High NHLRC2 expression is associated with shortened survival in lung adenocarcinoma.

Background: Certain variants of NHL repeat (named after NCL-1, HT2A and LIN-41)-containing protein 2 (NHLRC2) gene have been linked to severe fibrotic interstitial lung disease in children. The aim of the current study was to evaluate the expression of NHLRC2 in lung cell and tissue samples from patients with lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC). Methods: The expression of NHLRC2 in lung tissue samples was studied by immunohistochemistry (102 ADC, 111 SCC), mRNA in situ hybridization (4 ADC, 3 SCC), and Western blot analysis (3 ADC, 2 SCC). The immunohistochemical NHLRC2 expression was measured by image analysis software and the percentage of NHLRC2-positive cancer cells was evaluated by semiquantitative analysis. The immunohistochemical results of NHLRC2 were compared with the clinical and histological characteristics of the patients. NHLRC2 protein levels in primary stromal and epithelial lung cancer cell lines were measured by Western blot analysis. Results: NHLRC2 was mainly expressed in cancer cells and inflammatory cells within the tumor. The NHLRC2 expression evaluated by image analysis method was significantly higher in ADC compared with that in SCC (P<0.001). High NHLRC2 expression was associated with reduced disease specific survival (P=0.002), overall survival (P=0.001), and high mitotic activity (P=0.042) in ADC. Additionally, the proportion of NHLRC2-positive cancer cells analyzed by the semiquantitative method was significantly higher in ADC than in SCC (P<0.001). Conclusions: NHLRC2 expression was higher in lung ADC than in SCC and its expression was associated with poor survival in ADC patients. Further studies are required to clarify the pathogenetic role of NHLRC2 in lung cancer.

Inflammatory myositis-associated interstitial lung disease can be distinguished from that associated with other connective tissue diseases.

Background: Acute onset of interstitial lung disease (ILD) has been described in patients with idiopathic inflammatory myositis (IIM), but controlled studies about this issue are sparse. The aim of this study was to compare disease onset, demographics, and high-resolution computed tomography (HRCT) patterns in IIM-ILD and other connective tissue disease (CTD)-ILDs. Methods: Clinical and radiological data of 22 IIM-ILD and 132 other CTD-ILD patients was retrospectively gathered from hospital registries between January 2000 and November 2019. Data was re-assessed and compared using a multivariate analysis. Results: Compared to other CTD-ILDs, the patients with IIM-ILD were younger (59.7 vs. 68.0 years, P=0.023), more often non-smokers (71.4% vs. 45.7%, P=0.029) and displayed radiological nonspecific interstitial pneumonia/organizing pneumonia (NSIP/OP) overlap pattern more frequently (27.3% vs. 1.5%, P<0.001). The onset of ILD was acute with patients needing intensive care significantly more often in IIM-ILD than in other CTD-ILDs (22.7% vs. 2.3%, P<0.001). In most patients ILD was diagnosed before or simultaneously with IIM presentation unlike in other CTD-ILDs (90.9% vs. 47.7%, P<0.001). In multivariate analysis, NSIP/OP overlap pattern, acute onset disease treated in intensive care unit and ILD preceding or being diagnosed simultaneously with CTD were significantly associated with IIM-ILD. The multivariate model, supplemented with age, had excellent diagnostic performance identifying IIM-ILD [area under curve (AUC) 0.845]. Conclusions: Unlike other CTD-ILDs, IIM-ILD often develops acutely, simultaneously with the systemic disease. Therefore, clinicians should consider IIM-ILD as an option of differential diagnosis in patients with acute ILD and promptly test muscle enzymes as well as comprehensive autoantibody tests.

Several specific high-resolution computed tomography patterns correlate with survival in patients with idiopathic pulmonary fibrosis.

