Quantitative analysis of lung elastic fibers in idiopathic pleuroparenchymal fibroelastosis (IPPFE): comparison of clinical, radiological, and pathological findings with those of idiopathic pulmonary fibrosis (IPF)

Six consecutive patients with IPPFE who underwent surgical lung biopsy or autopsy in our hospital or affiliated hospitals from 2010 to 2011 were evaluated. The pathological diagnosis of IPPFE was based on a previous report by Frankel et al. [2]. Briefly, the diagnosis of IPPFE was based on the presence of prominent subpleural fibroelastosis, sparing of the parenchyma distant from the pleura, and a relatively abrupt border between the fibroelastosis and the underlying normal parenchyma (Figure 1B and C). Clinical, radiological, and pathological data of these patients were retrospectively reviewed. Patients who met the criteria for any connective tissue disorders were excluded from this study.

Twenty-eight consecutive patients with IPF who underwent surgical lung biopsy in our hospital from 1997 to 2007 were also evaluated in this study. The initial diagnosis of IPF was based on the latest diagnostic criteria of that period. All 28 patients also met the 2011 IPF consensus criteria of the ATS, ERS, Japanese Respiratory Society (JRS), and Latin American Thoracic Association (ALAT) [5]. The histologic features of UIP were based on a previously published reports [5, 9]. No lung specimens had a “not UIP pattern” as defined by the IPF consensus criteria (i.e., presence of hyaline membranes, organizing pneumonia, granulomas, marked interstitial inflammatory cell infiltrate away from honeycombing, or predominant airway-centered changes) [5]. The study protocol was approved by the Ethics Committee of Hamamatsu University School of Medicine (approval number 24-167).

All available lung specimens were examined. Samples were fixed in neutral buffered 10% formalin and embedded in paraffin. Staining was performed on 4-μm sections mounted on glass slides. Sections were stained by HE or Weigert’s resorcin-fuchsin solution. Images of sections stained with Weigert’s elastic staining were collected using a microscope with a CCD camera (DP21; Olympus, Tokyo, Japan). Images of fibrotic lesions at × 40 magnification were obtained from all slides (Figure 2A). For each image, the area occupied by EF was quantified using imaging software (Image J; National Institutes of Health, Bethesda, MD and Photoshop Elements; Adobe, San Jose, CA), according to our previous report [8]. Images were converted to gray scale, then binarized with two different thresholds to quantify the area of the whole fibrotic lung lesion (Figure 2B, blue area) and the area of EF (Figure 2C, red area). The proportion of EF in the fibrotic interstitial area was calculated by dividing the number of pixels containing EF (red area) by that of the whole fibrotic lesion (blue area). For each patient, the mean proportion of EF in all histological images was calculated and expressed as a percentage (the EF score).

The investigators examining the histological specimens also evaluated the collagen deposition, cellularity, and organization in the air spaces. The severity of each of these findings was scored on a scale from 0 to 3 (0, none; 1, mild; 2, moderate; 3, severe). The presence of honeycombing, lymphoid hyperplasia, and pleuritis was also recorded. These findings were reviewed by two observers and the agreement rates between the observers were evaluated by weighted-kappa coefficients. The coefficients ranged from 0.34 to 0.68. When the score differed between the observers, a consensus was reached after discussion.

The extent of lung fibrosis was measured using high-resolution computed tomography (HRCT) on slices taken at the tracheal bifurcation, the base of the lower lobes, and at the midpoint. The extent of fibrosis in each lobe was scored using the following system: 0, none; 1, 1–10%; 2, 11–25%; 3, 26–50%; 4, 51–75%; and 5, 76–100%. The sum of the scores from five lobes (0–25) was used to express the extent of fibrosis throughout each patient’s lungs. The severity of ground-glass opacity and consolidation was scored using the same scale as that used for the pathologic evaluation. Finally, the presence of honeycombing on HRCT was also evaluated. These findings were reviewed by two observers; the agreement rates between them were evaluated by weighted-kappa coefficients. The coefficients ranged from 0.34 to 0.83. When the score differed between the observers, a consensus was reached after discussion.

Clinical data, including sex, age, smoking history, symptoms, treatment, and survival were obtained from the patients’ medical records. Laboratory findings, pulmonary function tests results, and bronchoalveolar lavage (BAL) data at the time of surgical lung biopsy or before autopsy were also recorded.

Statistical analyses were performed using StatView J-4.5 (SAS Institute, Inc., Cary, NC, US). Categorical data were compared using the chi-square test or Fisher’s exact probability test for independence. Continuous data were compared using a paired or unpaired Student’s t-test. Discontinuous data were compared using Mann-Whitney’s U-test. The relationship between the EF score and serial data was analyzed using Pearson’s correlation coefficient, and that between the EF score and discrete variable data was analyzed using Spearman’s rank correlation coefficient. The overall survival of each group was estimated by Kaplan-Meier curves. The log-rank test was used to compare survival between the two groups. All tests were two-sided, and statistical significance was defined as p < 0.05.

Six consecutive patients with IPPFE who underwent surgical lung biopsy or autopsy were evaluated. One patient was autopsied after acute exacerbation of lung disease. Antinuclear antibody was positive at 1:40 in three patients, and rheumatoid factor was positive in two patients. Anti-cyclic citrullinated peptide antibody was positive in one patient. Anti-double-strand DNA antibody and anti-cardiolipin β2 glycoprotein 1 antibody were positive in one patient. However, no patients met the criteria for any connective tissue disorders. No patients had a family history of interstitial lung disease or a history of dust exposure. One patient had undergone chemotherapy for gingival carcinoma. Although repeated bacterial pneumonia had occurred in one patient, no patients had a history of fungal infection such as pulmonary aspergillosis. Refractory pneumothorax appeared shortly after the surgical lung biopsy in one patient.

