTX 15 982 24 1 - - - - - hemorrhage PNS, skin Lung, eye, M/71 GPA 24 Nodules, ILD RTX GCs 5 1528 9 3 - - - - - joints F/64 MPA 24 Lung ILD - - - 756 4 - - - 1003 0 - Lung, M/48 GPA 1 kidney, Nodules - GCs 50 352 18 0 RTX 25 279 2 0 nose, eye CYC, Lung, F/55 GPA 252 Nodules MMF, GCs, MTX 25 401 15 3 GCs, RTX 12.5 602 3 3 skin AZA F/38 MPA 1 Skin - - - - 347 6 0 - - - - - Kidney, F/74 MPA 180 - CYC GCs 2.5 368 11 5 GCs, MTX 5 414 0 5 PNS GCs, RTX, GCs, RTX, F/49 GPA 60 Nose, eye - MTX 5 122 6 2 5 116 0 2 AZA AZA M/60 MPA 40 Skin, PNS - GCs MTX - 205 9 1 - - - - - F/39 GPA 4 Nose - - GCs 50 310 6 3 - - - - - Nodules, CT F/59 GPA 72 Lung consolidation, - - - 338 4 - - - - 0 - biopsy confirmed GPA Biology 2022, 11, 94 5 of 10 ROC curve analysis between ILD and non-ILD in AAV patients (Figure 1a) showed the area under curve (AUC) of 100% with the best cut-off value of 368 U/mL (sensitivity 100% and specificity 87.5%, accuracy of 91.7%, LR+ 8.0 and LR- 0.0). In order to improve predictive power, we used a decision-tree model (with cross-validation by confusion matrix). The model (Figure 1b) obtained using KL-6 concentrations at T0 showed a 33% Biology 2022, 11, x FOR PEER REVIEW 4 of 11 improvement in class purity using a cut-off value of 513 U/mL to distinguish AAV patients with (lung) and without (non-lung) ILD (p = 0.034). Figure 1.1. (a) Figure (a)Receiver Receiveroperating operatingcharacteristic characteristic (ROC) (ROC) curves curves were were firstfirst employed employed to analyze to analyze the the diag- diagnostic performance of KL-6 for identifying the ILD involvement and to select the best nostic performance of KL-6 for identifying the ILD involvement and to select the best cutoff threshold cutoff threshold with with high and high sensitivity sensitivity and (b) specificity. specificity. Decision (1b) Decision tree model tree model obtained usingobtained using KL-6 KL-6 concentrations concentrations at T0 showed a 33% improvement in class purity using a cut-off value at T0 showed a 33% improvement in class purity using a cut-off value of 513 U/mL to distinguish of 513 U/mL to distinguish AAV patients with and without ILD (p = 0.034). Lung and non-lung are referred to AAV patients with and without ILD (p = 0.034). Lung and non-lung are referred to the presence and the presence and absence of ILD, respectively. Abbreviations: TP, true positive; TN, true negative; absence of ILD, respectively. Abbreviations: TP, true positive; TN, true negative; FP, false positive; FP, false positive; FN, false negative. FN, false negative. As expected, a higher prevalence of ILD was found in the MPA (20%) than in the GPA (10%) group. A decision tree model (and cross-validation by confusion matrix) was applied to T0 and T1 KL-6 concentrations in AAV patients stratified as MPA and GPA with and without ILD. The model (Figure 2) obtained an improvement of 57.1% for classifying MPA-ILD patients with a serum T0 KL-6 cut-off of 513 U/mL and 40% of improvement for classifying GPA non-ILD with a T0 serum KL-6 cut-off value of 513 U/mL and a T1 KL-6 cut-off of 301 U/mL. As expected, a higher prevalence of ILD was found in the MPA (20%) than in the GPA (10%) group. A decision tree model (and cross-validation by confusion matrix) was applied to T0 and T1 KL-6 concentrations in AAV patients stratified as MPA and GPA with and without ILD. The model (Figure 2) obtained an improvement of 57.1% for classifying MPA-ILD patients with a serum T0 KL-6 cut-off of 513 U/mL and 40% of Biology 2022, 11, 94 6 of 10 improvement for classifying GPA non-ILD with a T0 serum KL-6 cut-off value of 513 U/mL and a T1 KL-6 cut-off of 301 U/mL. Figure 2. A Figure 2. decision tree A decision tree model model(and (andcross-validation