BACKGROUND: Evidence of honeycombing in high-resolution computed tomography (HRCT) is a recognized risk factor for shortened survival in patients with idiopathic pulmonary fibrosis (IPF), but few studies have evaluated the feasibility of exploiting other specific patterns for predicting survival. The aim of this study was to examine the extent of specific HRCT patterns in IPF and determine whether they correlate with clinical features, pulmonary function tests (PFT), and survival. METHODS: Both the presence and extent of specific HRCT patterns, such as traction bronchiectasis, honeycombing, architectural distortion, reticulation, emphysema, and ground glass opacity, in 129 HRCT examinations were scored semi-quantitatively in three zones of each lung. HRCT examinations were also re-classified according to the 2011 and 2018 international statements. Correlations were calculated between the scores of specific HRCT patterns, clinical features, PFT, and patient survival. RESULTS: The extent of traction bronchiectasis was found to be an independent risk factor of shortened survival (HR 1.227, P=0.001). Patients with a possible usual interstitial pneumonia (UIP) pattern had a better median survival than the patients with a definite UIP pattern (61 vs. 37 months, P=0.026). The extents of traction bronchiectasis, honeycombing, and architectural distortion displayed an inverse correlation with all PFT values at the time of diagnosis. There were few differences between the radiological classifications of the 2011 and 2018 international statements. CONCLUSIONS: We conclude that several specific HRCT patterns displayed a correlation with shortened survival in IPF; these may help in evaluating the risk of death in IPF patients.

Several high-resolution computed tomography findings associate with survival and clinical features in rheumatoid arthritis-associated interstitial lung disease.

OBJECTIVE: To compare the presence and extent of several high-resolution computed tomography (HRCT) observations in different subtypes of rheumatoid arthritis-related interstitial lung disease (RA-ILD) and to examine associations between radiological findings, hospitalization, age, RA duration, pulmonary function tests (PFT) and survival. MATERIALS AND METHODS: HRCTs from 60 RA-ILD patients were independently evaluated and re-categorized into usual interstitial pneumonia (UIP), nonspecific interstitial pneumonia (NSIP), organizing pneumonia (OP), diffuse alveolar damage (DAD) and unclassified subtypes by two radiologists. The presence and extent, which was reported using a semi-quantitative scoring system, of e.g. reticulation, ground-glass opacity, honeycombing, emphysema, traction bronchiectasis and architectural distortion were further evaluated and compared between the subtypes. Associations between radiological findings and survival were identified with the Kaplan-Meier method and Cox's univariate model. The correlations between radiological findings, hospitalization, age, pack years, RA duration and PFT were calculated using Spearman's correlation coefficient. RESULTS: The extents of reticulation (HR 1.144, p = 0.041), traction bronchiectasis (HR 1.184, p = 0.030), architectural distortion (HR 1.094, p = 0.044) and the presence of pleural fluid (HR 14.969, p < 0.001) were associated with decreased survival. A negative correlation was observed between ground-glass opacity (GGO) and the duration of RA (r = -0.308, p = 0.023). The extents of honeycombing (r = 0.266, p = 0.046), traction bronchiectasis (r = 0.333, p = 0.012) and architectural distortion (r = 0.353, p = 0.007) correlated with hospitalizations due to respiratory reasons. CONCLUSIONS: Many radiological findings associate with the course of the disease of RA-ILD and could potentially be useful when planning the RA treatment or evaluating the risk of death in these patients.

Are risk predicting models useful for estimating survival of patients with rheumatoid arthritis-associated interstitial lung disease?

BACKGROUND: Risk predicting models have been applied in idiopathic pulmonary fibrosis (IPF), but still not validated in patients with rheumatoid arthritis-associated interstitial lung disease (RA-ILD). The purpose of this study was to test the suitability of three prediction models as well as individual lung function and demographic factors for evaluating the prognosis of RA-ILD patients. METHODS: Clinical and radiological data of 59 RA-ILD patients was re-assessed. GAP (gender, age, physiologic variables) and the modified interstitial lung disease (ILD)-GAP as well as the composite physiologic indexes (CPI) were tested for predicting mortality using the goodness-of-fit test and Cox model. Potential predictors of mortality were also sought from single lung function parameters and clinical characteristics. RESULTS: The median survival was 152 and 61 months in GAP / ILD-GAP stages I and II (p = 0.017). Both GAP and ILD-GAP models accurately estimated 1-year, 2-year and 3-year mortality. CPI (p = 0.025), GAP (p = 0.008) and ILD-GAP (p = 0.028) scores, age (p = 0.002), baseline diffusion capacity to carbon monoxide (DLCO) (p = 0.014) and hospitalization due to respiratory reasons (p = 0.039), were significant predictors of mortality in the univariate analysis, whereas forced vital capacity (FVC) was not predictive. CPI score (HR 1.03, p = 0.018) and baseline DLCO (HR 0.97, p = 0.011) remained significant predictors of mortality after adjusting for age. CONCLUSIONS: GAP and ILD-GAP are applicable for evaluating the risk of death of patients with RA-ILD in a similar manner as in those with IPF. Baseline DLCO and CPI score also predicted survival.