Representative HRCT and surgical lung biopsy findings specimens from one patient with IPPFE are shown in Figure 1. HRCT showed pleural thickening and subpleural consolidation opacities in the upper lobes (Figure 1A). An HE-stained lung section revealed subpleural fibrosis with abrupt transition to underlying normal lung parenchyma and fibroblastic foci, similar to UIP (Figure 1B). Pleural fibrosis was also seen. Deposition of dense EF (elastosis) in subpleural fibrotic lung lesions, which is a specific histological finding of IPPFE, was seen on a section with Elastica-van Gieson (EVG) staining (Figure 1C).

The clinical characteristics of patients with IPPFE and IPF are compared in Table 1. Five of the six patients with IPPFE were male, and their mean age of the six patients was 69.2 years. The observation period was 39.8 ± 29.2 (mean ± SD) months. Patients with IPPFE had a higher proportion of never-smokers (50% vs. 7.1%, p = 0.011) and a lower mean body mass index (17.9 vs. 24.3 (mean), p < 0.0001) than did patients with IPF. Fewer patients with IPPFE than IPF had fine crackles at the time of diagnosis (50.0% vs. 96.4%, p = 0.012). In addition, patients with IPPFE had more pneumothoraces during the observation period (66.7% vs. 3.6%, p = 0.002). Refractory pneumothorax appeared shortly after surgical lung biopsy in one patient with IPPFE. Although the proportion of patients who underwent medical check-ups to detect interstitial pneumonia was not different between the two groups, the period from detection of interstitial pneumonia to acquisition of lung specimens was significantly longer in patients with IPPFE than in those with IPF (73.3 vs. 29.7 months, p = 0.012). Four of the six patients with IPPFE received therapeutic interventions. Steroid treatment was started in one patient, whilst pirfenidone was administrated to the other three patients. However, these treatments showed no therapeutic effects, and three of the six patients with IPPFE died during the study period. One died of acute exacerbation of IPPFE, and the other two died of chronic disease progression.

The laboratory, physiologic, and BAL data between the patients with IPPFE and IPF are compared in Table 2. The serum levels of interstitial pneumonia markers, such as lactate dehydrogenase (LDH), Krebs von den Lungen-6 (KL-6), and surfactant protein D (SP-D), were not different between the two groups. The forced vital capacity (FVC) was significantly lower in patients with IPPFE than in those with IPF (62.7% vs. 88.6% predicted, p = 0.009), although the diffusion lung capacity for carbon monoxide (DLCO) was not significantly different (p = 0.395). Furthermore, the proportions of neutrophils and eosinophils in the BAL were higher in patients with IPPFE than in those with IPF (2.5% vs. 0.6%, p = 0.018 and 1.7% vs. 0.4%, p = 0.011, respectively).

The HRCT and lung specimen findings in patients with IPPFE or IPF are summarized in Table 3. All patients with IPPFE showed upper lobe predominance and pleural thickening on HRCT (p < 0.0001). Higher consolidation scores on HRCT were found in patients with IPPFE than in those with IPF (1.7 vs. 0.3, p = 0.004). No significant differences were found in the other HRCT findings, such as the extent score or ground glass score.

HE-stained lung specimens showed pleural fibrosis or pleuritis underneath the dense subpleural fibroelastosis in five of the six patients with IPPFE (p < 0.0001). Significantly higher organizing pneumonia scores in the alveolar space were found in patients with IPPFE than in those with IPF (1.8 vs. 0.6, p = 0.003). Although the abrupt transition between the subpleural fibroelastosis and underlying normal lung area was more prominent in patients with IPPFE, no significant differences were found in the other pathological findings, such as the collagen deposition score or cellularity score.

Using a CCD-camera and analytic software, abundant EF were found in the fibrotic lung lesions with EVG staining, and the amount of EF was readily evaluated by our quantitative analytic method (Figure 2). The proportion of EF in fibrotic areas (the EF score) was assessed according to the procedure described in the Methods section. All EF scores are shown in Figure 3. EF scores in patients with IPPFE were significantly higher than those in patients with IPF (median, 28.3% [range, 25.1-35.6%] vs. 11.0% [range, 5.1-23.3%], p < 0.0001). In patients with IPPFE, no correlations were found between EF scores and %FVC (r = -0.058, p = 0.919) (Additional file 1: Figure S1A), between EF scores and %DLCO (r = -0.548, p = 0.384) (Additional file 1: Figure S1B), between EF scores and the change in FVC 12 months after biopsy (r = 0.446, p = 0.631) (Additional file 1: Figure S1C), or between EF scores and the period from detection of interstitial pneumonia until acquisition of lung specimens (r = 0.424, p = 0.433) (Additional file 1: Figure S1D).

Lung specimens were obtained from two different lobes in five of the six patients with IPPFE. EVG-stained sections showed fewer EF in the right lower lobe (Figure 4D-F) than that in the right upper lobe (Figure 4A-C) in the same patient with IPPFE. In this patient, the EF score in the lower lobe was 20.3% (Figure 4F), whilst that in the upper lobe was 32.4% (Figure 4C). All EF scores in both lobes are shown in Figure 5A. The EF scores in the lower lobes were significantly lower than those in the upper lobes in patients with IPPFE (median, 24.2% [range, 20.3-26.5%] vs. 32.4% [range, 24.9-39.6%], p = 0.048). Conversely, in patients with IPF, the difference in EF scores between the lower lobes and upper lobes was not significant (14.6 vs. 17.2%, p = 0.154) (Figure 5B and C). However, even in the lower lobes, EF scores in patients with IPPFE were still higher than those in patients with IPF (p < 0.0001) (Figure 5C) as well as those in the upper lobes (p = 0.0007) (Figure 5B).