Clinical Characteristics of Interstitial Pneumonia With Autoimmune Features (IPAF) - a Multicenter Prospective Study
Clinical Characteristics of Interstitial Pneumonia With Autoimmune Features (IPAF) - a Multicenter Prospective Study
Interstitial pneumonia with autoimmune features (IPAF) was defined in 2015 by the Working
 Group of the European Respiratory Society (ERS) and the American Thoracic Society (ATS) as
 interstitial pneumonia with some clinical and/or serological features suggesting presence of
 an underlying autoimmune disorder. However, ofiicial criteria for diagnosis of an autoimmune
 disease are not met.

 Aims of the study:

 1. Determine the incindence of IPAF in comparison with interstitial lung diseases (ILDs)
 and classic autoimmune diseases (ADs) in polish pulmonological centers.

 2. Clinical, serological, functional and radiological and histopathological characteristics
 of IPAF patients.

 3. Analysis of diagnostic strategies towards specific IPAF subgroups.

 4. Characterictics of potencial diagnostic, predictive and prognostic features of IPAF.

 5. Prospective assessment of IPAF patients in the courseof 5 years in order to determine
 stability of the diagnosis and potential progression to other diseases, e.g. ADs.
The following patient groups will be studied:

 1. Study group - IPAF.

 2. Control groups:

 1. Connective tissue disease associated interstitial lung disease (CTD-ILD) patients:
 .rheumatoid arthritis - RA, systemic sclerosis - SSc, polymyositis - PM,
 dermatomyositis - DM, (anti-synthetase syndrome - AS, Sjoegren's syndrome - SjS,
 mixed connective tissue disease - MCTD ,systemic lupus erythematosus - SLE,
 diagnosed according to diagnostic criteria issued by European League Against
 Rheumatism (EULAR) and/or American College of Rheumatology (ACR)

 2. Idiopathic interstitial pneumonia group: idiopathic pulmonary fibrosis - IPF,
 nonspecific interstitial pneumonia - NSIP, cryptogenic organizing pneumonia - COP,
 acute interstitial pneumonia - AIP; respiratory bronchiolitis associated
 interstitial lung disease - RB-ILD, desquamative interstitial pneumonia - DIP,
 lymphocytic interstitial pneumonia - LIP).

 Methods Patients will be assessed by a pulmonologist and rheumatologist.
 Questionnaires regarding clinical symptoms, concomitant diseases and their
 treatment, disease activity will be fulfilled.

 The majority of diagnostic tests and procedures are routinely performed during
 clinical care.

 The study will be divided into 2 phases:

 1. Phase 1 - visit 1

 2. Phase 2 - observation of selected subjects from visit 1 to 5

 Phase 1:

 30 patients meeting the eligibility criteria, including individuals with a long
 medical history and currently undergoing treatment for interstitial lung disease/
 autoimmune disorder are going to be recruited to the study in every clinical
 center, totaling up to:

 -  210 patients in IPAF group

 -  210 patients in CTD-ILD group

 -  210 patients in IIP group

 Phase 2 Only newly diagnosed patients (individuals who have not been previously
 diagnosed with interstitial lung disease, and such diagnosis was set during phase 1
 of the study) will be recruited to this phase. Due to rare incidence, the
 recruitment phase may take up to 36 months.

 The total number of patients will be

 -  70 in IPAF group

 -  70 in CTD-ILD group (RA, SSc, PM, DM)

 -  70 in IIP group (35 with IPF and 35 with NSIP, COP and LIP)

 Visit 1:

 • The following tests and procedures will be performed:

 -  Detailed anamnesis obtained through 'visit 1 questionnaire' [non-routine
 procedure]

 -  Lung function tests (spirometry with reversibility testing, plethysmography,
 diffusion lung capacity for carbon monoxide) [routine procedure]. Absolute
 value and % of the reference value will be assessed.

 FEV1 - Forced expiratory volume in 1 second FVC - Forced vital capacity VC - Vital
 capacity FEV1/VC, ratio TLC - Total lung capacity, DLCO SB - The carbon monoxide
 diffusion capacity (single breath method) ITGV - intrathoracic gas volume RV/TLC -
 Residual volume/ Total lung capacity ratio

 -  validated methods of cough assessment [routine procedure]: Chung cough
 questionnaire and Visual analogue scale (VAS).

 -  validated methods of dyspnea assessment [routine procedure]: Likert dyspnea
 questionnaire and modified Borg scale.