cross-validationbybyconfusion confusionmatrix) matrix) was was applied applied to to T0 T0 andand T1 T1 KL-6 concentrations in AAV patients stratified as MPA and GPA with and without ILD. The KL-6 concentrations in AAV patients stratified as MPA and GPA with and without ILD. The model model obtained an improvement of 57.1% for classifying MPA-ILD patients with a serum T0 KL-6 obtained an improvement of 57.1% for classifying MPA-ILD patients with a serum T0 KL-6 cut-off of cut-off of 513 U/mL and 40% of improvement for classifying GPA non-ILD with a T0 serum KL-6 513 U/mL cut-off and value of40% of improvement 513 U/mL for classifying and a T1 KL-6 GPAU/mL. cut-off of 301 non-ILD with a T0 serum KL-6 cut-off value of 513 U/mL and a T1 KL-6 cut-off of 301 U/mL. Previous treatment did not influence serum concentrations of KL-6, as demonstrated Previous treatment did not influence serum concentrations of KL-6, as demonstrated by the decision tree model (and cross-validation by confusion matrix) (Figure 3), obtaining by the decision tree model (and cross-validation by confusion matrix) (Figure 3), obtaining aa 20% improvement in 20% improvement class purity in class purity for for treated treated patients patients with with aa cut-off cut-off value value of of 360 360 U/mL U/mL and Biology 2022, 11, x FOR PEER REVIEW also with a further cut-off of 803.5 U/mL. The non-treated group used for the and also with a further cut-off of 803.5 U/mL. The non-treated group used for the model model 7 of 11 obtained a 40% improvement in purity with a serum KL6 cut-off value of 257.5 obtained a 40% improvement in purity with a serum KL6 cut-off value of 257.5 U/mL. U/mL. Figure 3. Decision tree model obtained from KL6 concentrations in AAV patients stratified Figure 3. Decision tree model obtained from KL6 concentrations in AAV patients stratified according according to previous treatment: 20% improvement in class purity for treated patients with a cut- to previous off treatment: value of 360 20%also U/mL and improvement in class with a further purity cut-off for treated of 803.5 patients U/mL. The with a cut-off non-treated valuefor group used of 360 model the U/mL obtained and also with a 40%a improvement further cut-offin ofpurity 803.5 U/mL. The non-treated with a serum group KL6 cut-off valueused for the of 257.5 model U/mL. obtained a 40% improvement in purity with a serum KL6 cut-off value of 257.5 U/mL. For clinical parameters, a direct correlation was found between serum concentrations For clinical parameters, a direct correlation was found between serum concentrations of KL-6 at T0 and T0 BVAS (r = 0.578, p = 0.044) (Figure 4). of KL-6 at T0 and T0 BVAS (r = 0.578, p = 0.044) (Figure 4). 2000 according to previous treatment: 20% improvement in class purity for treated patients with a cut- off value of 360 U/mL and also with a further cut-off of 803.5 U/mL. The non-treated group used for the model obtained a 40% improvement in purity with a serum KL6 cut-off value of 257.5 U/mL. For clinical parameters, a direct correlation was found between serum concentrations Biology 2022, 11, 94 7 of 10 of KL-6 at T0 and T0 BVAS (r = 0.578, p = 0.044) (Figure 4). 2000 1500 T0 KL6 levels 1000 500 0 0 10 20 30 T0 BVAS Figure 4. The correlation between serum concentrations of KL-6 at T0 and T0 BVAS (r = 0.578, Figure 4. The correlation between serum concentrations of KL-6 at T0 and T0 BVAS (r = 0.578, p = p = 0.044) in AAV patients. 0.044) in AAV patients. 