Autoimmune Markers in Pulmonary Fibrosis and Emphysema
This multicentre study enrolled consecutive patients meeting the criteria for CPFE, as described by Cottin et al. and patients with IPF according to the new ATS/ERS/JRS/ALAT 2011 criteria diagnosed in three different hospitals, (University Hospital of Alexandroupolis, General Hospital of Kavala and General Hospital of Serres, Greece), during the period of time between September 2004 and August 2010. The latter group served as control. Disease diagnosis was based on a multidisciplinary approach (chest physician, radiologist and pathologist). Clinical and serological data for cases included in the study were collected and analyzed on a retrospective basis. Patients were followed-up by August 2012 where the last data entry (clinical, serological, survival) in our retrospective analysis occurred. According to Greek legislation informed consent is not needed for retrospective analyses of data corresponding to current practice. This study was approved by Local Ethics Committee and the institutional review board of the University Hospital of Alexandroupolis, Democritus University of Thrace (579-03-2012).
All patients (n = 100) enrolled in the study underwent a complete clinical and serum rheumatologic review based on the following criteria: based on the following criteria: 1) American College of Rheumatology (ACR) 1987 revised criteria for the classification of rheumatoid arthritis (RA); 2) the preliminary ACR criteria for systemic sclerosis (SSc); 3) the criteria proposed by Kahn et al. for mixed CTD; 4) the American–European Consensus Group criteria for Sjogren's syndrome; and 5) the criteria for polymyositis and dermatomyositis described by Troyanov et al.. Serum immunologic included the following: 1) antinuclear antibodies (ANA) 2) anti-double strand (anti-ds) DNA antibodies 3) anti-extractable nuclear antigens (ENA) panel including anti-scl70, anti-Ro (SSA), anti-La (SSB), anti-Sm, anti-RNP, 4) rheumatoid factor and anti-CCPs, 5) myositis panel including anti-Jo1 antibodies 6) complement 3 and 4 (C3,4) levels 7) antineutrophil cytoplasm antibodies (ANCAs) against myeloperoxidase (MPO) and proteinase-3 (PR-3). Assessment of the above panel of immunologic markers was performed at the following serial time points: 1) At time of diagnosis as a part of routine work-up in order to exclude collagen vascular diseases as a cause of lung fibrosis, and 2) At regular follow-up every 6 months or earlier than 6 months if clinical signs compatible with autoimmune disease (arthralgia, muscle weakness, dysphagia, Raynaud's phenomenon, photosensitivity) or disease exacerbation (dyspnea deterioration, hemoptysis, hematuria) emerged.
All patients were subjected to HRCT of the thorax to set the diagnosis of CPFE and IPF. One mm thick- HRCT sections were acquired supine at full inspiration, at 10 mm intervals with a helical CT scanner (General Electric Prospeed Series). HRCT studies at the time of disease diagnosis were evaluated independently by 2 radiologists who were blinded to immunology and histopathology profile but were aware that the patients had CPFE syndrome. Discrepancies were resolved by consensus. HRCT studies were evaluated at five predetermined levels using a modification of a semiquantitative HRCT scoring system previously described. HRCT scans were evaluated for the presence and extent of emphysema and the total extent of interstitial lung disease including the presence of reticular pattern, ground glass pattern and honeycombing. The above mentioned HRCT findings were analyzed and evaluated according to the Fleischner glossary of terms. HRCT images were scored at five predetermined levels: 1) origin of great vessels; 2) main carina; 3) pulmonary venous confluence; 4) halfway between the third and fifth section; 5) immediately above the right hemi-diaphragm.
Based on ATS/ERS/JRS/ALAT 2011 criteria for IPF diagnosis video-assisted thoracoscopic surgery (VATS) procedure was performed and biopsy samples from two different lobes were obtained in selective number of cases in order to establish a more rigid diagnosis of IPF and to exclude other patterns of IIPs. Definite usual interstitial pneumonia (UIP) pattern was based on the presence of the following 4 criteria: 1) evidence of marked fibrosis, architectural distortion and honeycombing in a predominantly subpleural distribution, 2) presence of patchy involvement of lung parenchyma by fibrosis, 3) presence of fibroblastic foci, 4) absence of features against a diagnosis of UIP including hyaline membranes, organizing pneumonia, granulomas, predominant airway centered changes and marked interstitial inflammatory cell infiltrates away from honeycombing. Probable UIP pattern was defined by the presence of criteria numbers 1 and 4 and the absence of criteria numbers either 2 or 3, but not both. On the other hand NSIP histopathological diagnosis was defined as the presence of marked interstitial chronic inflammation, and/or interstitial fibrosis lacking the temporal heterogeneity pattern and the patchy features of UIP and the absence of fibroblastic foci.
