Clinical characteristics, proteins, and genes related to interstitial pneumonia-associated squamous cell carcinoma of the lungs.

Squamous cell carcinoma (SCC) is a common histological type of lung carcinoma that is associated with interstitial pneumonia (IP). We hypothesized that identifying specific genetic alterations or molecular markers of SCC with IP may aid the development of novel therapeutic strategies for the same. Therefore, in the present study, we aimed to identify tumorigenic genetic alterations and molecular markers in cases of SCC with IP. We included 28 lung SCC cases (14 cases with IP and 14 cases without IP). We performed immunohistochemistry for STAT3, STAT5, and TLE1, and next-generation sequencing was performed using an iSeq 100 system. The panel used in this study targeted 50 cancer-associated genes. Immunohistochemically, the rate of TLE1 positivity was higher in the SCC without IP group (93 %) than in the SCC with IP group (29 %), while that of STAT5 was higher in the SCC with IP group (79 %) than in the SCC without IP group (14 %). STAT3 expression was high in both the groups (SCC with IP, 64 %; SCC without IP, 71 %). Eighteen genes were mutated in more than six samples, and FBXW7 mutation was mainly observed in the SCC with IP group (p < 0.01). Mechanisms underlying tumorigenesis in SCC with IP included STAT5 activation via inflammation, while that in SCC without IP included squamous TLE1-mediated metaplasia. These findings are based on smoking-induced STAT3 activation; therefore, patients with IP who smoke are more likely to have progressive SCC. We also found that FBXW7 mutations may be associated with SCC with IP and keratinization. ERBB4 and KDR mutations were observed in both with or without IP, and these genes may be tumor-related genes in SCC. These molecular markers may help determine the prognoses of patients with SCC with IP and direct the development of treatment approaches.

Endothelial cell expression of a STING gain-of-function mutation initiates pulmonary lymphocytic infiltration.

Patients afflicted with Stimulator of interferon gene (STING) gain-of-function mutations frequently present with debilitating interstitial lung disease (ILD) that is recapitulated in mice expressing the STINGV154M mutation (VM). Prior radiation chimera studies revealed an unexpected and critical role for non-hematopoietic cells in initiating ILD. To identify STING-expressing non-hematopoietic cell types required for the development of ILD, we use a conditional knockin (CKI) model and direct expression of the VM allele to hematopoietic cells, fibroblasts, epithelial cells, or endothelial cells. Only endothelial cell-targeted VM expression results in enhanced recruitment of immune cells to the lung associated with elevated chemokine expression and the formation of bronchus-associated lymphoid tissue, as seen in the parental VM strain. These findings reveal the importance of endothelial cells as instigators of STING-driven lung disease and suggest that therapeutic targeting of STING inhibitors to endothelial cells could potentially mitigate inflammation in the lungs of STING-associated vasculopathy with onset in infancy (SAVI) patients or patients afflicted with other ILD-related disorders.

High risk of lung cancer in surfactant-related gene variant carriers.

BACKGROUND: Several rare surfactant-related gene (SRG) variants associated with interstitial lung disease are suspected to be associated with lung cancer, but data are missing. We aimed to study the epidemiology and phenotype of lung cancer in an international cohort of SRG variant carriers. METHODS: We conducted a cross-sectional study of all adults with SRG variants in the OrphaLung network and compared lung cancer risk with telomere-related gene (TRG) variant carriers. RESULTS: We identified 99 SRG adult variant carriers (SFTPA1 (n=18), SFTPA2 (n=31), SFTPC (n=24), ABCA3 (n=14) and NKX2-1 (n=12)), including 20 (20.2%) with lung cancer (SFTPA1 (n=7), SFTPA2 (n=8), SFTPC (n=3), NKX2-1 (n=2) and ABCA3 (n=0)). Among SRG variant carriers, the odds of lung cancer was associated with age (OR 1.04, 95% CI 1.01-1.08), smoking (OR 20.7, 95% CI 6.60-76.2) and SFTPA1/SFTPA2 variants (OR 3.97, 95% CI 1.39-13.2). Adenocarcinoma was the only histological type reported, with programmed death ligand-1 expression ≥1% in tumour cells in three samples. Cancer staging was localised (I/II) in eight (40%) individuals, locally advanced (III) in two (10%) and metastatic (IV) in 10 (50%). We found no somatic variant eligible for targeted therapy. Seven cancers were surgically removed, 10 received systemic therapy, and three received the best supportive care according to their stage and performance status. The median overall survival was 24 months, with stage I/II cancers showing better survival. We identified 233 TRG variant carriers. The comparative risk (subdistribution hazard ratio) for lung cancer in SRG patients versus TRG patients was 18.1 (95% CI 7.1-44.7). CONCLUSIONS: The high risk of lung cancer among SRG variant carriers suggests specific screening and diagnostic and therapeutic challenges. The benefit of regular computed tomography scan follow-up should be evaluated.