4. Discussion 4. Discussion This is the first time that serial measurements of serum KL-6 have been made in AAV This isinthe patients first to order time that serial assess measurements the utility of serum KL-6 of this glycoprotein have been made for diagnosis, in AAV prognosis, and patients in order monitoring to assess of these the utility patients. of this tract Respiratory glycoprotein involvementfor diagnosis, in AAV mayprognosis, and be extremely monitoring varied [23],ofranging these patients. Respiratory from diffuse alveolar tract involvement hemorrhage (DAH), intypically AAV may be extremely occurring in MPA, varied [23], ranging to nodules, usuallyfromfounddiffuse in GPA,alveolar hemorrhagepneumonia, and eosinophilic (DAH), typically which occurring in together with MPA, to nodules, usually found in GPA, and eosinophilic asthma and transient infiltrates is the hallmark of EGPA. pneumonia, which together with asthma Althoughandnottransient infiltrates considered is the a typical hallmark feature of EGPA. of AAV, ILD is reported in a certain percent- age of patients: up to 45% and 23% of MPA and GPA patients, respectively, have various forms of ILD [4], while only one case of EGPA and pulmonary fibrosis is reported in the literature. In line with these previous observations, interstitial lung involvement in our cohort was generally detected in MPA patients: significantly, the only GPA patient with concomitant ILD was ANCA-MPO-positive. ILD usually occurs months to years before diagnosis of AAV. Occasionally, the diagno- sis of AAV is concomitant or precedes that of ILD. ANCA themselves seem related to the development of ILD, even without concomitant vasculitis: several papers have reported a significant variable percentage of ANCA-positive patients with ILD but with no extra-pulmonary signs or symptoms of AAV [24]. To date, there is no clear-cut diagnostic workup for this condition, and no imaging technique or laboratory analysis seems capable of predicting the onset of AAV-ILD or how it will respond to treatment. Among diagnostic procedures, HRCT has a well-established role, but as it exposes the patient to a significant dose of ionizing radiation and it is less suitable for close follow-up of patients with no definite ILD at baseline. KL-6 is a high molecular weight glycoprotein, originally suggested as a serum biomarker for lung, breast, and pancreatic cancers, albeit with low diagnostic accuracy. Serum con- centrations of KL-6 were found to be elevated in ILDs characterized by alveolar epithelial cell damage and progressive interstitial thickening [10]. A cut-off value of 465 U/mL was recently established to distinguish fibrotic ILD patients from healthy subjects and patients with other non-fibrotic lung diseases [22]. Elevated serum concentrations of KL-6 have been found in idiopathic pulmonary fibrosis (IPF) patients, a disease characterized by alveolar epithelial cell damage and pro- gressive interstitial thickening. A prognostic role of KL-6 was suggested to predict survival in IPF patients evaluating its serum concentration at disease onset (cut-off 1000 U/mL) [25]. Fluctuations in KL-6 during follow-up of IPF patients have also been reported to have potential for predicting functional progression of the disease [26–29]. Biology 2022, 11, 94 8 of 10 Only two studies have analyzed KL-6 concentrations in AAV patients with and without ILD, and one was a case report: both used an ELISA kit and neither considered overall disease activity or extra-pulmonary manifestations. In line with these data, our multicenter study, which included eight female and five male patients (in line with most recent epidemiologic data [30]), confirmed the higher serum concentrations of KL-6 in AAV patients with ILD compared to those without ILD. On the other hand, no significant correlation was found for KL6 and extrapulmonary manifestations of vasculitis, or for lung involvement other than ILD. Likewise, KL6 concen- trations did not appear to be higher in patients