Variable course of disease of rheumatoid arthritis-associated usual interstitial pneumonia compared to other subtypes.

BACKGROUND: In rheumatoid arthritis-associated interstitial lung disease (RA-ILD), occurring in 10 % of patients with patients with RA, usual interstitial pattern (UIP) has shown to associate with poor prognosis but more detailed data about the course of the disease in different subtypes is limited. Our aim was to compare the disease course of patients with RA-ILD categorized into either UIP or other types of ILDs. METHODS: Clinical and radiological information of 59 patients with RA-ILD were re-assessed and re-classified into UIP or non-UIP groups, followed by a between-group comparison of demographic data, lung function, survival, cause of death and comorbidities. RESULTS: The majority of patients (n = 35/59.3 %) showed a radiological UIP-like pattern in high resolution computed tomography. The median survival was 92 months (95 % CI 62.8-121.2) in the UIP-group and 137 months (95 % CI 31.0-243.0) in the non-UIP-group (p = 0.417). Differences in course of disease were found in the number of hospitalizations for respiratory reasons (mean 1.9 ± 2.6 in UIP vs. 0.5 ± 0.9 in non-UIP group, p = 0.004), the use of oxygen therapy (8/22.9 % UIP patients vs. 0 non-UIP patients, p = 0.016), number of deaths (23/65.7 % vs. 10/41.7 %, p = 0.046) and decline in diffusion capacity (56 ± 20.6 vs. 69 ± 20.2, p = 0.021). Dyspnea and inspiratory crackles were detected more often in the UIP group. RA-ILD was the most common primary cause of death (39.4 % of cases). Hypertension, coronary artery disease, chronic obstructive pulmonary disease, heart insufficiency, diabetes and asthma were common comorbidities. ILD preceded RA diagnosis in 13.6 % of patients. CONCLUSIONS: The course of the disease in RA-UIP patients is different from the other RA-ILD subtypes. Several comorbidities associated commonly with RA-ILD, although ILD was the predominant primary cause of death.

Lung cancer-associated myofibroblasts reveal distinctive ultrastructure and function.

BACKGROUND: Cancer-associated stromal cells interact with carcinoma cells and thus participate in tumor growth. Our aim was to characterize the ultrastructure and contractile properties of stromal cells in collagen gel cultured from lung cancer of various histological types and from tumor-free lung. METHODS: Cells cultured from lung cancer (13 adenocarcinomas, six squamous cell carcinomas, one adenosquamous carcinoma, and one pleomorphic carcinoma) and tumor-free lung were analyzed by transmission electron microscopy and three-dimensional collagen gel contraction assays. The expression of α-smooth muscle actin (α-SMA), a recognized myofibroblast marker, was examined by immunoelectron microscopy from individual cells and by Western blotting from the whole cultured cell population. RESULTS: According to their ultrastructure, the cell lines were composed of fibroblastic and myofibroblastic cells. In electron microscopy, cells of lung cancer exhibited more myofibroblastic features displaying higher amounts of actin belts (p = 0.057) and α-SMA labeling (p = 0.010) than cells from tumor-free lung. Myofibroblasts cultured from lung cancer of smokers expressed less α-SMA labeling (p = 0.013) than counterparts from nonsmokers. Western blotting revealed that cancer-associated fibroblasts expressed more α-SMA (p = 0.006) than cells from tumor-free lung, whereas cells from tumor-free central lung of smokers showed less α-SMA (p = 0.039) than counterparts from nonsmokers. Cells cultured from cancer contracted more in collagen gel than those from tumor-free lung. The contractile capacity in collagen gel correlated with the frequency of extracellular component of fibronexus by transmission electron microscopy. CONCLUSIONS: Lung cancer-associated myofibroblasts are different both ultrastructurally and functionally when compared with cells cultured from tumor-free lung. Smoking altered myofibroblastic phenotype, regardless of their origin.