Dynamic diversity of SARS-CoV-2 genetic mutations in a lung transplantation patient with persistent COVID-19.

Numerous SARS-CoV-2 variant strains with altered characteristics have emerged since the onset of the COVID-19 pandemic. Remdesivir (RDV), a ribonucleotide analogue inhibitor of viral RNA polymerase, has become a valuable therapeutic agent. However, immunosuppressed hosts may respond inadequately to RDV and develop chronic persistent infections. A patient with respiratory failure caused by interstitial pneumonia, who had undergone transplantation of the left lung, developed COVID-19 caused by Omicron BA.5 strain with persistent chronic viral shedding, showing viral fusogenicity. Genome-wide sequencing analyses revealed the occurrence of several viral mutations after RDV treatment, followed by dynamic changes in the viral populations. The C799F mutation in nsp12 was found to play a pivotal role in conferring RDV resistance, preventing RDV-triphosphate from entering the active site of RNA-dependent RNA polymerase. The occurrence of diverse mutations is a characteristic of SARS-CoV-2, which mutates frequently. Herein, we describe the clinical case of an immunosuppressed host in whom inadequate treatment resulted in highly diverse SARS-CoV-2 mutations that threatened the patient's health due to the development of drug-resistant variants.

Drug-Gene Risk Stratification in Patients with Suspected Drug-Induced Interstitial Lung Disease.

BACKGROUND: Pulmonary toxicity has been associated with drug use. This is often not recognized in clinical practice, and underestimated. OBJECTIVE: We aimed to establish whether polymorphisms in certain genes corresponding with a metabolic pathway of drug(s) used are associated with pulmonary toxicity in patients with suspected drug-induced interstitial lung disease (DI-ILD). METHODS: This retrospective observational study explored genetic variations in three clinically relevant cytochrome P450 (CYP) iso-enzymes (i.e., CYP2D6, CYP2C9, and CYP2C19) in a group of patients with a fibroticinterstitial lung disease, either non-specific interstitial pneumonia (n = 211) or idiopathic pulmonary fibrosis (n = 256), with a suspected drug-induced origin. RESULTS: Of the 467 patients, 79.0% showed one or more polymorphisms in the tested genes accompanied by the use of drug(s) metabolized by a corresponding affected metabolic pathway (60.0% poor metabolizers and/or using two or more drugs [likely DI-ILD], 37.5% using three or more [highly likely DI-ILD]). Most commonly used drugs were statins (63.1%) with a predominance among men (69.4 vs 47.1%, p < 0.0001). Nitrofurantoin, not metabolized by the tested pathways, was prescribed more frequently among women (51.9 vs 4.5%, p < 0.00001). CONCLUSIONS: In our cohort with suspected DI-ILD, 79% carried one or more genetic variants accompanied by the use of drugs metabolized by a corresponding affected pathway. In 60%, the diagnosis of DI-ILD was likely, whereas in 37.5%, it was highly likely, based on CYP analyses. This study underlines the importance of considering both drug use and genetic make-up as a possible cause, or at least a contributing factor, in the development and/or progression of fibrotic lung diseases. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT00267800, registered in 2005.

Gene expression meta-analysis reveals aging and cellular senescence signatures in scleroderma-associated interstitial lung disease.

Aging and cellular senescence are increasingly recognized as key contributors to pulmonary fibrosis. However, our understanding in the context of scleroderma-associated interstitial lung disease (SSc-ILD) is limited. To investigate, we leveraged previously established lung aging- and cell-specific senescence signatures to determine their presence and potential relevance to SSc-ILD. We performed a gene expression meta-analysis of lung tissues from 38 SSc-ILD and 18 healthy controls and found that markers (GDF15, COMP, and CDKN2A) and pathways (p53) of senescence were significantly increased in SSc-ILD. When probing the established aging and cellular senescence signatures, we found that epithelial and fibroblast senescence signatures had a 3.6- and 3.7-fold enrichment, respectively, in the lung tissue of SSc-ILD and that lung aging genes (CDKN2A, FRZB, PDE1A, and NAPI12) were increased in SSc-ILD. These signatures were also enriched in SSc skin and associated with degree of skin involvement (limited vs. diffuse cutaneous). To further support these findings, we examined telomere length (TL), a surrogate for aging, in the lung tissue and found that, independent of age, SSc-ILD had significantly shorter telomeres than controls in type II alveolar cells in the lung. TL in SSc-ILD was comparable to idiopathic pulmonary fibrosis, a disease of known aberrant aging. Taken together, this study provides novel insight into the possible mechanistic effects of accelerated aging and aberrant cellular senescence in SSc-ILD pathogenesis.

Novel endotypes of antisynthetase syndrome identified independent of anti-aminoacyl transfer RNA synthetase antibody specificity that improve prognostic stratification.