with a longer duration of disease or in those with multi-systemic involvement: this also confirms the high specificity of the biomarker in patients with complex, long-standing vasculitis who are more difficult to assess. At the same time, we found that previous pharmacological treatment did not influence elevated concentrations of KL-6 in AAV-ILD patients. Similarly, no correlation was found between serum KL6 and VDI, which is not sur- prising, since lung fibrosis, chronic breathlessness, and impaired lung function are only 3 out of 63 items of this index. Conversely, and more importantly, a direct correlation was demonstrated between serum concentrations of KL6 at T0 and BVAS at T0. This con- firms the strong link between KL6 and disease activity, which is of paramount importance as biomarker needs to be an easy diagnostic and prognostic tool, not merely useful for assessing organ or system damage. The present study suggests a KL-6 cut-off value below 513 U/mL of serum, which may be particularly useful in patients in whom clinical (such as GPA) and serological features are not strongly associated with the onset of ILD. Our study has some limitations: First, there was a limited number of patients who underwent KL-6 measurements over time that presumably underpowered its specificity at follow-up. Moreover, we were not able to establish a definite diagnostic cut-off value due to the limited sample size of patients with the concomitant ILD. Nevertheless, considering the rarity of disease and the relevant diagnostic issues due to the lack of definite imaging or laboratory findings for this condition, the demonstration of elevated serum concentrations of KL-6 in AAV patients with ILD is of interest. Further research lines should be addressed in order to assess whether KL-6 changes may be related to disease clinical course or response to treatment. 5. Conclusions In conclusion, KL-6 was demonstrated to be a reliable, non-invasive, and easy-to- perform marker of interstitial lung involvement in AAV patients, being helpful for assess- ment of disease activity. Changes in serum concentrations of KL-6 over time could be useful for monitoring AAV patients. Further study of KL-6 as a marker of response to therapy during long-term follow-up would also be worthwhile. Author Contributions: Conceptualization, E.C., M.d.; methodology, M.d., L.B.; software, M.d.; validation, M.d., P.C., B.F., D.C., L.C., E.B., L.B.; writing—original draft preparation, E.C., M.d., L.B., D.C., P.C., B.F., L.C., E.B. All authors have read and agreed to the published version of the manuscript. Funding: This research project is not funded. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee (Siena, Markerlung 17431 and Barcelona, IIBSP-KLS-2016-39). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Acknowledgments: Not applicable. Biology 2022, 11, 94 9 of 10 Conflicts of Interest: The authors declare no conflict of interest. Abbreviations AZA azathioprine BVAS Birmingham vasculitis activity scale F female GCs glucocorticoids GPA granulomatosis with polyangiitis KL6 Krebs von den Lungen M male MMF mycophenolate mofetil MPA microscopic polyangiitis MTX methotrexate PNS peripheral nervous system RTX rituximab VDI vasculitis damage index * at T0 References 1. Jennette, J.C.; Falk, R.J.; Bacon, P.A.; Basu, N.; Cid, M.C.; Ferrario, F.; Flores-Suarez, L.F.; Gross, W.L.; Guillevin, L.; Hagen, E.C.; et al. 2012 Revised International Chapel Hill Consensus Conference Nomenclature of Vasculitides. Arthritis Rheumatol. 2013, 65, 1–11. [CrossRef] [PubMed] 2. Sinico, R.A.; Di Toma, L.; Maggiore, U.; Bottero, P.; Radice, A.; Tosoni, C.; Grasselli, C.; Pavone, L.; Gregorini, G.; Monti, S.; et al. Prevalence and Clinical Significance of Antineutrophil Cytoplasmic Antibodies in Churg-Strauss Syndrome. Arthritis Rheumatol. 