Two tissue microarray blocks comprising of a total of 58 lung tissue samples including 15 lung fibrosis biopsy samples of different histopathologic patterns derived from patients with CPFE, 28 lung samples from patients with IPF and 15 control tissues extracted from the normal part of the lung removed for benign lesions, was constructed as previously reported. Briefly, tissue cylinders of 2.0 mm diameter were punched from selected areas of each "donor" block by utilizing a thin-wall stainless tube from a precision instrument (TMA-100, Chemicon, USA) and were transferred by a solid stainless stylet into defined array coordinates in a 45 * 20 mm new recipient paraffin block. Each tissue element in the array was 2.0 mm in diameter and spacing between two adjacent elements was 0.1 mm. After the tissue microarray construction 3 μm sections for immunohistochemical analysis were cut from the "donor" blocks and were transferred to glass slides using an adhesive-coated tap sectioning system.
Immunohistochemistry (IHC) was performed by using specific monoclonal antibody for CD20 (mouse anti-human, DAKO), as previously reported. The number of CD20 positive cells in 5 fields per case was counted by two independent pathologists - observers using the high-resolution DUET, BioView scanning system for IHC morphology and immunocytochemistry applications, at ×100 magnification. All cell counts were expressed as cells/mm.
Statistical analysis was carried out using SPSS 14.0 software and Origin pro 8. Results are expressed as mean ± SD, or median (range), unless otherwise indicated. Pearson's chi-square was used to compare frequencies of ANA and ANCA positivity among two studied populations (CPFE and IPF). Independent t-test was used to compare serological, pulmonary functional parameters between patients with CPFE and IPF, as well as CD20 positive cells/mm between CPFE and control lung samples. Spearman's correlation was performed to find relationship between CD20 positive cells/mm and median survival in patients with CPFE and IPF. A p-value of < 0.05 was considered as statistically significant.
Methods
Study Design
This multicentre study enrolled consecutive patients meeting the criteria for CPFE, as described by Cottin et al. and patients with IPF according to the new ATS/ERS/JRS/ALAT 2011 criteria diagnosed in three different hospitals, (University Hospital of Alexandroupolis, General Hospital of Kavala and General Hospital of Serres, Greece), during the period of time between September 2004 and August 2010. The latter group served as control. Disease diagnosis was based on a multidisciplinary approach (chest physician, radiologist and pathologist). Clinical and serological data for cases included in the study were collected and analyzed on a retrospective basis. Patients were followed-up by August 2012 where the last data entry (clinical, serological, survival) in our retrospective analysis occurred. According to Greek legislation informed consent is not needed for retrospective analyses of data corresponding to current practice. This study was approved by Local Ethics Committee and the institutional review board of the University Hospital of Alexandroupolis, Democritus University of Thrace (579-03-2012).
Serum Immunologic Profile
All patients (n = 100) enrolled in the study underwent a complete clinical and serum rheumatologic review based on the following criteria: based on the following criteria: 1) American College of Rheumatology (ACR) 1987 revised criteria for the classification of rheumatoid arthritis (RA); 2) the preliminary ACR criteria for systemic sclerosis (SSc); 3) the criteria proposed by Kahn et al. for mixed CTD; 4) the American–European Consensus Group criteria for Sjogren's syndrome; and 5) the criteria for polymyositis and dermatomyositis described by Troyanov et al.. Serum immunologic included the following: 1) antinuclear antibodies (ANA) 2) anti-double strand (anti-ds) DNA antibodies 3) anti-extractable nuclear antigens (ENA) panel including anti-scl70, anti-Ro (SSA), anti-La (SSB), anti-Sm, anti-RNP, 4) rheumatoid factor and anti-CCPs, 5) myositis panel including anti-Jo1 antibodies 6) complement 3 and 4 (C3,4) levels 7) antineutrophil cytoplasm antibodies (ANCAs) against myeloperoxidase (MPO) and proteinase-3 (PR-3). Assessment of the above panel of immunologic markers was performed at the following serial time points: 1) At time of diagnosis as a part of routine work-up in order to exclude collagen vascular diseases as a cause of lung fibrosis, and 2) At regular follow-up every 6 months or earlier than 6 months if clinical signs compatible with autoimmune disease (arthralgia, muscle weakness, dysphagia, Raynaud's phenomenon, photosensitivity) or disease exacerbation (dyspnea deterioration, hemoptysis, hematuria) emerged.