Myofibroblast expression in airways and alveoli is affected by smoking and COPD.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by structural changes in alveoli and airways. Our aim was to analyse the numbers of alpha-smooth muscle actin (α-SMA) positive cells, as a marker of myofibroblasts, in different lung compartments in non-smokers and smokers with normal lung function or COPD. METHODS: α-SMA, tenascin-C (Tn-C) and EDA-fibronectin in alveolar level and airways were assayed by immunohistochemistry and quantified by image analysis. Immunohistochemical findings were correlated with clinical data. α-SMA protein was also analysed by Western blotting from fibroblastic cells cultured from peripheral lung of non-smokers, smokers without COPD and smokers with COPD. RESULTS: In many cases, the endings of the detached alveolar walls were widened, the structures of which were named as widened alveolar tips. Widened alveolar tips contained α-SMA positive cells, which were obviously myofibroblasts. There were less alveolar tips containing positive cells for α-SMA in alveoli and α-SMA positive cells in bronchioles in smokers and in COPD compared to non-smokers. The quantity of α-SMA positive cells was increased in bronchi in COPD. Tn-C was elevated in bronchi in COPD and smokers' lung. The α-SMA protein level was 1.43-fold higher in stromal cells cultured from non-smokers than in those of smokers. CONCLUSIONS: Myofibroblasts are localized variably in normal and diseased lung. This indicates that they have roles in both regeneration of lung and pathogenesis of COPD. The widened alveolar tips, these newly characterized histological structures, seemed to be the source of myofibroblasts at the alveolar level.

Divergence of tight and adherens junction factors in alveolar epithelium in pulmonary fibrosis.

It has been proposed that an epithelial injury may be one of the multiple primary events in the pathogenesis of idiopathic pulmonary fibrosis (IPF). The aim of this study was to characterize the tight junction and adherens junction proteins in normal human lung, IPF, cryptogenic organizing pneumonia, and asbestosis. We determined the immunohistochemical cell-specific expression of tight junction proteins claudin-1, claudin-2, claudin-3, claudin-4, claudin-5, and claudin-7, as well as 3 adherens junction proteins, E-cadherin, N-cadherin, and ß-catenin. We further analyzed the expression of claudin-1, claudin-3, and claudin-4 and E-cadherin, N-cadherin, and ß-catenin at the transcriptional level by quantitative real-time reverse transcriptase polymerase chain reaction. The expression levels of both tight junction and adherens junction proteins were elevated in regenerative alveolar epithelium in pulmonary fibrosis as compared with the expression of these proteins in normal alveolar epithelium. In particular, the expression levels of claudins-1 and claudin-3 were clearly elevated in all diseases. Furthermore, the amounts of adherens junction proteins messenger RNAs (mRNAs) were also all increased in pulmonary fibroses in comparison with healthy controls, with N-cadherin showing the greatest increase in mRNA levels in all diseases. However, the amounts of claudin-1, claudin-3, and claudin-4 mRNAs in fibrotic lung were similar to or even lower than those measured in the healthy controls. It is possible that the diminished capacity to produce claudin mRNAs may be one explanation for poor repair capacity of alveolar epithelial cells in IPF.

Myofibroblasts in interstitial lung diseases show diverse electron microscopic and invasive features.

The characteristic features of myofibroblasts in various lung disorders are poorly understood. We have evaluated the ultrastructure and invasive capacities of myofibroblasts cultured from small volumes of diagnostic bronchoalveolar lavage (BAL) fluid samples from patients with different types of lung diseases. Cells were cultured from samples of BAL fluid collected from 51 patients that had undergone bronchoscopy and BAL for diagnostic purposes. The cells were visualized by transmission electron microscopy and immunoelectron microscopy to achieve ultrastructural localization of alpha-smooth muscle actin (α-SMA) and fibronectin. The levels of α-SMA protein and mRNA and fibronectin mRNA were measured by western blot and quantitative real-time reverse transcriptase polymerase chain reaction. The invasive capacities of the cells were evaluated. The cultured cells were either fibroblasts or myofibroblasts. The structure of the fibronexus, and the amounts of intracellular actin, extracellular fibronectin and cell junctions of myofibroblasts varied in different diseases. In electron and immunoelectron microscopy, cells cultured from interstitial lung diseases (ILDs) expressed more actin filaments and α-SMA than normal lung. The invasive capacity of the cells obtained from patients with idiopathic pulmonary fibrosis was higher than that from patients with other type of ILDs. Cells expressing more actin filaments had a higher invasion capacity. It is concluded that electron and immunoelectron microscopic studies of myofibroblasts can reveal differential features in various diseases. An analysis of myofibroblasts cultured from diagnostic BAL fluid samples may represent a new kind of tool for diagnostics and research into lung diseases.