2005, 52, 2926–2935. [CrossRef] 3. Papo, M.; Sinico, R.A.; Teixeira, V.; Venhoff, N.; Urban, M.-L.; Iudici, M.; Mahrhold, J.; Locatelli, F.; Cassone, G.; Schiavon, F.; et al. Significance of PR3-ANCA Positivity in Eosinophilic Granulomatosis with Polyangiitis (Churg-Strauss). Rheumatology (Oxford) 2021, 60, 4355–4360. [CrossRef] [PubMed] 4. Sebastiani, M.; Manfredi, A.; Vacchi, C.; Cassone, G.; Faverio, P.; Cavazza, A.; Sverzellati, N.; Salvarani, C.; Luppi, F. Epidemiology and Management of Interstitial Lung Disease in ANCA-Associated Vasculitis. Clin. Exp. Rheumatol. 2020, 38 (Suppl. 124), 221–231. 5. Bargagli, E.; Conticini, E.; Mazzei, M.A.; Cameli, P.; Guerrini, S.; d’Alessandro, M.; Frediani, B.; Pleuroparenchymal Fibroelastosis (PPFE) Siena Unit, Italy; Rheumatology Unit Department of Medical Sciences, Surgery and Neurosciences, University of Siena, Italy; Respiratory Diseases and Lung Transplantation, Department of Medical and Surgical Sciences & Neurosciences, Siena University Hospital, Siena, Italy. Pleuroparenchymal Fibroelastosis in Interstitial Lung Disease with Antineutrophil Cytoplasmic Antibody-Associated Vasculitis. Clin. Exp. Rheumatol. 2021, 39 (Suppl. 129), 190. 6. Kadura, S.; Raghu, G. Antineutrophil Cytoplasmic Antibody-Associated Interstitial Lung Disease: A Review. Eur. Respir. Rev. 2021, 30, 210123. [CrossRef] 7. Khan, I.; Watts, R.A. Classification of ANCA-Associated Vasculitis. Curr. Rheumatol. Rep. 2013, 15, 383. [CrossRef] 8. Kohno, N.; Kyoizumi, S.; Awaya, Y.; Fukuhara, H.; Yamakido, M.; Akiyama, M. New Serum Indicator of Interstitial Pneumonitis Activity. Sialylated Carbohydrate Antigen KL-6. Chest 1989, 96, 68–73. [CrossRef] 9. Kohno, N. Serum marker KL-6/MUC1 for the diagnosis and management of interstitial pneumonitis. J. Med. Invest. 1999, 46, 151–158. [PubMed] 10. Ishikawa, N.; Hattori, N.; Yokoyama, A.; Kohno, N. Utility of KL-6/MUC1 in the Clinical Management of Interstitial Lung Diseases. Respir. Investig. 2012, 50, 3–13. [CrossRef] 11. Bonella, F.; Long, X.; Ohshimo, S.; Horimasu, Y.; Griese, M.; Guzman, J.; Kohno, N.; Costabel, U. MUC1 Gene Polymorphisms Are Associated with Serum KL-6 Levels and Pulmonary Dysfunction in Pulmonary Alveolar Proteinosis. Orphanet J. Rare Dis. 2016, 11, 48. [CrossRef] 12. Kennedy, B.; Branagan, P.; Moloney, F.; Haroon, M.; O’Connell, O.J.; O’Connor, T.M.; O’Regan, K.; Harney, S.; Henry, M.T. Biomarkers to Identify ILD and Predict Lung Function Decline in Scleroderma Lung Disease or Idiopathic Pulmonary Fibrosis. Sarcoidosis Vasc. Diffus. Lung Dis. Off. J. WASOG 2015, 32, 228–236. 13. Yokoyama, A.; Kohno, N.; Hamada, H.; Sakatani, M.; Ueda, E.; Kondo, K.; Hirasawa, Y.; Hiwada, K. Circulating KL-6 Predicts the Outcome of Rapidly Progressive Idiopathic Pulmonary Fibrosis. Am. J. Respir. Crit. Care Med. 1998, 158, 1680–1684. [CrossRef] 14. d’Alessandro, M.; Bellisai, F.; Bergantini, L.; Cameli, P.; D’Alessandro, R.; Mazzei, M.A.; Gentili, F.; Conticini, E.; Selvi, E.; Frediani, B.; et al. Prognostic Role of KL-6 in SSc-ILD Patients with Pleuroparenchymal Fibroelastosis. Eur. J. Clin. Investig. 2021, 51, e13543. [CrossRef] [PubMed] Biology 2022, 11, 94 10 of 10 15. Remuzgo-Martínez, S.; Atienza-Mateo, B.; Ocejo-Vinyals, J.G.; Genre, F.; Pulito-Cueto, V.; Mora-Cuesta, V.M.; Iturbe-Fernández, D.; Lera-Gómez, L.; Pérez-Fernández, R.; Prieto-Peña, D.; et al. Role of MUC1 Rs4072037 Polymorphism and Serum KL-6 Levels in Patients with Antisynthetase Syndrome. Sci. Rep. 2021, 11, 22574. [CrossRef] 16. Stock, C.J.W.; Hoyles, R.K.; Daccord, C.; Kokosi, M.; Visca, D.; De Lauretis, A.; Alfieri, V.; Kouranos, V.; Margaritopoulos, G.; George, P.M.; et al. Serum Markers of Pulmonary Epithelial Damage in Systemic Sclerosis-Associated Interstitial Lung Disease and Disease Progression. Respirology 2020, 26, 461–468. [CrossRef] 17. Hk, L.; Ea, R. Circulating Biomarkers of Interstitial Lung Disease in Systemic Sclerosis. Int. J. Rheumatol. 