 Blood testing. In total 30 ml blood will be drawn (15 ml blood into a plain
 microtube without anticoagulant and 15ml blood into anticoagulation microtube).
 Detailed description below:

 NT-proBNP, laboratory tests performed during diagnostic process of rheumatologic
 diseases and potential IPAF markers [routine procedure]. 15 ml venous blood will be
 drawn and divided into 2 microtubes (10 ml and 5 ml) without anticoagulant 10 ml
 microtube will be expedited to a local laboratory in order to measure plasma levels
 of NT-proBNP and the following autoantibodies' titer:

 -  rheumatoid factor (RF),

 -  Anti-Citrullinated Protein Antibodies (ACPA; anti-CCP),

 -  Antinuclear Antibodies (ANA) (ANA 1 test performed as screening); in case of
 ANA presence and further staining pattern identification (e.g. homogenous,
 speckled, peripheral, nucleolar, centromere), further tests will be performed
 (ANA 2 and ANA 3):

 -  anti-dsDNA,

 -  anti-Sm,

 -  anti-SS-A (Ro),

 -  anti-SS-B (La),

 -  anti-Scl-70,

 -  anti-RNP,

 -  anti-Jo-1 and anti-PL-7, anti-PL12,

 -  anti-Mi-2,

 -  anti-PM-Scl,

 -  anti-MDA5. From the second 5 ml microtube, after clot has formed, ca
 2,5ml serum will be drawn away with a pipette; serum will be later
 divided into 5 Eppendorf type microtubes (0,5 ml each). These microtubes
 should be labeled according to the following: first letter of the
 patient's name- first letter of the patient's surname/ gender F or M/date
 of birth DD-MM-YY/date of sample collection DD-MM-YY, e.g.
 AL/K/12.05.45/01.02.18. The samples will be stored in -70 °C. The samples
 will be collected by a courier and transported to the clinical center
 responsible for this part of the study. This sample will be used to test
 for potential IPAF markers, e.g. chemokine C-C motif ligand 18 (CXCL18),
 Surfactant Protein A- (SP-A), Surfactant Protein D (SP-D), Krebs von den
 Lungen-6 protein (KL-6) and chitotrisidase 1 (CHIT1) [non-routine
 procedure].

 Blood sample collection [procedure not routinely performed during clinical care].
 15 ml of whole venous blood will be drawn and then divided into 10 Eppendorf tubes
 1, 5 ml each. The tubes will be labeled as described above. The specimens will be
 stored in -70 °C. The samples will be transported to the clinical center
 responsible for this part of the study.

 Optionally, additional tests (testing for proteomic and metabolomic biomarkers)
 will be performed in samples of biological material collected beforehand (BALF,
 serum, urine [non-routine procedure].

 o Bronchofiberoscopy with bronchalveolar lavage (BF + BAL), performing biopsy of
 the bronchi mucosa: BF + BAL [routine procedure]: before starting BF, regional
 anesthesia and sedation with use of lidocaine and midazolam will be performed,
 according to anesthesia protocols applied in respective endoscopy units.
 Intravenous cannula will be inserted prior to BF. During endoscopy, the patient
 will be monitored according to safety protocols applied in respective endoscopy
 units.

 Bronchoalveolar lavage will be performed in the bronchus from the middle robe of
 right lung or the lingula of left lung (B4, B5). Localization will be chosen based
 on HRCT results and will be recorded in patient's medical history. Sterile solution
 of 0, 9% NaCl will be instilled. 200 ml fluid will be applied with a syringe in
 portions of 25 ml or 50 ml. It is recommended that minimum 60 % of the lavage fluid
 is retrieved.

 Bronchoalveolar lavage fluid (BALF) testing. Preparation of microscopic slides,
 analysis of the cellular components of the specimen (kind of the most abundant
 cells retrieved, the cells' durability) will be performed according to a standard
 laboratory procedure (guidelines of the Polish Respiratory Society). Total cell
 count along with cells' durability will be measured in a sample taken from the
 filtrate or material after the first spin. Cytospin (routinely 10 min of 1200-2000
 rpm) will be used to prepare the material. May-Grunewald-Giemsa stain will be used
 to prepare the microscopic slides.

 BALF samples will be secured and stored in order to test for potential IPAF
 markers. 15 ml BALF will be divided into Eppendorf 1,5 ml microtubes and then
 stored in - 70 C. Samples will be checked for concentration of S1009A protein,
 chemokine (C-C motif) ligand 2 and chitotrisidase 1 (CHIT 1).

 Specimens of bronchial mucosa taken during BF [procedure not routinely performed
 during clinical care]. 5 mucosa specimens will be taken from the initial segment of
 the middle lobe bronchus, placed into Eppendorf microtubes and stored in -80 C.
 Specimen will be sent for histopathological assessment.

