Study Design and Population
This study was approved by the Institutional Review Board at the Tokyo Medical and Dental University (TMDU) in accordance with the Declaration of Helsinki (approval number M2019-010). The study consisted of two cohorts. First, a retrospective medical record-based observational cohort study, which served as a derivation cohort, was performed. One hundred two patients with ILD who were admitted to TMDU between June 2010 and July 2011 were included. Among the 102 patients, serum samples from 47 patients were stored. A total of 34 patients who were diagnosed with PF-ILD were analyzed (Figure 1A). Forty staff members from our hospital and department were evaluated as healthy volunteers (HVs). Second, a prospective observational cohort study, which served as a validation cohort, was performed. Informed consent from all of the patients was obtained. In addition to the patients recruited at the time of diagnosis, patients attending our outpatient department on a routine basis were recruited from December 2012 to July 2014. Patients with bronchial asthma, chronic obstructive pulmonary disease, and bronchiectasis were also recruited as disease controls during the same study period. Serum samples were collected from 39 patients with PF-ILD and 40 control subjects during the recruitment period (Figure1B). Patients with cancer at baseline and an estimated glomerular filtration rate (eGFR) of less than 50 ml/min/1.73m2 were excluded in the two cohorts. None of the patients experienced any episode of acute exacerbation of ILD prior to recruitment or overlapped in the two cohorts.
Clinical data of patients were included age, gender, smoking history, laboratory findings, physiological test results, radiological findings, and the etiology of ILD. The outcome of patients with PF-ILD was followed from baseline to 3 years. The clinical outcome assessed was all-cause death.
The diagnosis for IPF and non-IPF IIPs (NSIP and unclassified) was based on the criteria from the American Thoracic Society and European Respiratory Society [12, 13]. CTD was diagnosed based on the diagnostic criteria commonly used for each disease [14-16]. Chronic HP was diagnosed based on combinations of clinical, radiological, and histological criteria [17, 18]. Acute exacerbation of ILD was diagnosed based on diagnostic criteria for acute exacerbation of IPF .
Blood samples for measuring HE4 were taken using a routine procedure, centrifuged, aliquoted, and stored at -20°C or below until analysis. The concentration of serum HE4 was measured by chemiluminescent microparticle immunoassay on an ARCHITECT® i2000 analyzer (Abbott Japan LLC).
Evaluation of high-resolution computed tomography
HRCT scans were analyzed at the level of the aortic arch, carina trachea, and right pulmonary vein in each lung. The following 7 categories that were classified by parenchymal abnormalities were evaluated by one expert chest radiologist (M.K.) and one pulmonary specialist (Y.N.) with no knowledge of the clinical information: reticulation, honeycombing, centrilobular nodules, ground-glass opacity, consolidation, emphysema, and traction bronchiectasis (TBE). The extent of each radiologic abnormality, excluding TBE, was quantified as a percentage of lung parenchyma affected, to the nearest 5%, in each of the six lung zones. TBE was graded on a scale of 0-3 as follows: 0 = none; 1 = mild; 2 = moderate; and 3 = severe (Supplementary Figure S1) . The average total score for each variable was calculated as the mean score of the six zones. The readings of the two observers on the extent of each abnormality were combined by calculating the averages. Interobserver agreement on the extent of each abnormality was evaluated with the Spearman rank correlation coefficient.
Evaluation of histological findings
Lung tissues were obtained from 8 patients with PF-ILD who underwent SLB from 2005 to 2013 in our institution. The lung tissues were retrospectively selected and analyzed separately from this observational study. Control lung tissues were obtained from normal parts of lungs excised in lung cancer surgeries.
The lung tissue sections were subjected to antigen retrieval by autoclaving for 20 minutes in citrate buffer after deparaffinization. Endogenous peroxidase was quenched with 3% H2O2, and 5% goat serum and 1% BSA were used for blocking. After the addition of rabbit monoclonal antibody to HE4 (Abcam, Japan, Cat. No. ab200828) overnight at 4°C, the sections were incubated with a biotinylated secondary antibody (Vector Laboratories) for 60 minutes followed by ABC reagent (VECTASATAIN) for 30 minutes. Next, the sections were incubated in 3,3’-diaminobenzidine tetrahydrochloride (DAB; VECTOR) solution and counterstained with Meyers’ hematoxylin solution. Negative controls were incubated without primary antibody.
Differences between the groups were analyzed by Student’s t-test, Welch’s t test, Mann-Whitney U test, or Pearson’s chi-square test. Correlations were determined by the Spearman rank test. The most suitable cutoff level for serum HE4 levels was determined using a receiver operating characteristic (ROC) curve analysis with the highest Youden index (i.e., sensitivity＋specificity-1). The Kaplan-Meier method was used to assess survival curves with GraphPad Prism version 7 (Graph Pad Software Inc., La Jolla, CA, USA). The log-rank test was used to evaluate the statistical significance of differences between the higher HE4 and lower HE4 groups. Cox proportional hazards models were used to examine the influences of serum HE4 levels on the prognosis of patients with PF-ILD. Statistical analyses were performed using EZR software version 1.37 (Saitama Medical Center, Jichi Medical University, Saitama, Japan) . A P value < 0.05 was considered statistically significant.