2012, 2012, 121439. [CrossRef] 18. Takahashi, F.; Miura, E.; Shibukawa, K.; Chinda, J.; Miyokawa, N.; Imamoto, C.; Kikuchi, K.; Hasebe, N. Monitoring with Serum SP-A, SP-D, and KL-6 in a Patient with Interstitial Pneumonia Complicated with ANCA-Associated Glomerulonephritis. CEN Case Rep. 2014, 3, 5–9. [CrossRef] 19. Iwata, Y.; Wada, T.; Furuichi, K.; Kitagawa, K.; Kokubo, S.; Kobayashi, M.; Sakai, N.; Yoshimoto, K.; Shimizu, M.; Kobayashi, K.; et al. Serum Levels of KL-6 Reflect Disease Activity of Interstitial Pneumonia Associated with ANCA-Related Vasculitis. Intern. Med. 2001, 40, 1093–1097. [CrossRef] 20. Fujita, S.; Hirayama, K.; Ogawa, Y.; Shimohata, H.; Kobayashi, M. Mizoribine Reduces Serum KL-6 Levels in ANCA-Associated Vasculitis. Clin. Exp. Nephrol. 2010, 14, 203–204. [CrossRef] 21. Yates, M.; Watts, R.A.; Bajema, I.M.; Cid, M.C.; Crestani, B.; Hauser, T.; Hellmich, B.; Holle, J.U.; Laudien, M.; Little, M.A.; et al. EULAR/ERA-EDTA Recommendations for the Management of ANCA-Associated Vasculitis. Ann. Rheum. Dis. 2016, 75, 1583–1594. [CrossRef] 22. Ohnishi, H.; Yokoyama, A.; Kondo, K.; Hamada, H.; Abe, M.; Nishimura, K.; Hiwada, K.; Kohno, N. Comparative Study of KL-6, Surfactant Protein-A, Surfactant Protein-D, and Monocyte Chemoattractant Protein-1 as Serum Markers for Interstitial Lung Diseases. Am. J. Respir. Crit. Care Med. 2002, 165, 378–381. [CrossRef] [PubMed] 23. Makhzoum, J.-P.; Grayson, P.C.; Ponte, C.; Robson, J.; Suppiah, R.; Watts, R.A.; Luqmani, R.; Merkel, P.A.; Pagnoux, C. DCVAS collaborators Pulmonary Involvement in Primary Systemic Vasculitides. Rheumatology (Oxford) 2021, keab325. [CrossRef] 24. Sebastiani, M.; Luppi, F.; Sambataro, G.; Castillo Villegas, D.; Cerri, S.; Tomietto, P.; Cassone, G.; Bocchino, M.; Atienza-Mateo, B.; Cameli, P.; et al. Interstitial Lung Disease and Anti-Myeloperoxidase Antibodies: Not a Simple Association. J. Clin. Med. 2021, 10, 2548. [CrossRef] [PubMed] 25. Ohshimo, S.; Ishikawa, N.; Horimasu, Y.; Hattori, N.; Hirohashi, N.; Tanigawa, K.; Kohno, N.; Bonella, F.; Guzman, J.; Costabel, U. Baseline KL-6 Predicts Increased Risk for Acute Exacerbation of Idiopathic Pulmonary Fibrosis. Respir. Med. 2014, 108, 1031–1039. [CrossRef] [PubMed] 26. Ishii, H.; Kushima, H.; Kinoshita, Y.; Fujita, M.; Watanabe, K. The Serum KL-6 Levels in Untreated Idiopathic Pulmonary Fibrosis Can Naturally Decline in Association with Disease Progression. Clin. Respir. J. 2018, 12, 2411–2418. [CrossRef] [PubMed] 27. d’Alessandro, M.; Bergantini, L.; Cameli, P.; Pieroni, M.; Refini, R.M.; Sestini, P.; Bargagli, E. Serum Concentrations of KL-6 in Patients with IPF and Lung Cancer and Serial Measurements of KL-6 in IPF Patients Treated with Antifibrotic Therapy. Cancers 2021, 13, 689. [CrossRef] [PubMed] 28. Huang, T.-H.; Kuo, C.-W.; Chen, C.-W.; Tseng, Y.-L.; Wu, C.-L.; Lin, S.-H. Baseline Plasma KL-6 Level Predicts Adverse Outcomes in Patients with Idiopathic Pulmonary Fibrosis Receiving Nintedanib: A Retrospective Real-World Cohort Study. BMC Pulm. Med. 2021, 21, 165. [CrossRef] 29. Yokoyama, A.; Kondo, K.; Nakajima, M.; Matsushima, T.; Takahashi, T.; Nishimura, M.; Bando, M.; Sugiyama, Y.; Totani, Y.; Ishizaki, T.; et al. Prognostic Value of Circulating KL-6 in Idiopathic Pulmonary Fibrosis. Respirology (Carlton Vic.) 2006, 11, 164–168. [CrossRef] 30. Mohammad, A.J. An Update on the Epidemiology of ANCA-Associated Vasculitis. Rheumatology (Oxford) 2020, 59, iii42–iii50. [CrossRef] [PubMed]