 -  6 minute walking test - (6MWT) performed according to a protocol applied in
 respective clinical centers [routine procedure]. The following data should be
 recorded:

 • Whether the patient completed the test. If 6MWT was interrupted, the reason
 should be recorded (e.g. dyspnea, high blood pressure, cardiac arrhythmia,
 intermittent claudication, other)

 • Distance in meters

 -  Pulse oximetry measured before and directly after the test

 -  Dyspnea measurement - Borg scale

 -  High resolution computed tomography (HRCT) [routine procedure]. Current
 tomography, performed according to protocol applied in respective clinical
 centers or CT 6 months prior to the study will be eligible. Test results
 (saved on CD) and radiological description will be used. CD will be labeled in
 a way described above. OsiriX Lite software will be used to assess the scans.
 The following densitometric values will be measured: mean lung attenuation
 (MLA), kurtosis, skewness, and standard deviation of lung radiodensity (SD
 I.R). Data analysis will be performed with Statistica software. Data will be
 presented as median and interquartile ranges (IQR). Quantitative data will be
 analyzed with Kruskal-Willis' and Dunn's post hoc tests

 -  Pulse oximetry (SpO2) [routine procedure]. If SpO2 is measured to be < 92% or
 if there are indications for oxygen therapy, arterial blood gas will be
 performed.

 -  Transthoracic echocardiography (TTE) [routine procedure] with detailed right
 heart assessment:

 Pulmonary artery diameter Acceleration time (Act) and pulmonary regurgitation
 Tricuspid annulus systolic velocity Tricuspid regurgitation velocity. Tricuspid
 annular plate systolic excursion, (TAPSE) Basal right ventricle (RV) diameter, mid
 RV diameter and base-apex dimension Right ventricle inflow tract dimension Right
 ventricle outflow tract dimension Right ventricle diameter measured at the level of
 pulmonary valve Right atrial volume Superior vena cava dimension Inferior vena cava
 dimension Right ventricular wall thickness (RVWT) o Rheumatology consultation of
 every patient in IPAF and autoimmune disorder groups [procedure routinely performed
 during clinical care].

 Qualification for visit 2-5 - only patients qualified to phase 2 of the study

 Visit 2:

 • 6 +/- 3 months from visit 1:

 -  Visit 1-5 questionnaire [non-routine procedure]

 -  Lung function tests [routine procedure]

 -  6MWT [routine procedure]

 -  Pulse oximetry [routine procedure]

 Visit 3:

 • 12 +/- 3 months from visit 1:

 -  Visit 1-5 questionnaire [non-routine procedure]

 -  Lung function tests [routine procedure]

 -  6MWT [routine procedure]

 -  HRCT [routine procedure]

 -  Blood testing (basic laboratory tests [routine procedure], screening for
 autoantibodies and cytokine concentration [non-routine procedure]

 -  BF + BAL [optionally; routine procedure]

 -  Arterialized capillary blood gas [routine procedure]

 -  TTE with detailed right heart assessment [routine procedure]

 -  Rheumatology consultation [routine procedure]

 Visit 4:

 • 24 +/- 3 months from visit 1:

 -  Visit 1-5 questionnaire [non-routine procedure]

 -  Lung function tests [routine procedure]

 -  6MWT [routine procedure]

 -  HRCT [routine procedure]

 -  Blood testing (basic laboratory tests [routine procedure], screening for
 autoantibodies and cytokine concentration [non-routine procedure]

 -  BF + BAL (optionally; routine procedure]

 -  Arterialized capillary blood gas [routine procedure]

 Visit 5

 • 60 +/- 3 months from visit 1

 o Visit 1-5 questionnaire [non-routine procedure]

 o Lung function tests [routine procedure]

 o 6MWT [routine procedure]

 -  HRCT [routine procedure]

 -  Blood testing (basic laboratory tests [routine procedure], screening for
 autoantibodies and cytokine concentration [non-routine procedure]

 -  BF + BAL (optionally; routine procedure)

 -  Arterialized capillary blood gas testing [routine procedure]

 -  TTE with detailed right heart assessment [routine procedure]

 -  Rheumatology consultation [routine procedure]

 -  Optionally, in case of clinical and/or radiological progression, worsening of
 lung function tests' results, criobiopsy. may be repeated after obtaining
 informed patient consent.
Inclusion Criteria:

 -  written informed consent to participate in the study

 -  diagnosis of one of interstitial lung diseases/autoimmune diseases as described below

 Exclusion Criteria:

 -  age < 18 years

 -  withdrawal od consent

 -  pregnancy

 -  lactation

 -  infectious disease 4 weeks prior