HRCT Evaluation
All patients were subjected to HRCT of the thorax to set the diagnosis of CPFE and IPF. One mm thick- HRCT sections were acquired supine at full inspiration, at 10 mm intervals with a helical CT scanner (General Electric Prospeed Series). HRCT studies at the time of disease diagnosis were evaluated independently by 2 radiologists who were blinded to immunology and histopathology profile but were aware that the patients had CPFE syndrome. Discrepancies were resolved by consensus. HRCT studies were evaluated at five predetermined levels using a modification of a semiquantitative HRCT scoring system previously described. HRCT scans were evaluated for the presence and extent of emphysema and the total extent of interstitial lung disease including the presence of reticular pattern, ground glass pattern and honeycombing. The above mentioned HRCT findings were analyzed and evaluated according to the Fleischner glossary of terms. HRCT images were scored at five predetermined levels: 1) origin of great vessels; 2) main carina; 3) pulmonary venous confluence; 4) halfway between the third and fifth section; 5) immediately above the right hemi-diaphragm.
Extent of emphysema
The total extent of emphysema was estimated to the nearest five percent in each of the five sections, with global extent of emphysema on HRCT computed as the mean of the scores. To establish the diagnosis of CPFE on HRCT a global extent of emphysema higher than 5% was set as threshold.
Total extent of interstitial lung disease
The total extent of interstitial lung disease was estimated to the nearest five percent in each of the five sections, with global extent of disease on HRCT computed as the mean of the scores.
Histopathology Evaluation
Based on ATS/ERS/JRS/ALAT 2011 criteria for IPF diagnosis video-assisted thoracoscopic surgery (VATS) procedure was performed and biopsy samples from two different lobes were obtained in selective number of cases in order to establish a more rigid diagnosis of IPF and to exclude other patterns of IIPs. Definite usual interstitial pneumonia (UIP) pattern was based on the presence of the following 4 criteria: 1) evidence of marked fibrosis, architectural distortion and honeycombing in a predominantly subpleural distribution, 2) presence of patchy involvement of lung parenchyma by fibrosis, 3) presence of fibroblastic foci, 4) absence of features against a diagnosis of UIP including hyaline membranes, organizing pneumonia, granulomas, predominant airway centered changes and marked interstitial inflammatory cell infiltrates away from honeycombing. Probable UIP pattern was defined by the presence of criteria numbers 1 and 4 and the absence of criteria numbers either 2 or 3, but not both. On the other hand NSIP histopathological diagnosis was defined as the presence of marked interstitial chronic inflammation, and/or interstitial fibrosis lacking the temporal heterogeneity pattern and the patchy features of UIP and the absence of fibroblastic foci.
Tissue Microarrays
Two tissue microarray blocks comprising of a total of 58 lung tissue samples including 15 lung fibrosis biopsy samples of different histopathologic patterns derived from patients with CPFE, 28 lung samples from patients with IPF and 15 control tissues extracted from the normal part of the lung removed for benign lesions, was constructed as previously reported. Briefly, tissue cylinders of 2.0 mm diameter were punched from selected areas of each "donor" block by utilizing a thin-wall stainless tube from a precision instrument (TMA-100, Chemicon, USA) and were transferred by a solid stainless stylet into defined array coordinates in a 45 * 20 mm new recipient paraffin block. Each tissue element in the array was 2.0 mm in diameter and spacing between two adjacent elements was 0.1 mm. After the tissue microarray construction 3 μm sections for immunohistochemical analysis were cut from the "donor" blocks and were transferred to glass slides using an adhesive-coated tap sectioning system.
Immunohistochemistry and Semi-quantitative Image Analysis
Immunohistochemistry (IHC) was performed by using specific monoclonal antibody for CD20 (mouse anti-human, DAKO), as previously reported. The number of CD20 positive cells in 5 fields per case was counted by two independent pathologists - observers using the high-resolution DUET, BioView scanning system for IHC morphology and immunocytochemistry applications, at ×100 magnification. All cell counts were expressed as cells/mm.
Statistical Analysis
Statistical analysis was carried out using SPSS 14.0 software and Origin pro 8. Results are expressed as mean ± SD, or median (range), unless otherwise indicated. Pearson's chi-square was used to compare frequencies of ANA and ANCA positivity among two studied populations (CPFE and IPF). Independent t-test was used to compare serological, pulmonary functional parameters between patients with CPFE and IPF, as well as CD20 positive cells/mm between CPFE and control lung samples. Spearman's correlation was performed to find relationship between CD20 positive cells/mm and median survival in patients with CPFE and IPF. A p-value of < 0.05 was considered as statistically significant.
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