OBJECTIVES: To systemically analyse the heterogeneity in the clinical manifestations and prognoses of patients with antisynthetase syndrome (ASS) and evaluate the transcriptional signatures related to different clinical phenotypes. METHODS: A total of 701 patients with ASS were retrospectively enrolled. The clinical presentation and prognosis were assessed in association with four anti-aminoacyl transfer RNA synthetase (ARS) antibodies: anti-Jo1, anti-PL7, anti-PL12 and anti-EJ. Unsupervised machine learning was performed for patient clustering independent of anti-ARS antibodies. Transcriptome sequencing was conducted in clustered ASS patients and healthy controls. RESULTS: Patients with four different anti-ARS antibody subtypes demonstrated no significant differences in the incidence of rapidly progressive interstitial lung disease (RP-ILD) or prognoses. Unsupervised machine learning, independent of anti-ARS specificity, identified three endotypes with distinct clinical features and outcomes. Endotype 1 (RP-ILD cluster, 23.7%) was characterised by a high incidence of RP-ILD and a high mortality rate. Endotype 2 (dermatomyositis (DM)-like cluster, 14.5%) corresponded to patients with DM-like skin and muscle symptoms with an intermediate prognosis. Endotype 3 (arthritis cluster, 61.8%) was characterised by arthritis and mechanic's hands, with a good prognosis. Transcriptome sequencing revealed that the different endotypes had distinct gene signatures and biological processes. CONCLUSIONS: Anti-ARS antibodies were not significant in stratifying ASS patients into subgroups with greater homogeneity in RP-ILD and prognoses. Novel ASS endotypes were identified independent of anti-ARS specificity and differed in clinical outcomes and transcriptional signatures, providing new insights into the pathogenesis of ASS.

Similarities and differences of interstitial lung disease associated with pathogenic variants in SFTPC and ABCA3 in adults.

BACKGROUND AND OBJECTIVE: Variants in surfactant genes SFTPC or ABCA3 are responsible for interstitial lung disease (ILD) in children and adults, with few studies in adults. METHODS: We conducted a multicentre retrospective study of all consecutive adult patients diagnosed with ILD associated with variants in SFTPC or ABCA3 in the French rare pulmonary diseases network, OrphaLung. Variants and chest computed tomography (CT) features were centrally reviewed. RESULTS: We included 36 patients (median age: 34 years, 20 males), 22 in the SFTPC group and 14 in the ABCA3 group. Clinical characteristics were similar between groups. Baseline median FVC was 59% ([52-72]) and DLco was 44% ([35-50]). An unclassifiable pattern of fibrosing ILD was the most frequent on chest CT, found in 85% of patients, however with a distinct phenotype with ground-glass opacities and/or cysts. Nonspecific interstitial pneumonia and usual interstitial pneumonia were the most common histological patterns in the ABCA3 group and in the SFTPC group, respectively. Annually, FVC and DLCO declined by 1.87% and 2.43% in the SFTPC group, respectively, and by 0.72% and 0.95% in the ABCA3 group, respectively (FVC, p = 0.014 and DLCO , p = 0.004 for comparison between groups). Median time to death or lung transplantation was 10 years in the SFTPC group and was not reached at the end of follow-up in the ABCA3 group. CONCLUSION: SFTPC and ABCA3-associated ILD present with a distinct phenotype and prognosis. A radiologic pattern of fibrosing ILD with ground-glass opacities and/or cysts is frequently found in these rare conditions.

Transcriptomic and network analysis identifies shared and unique pathways and immune changes across fibrotic interstitial lung diseases.

BACKGROUND: Interstitial lung disease (ILD) encompasses a diverse group of disorders characterized by chronic inflammation and fibrosis of the pulmonary interstitium. Three ILDs, namely idiopathic pulmonary fibrosis (IPF), fibrotic hypersensitivity pneumonitis (fHP), and connective tissue disease-associated ILD (CTD-ILD), exhibit similar progressive fibrosis phenotypes, yet possess distinct etiologies, encouraging us to explore their different underlying mechanisms. METHODS: Transcriptome data of fibrotic lung tissues from patients with IPF, fHP, and CTD-ILD were subjected to functional annotation, network, and pathway analyses. Additionally, we employed the xCell deconvolution algorithm to predict immune cell infiltration in patients with fibrotic ILDs and healthy controls. RESULTS: We identified a shared progressive fibrosis-related module in these diseases which was related to extracellular matrix (ECM) degradation and production and potentially regulated by the p53 family transcription factors. In IPF, neuron-related processes emerged as a critical specific mechanism in functional enrichment. In fHP, we observed that B cell signaling and immunoglobulin A (IgA) production may act as predominant processes, which was further verified by B cell infiltration and the central role of CD19 gene. In CTD-ILD, active chemokine processes were enriched, and active dendritic cells (aDCs) were predicted to infiltrate the lung tissues. CONCLUSIONS: This study revealed shared and specific molecular and cellular pathways among IPF, fHP, and CTD-ILD, providing a basis for understanding their pathogenesis and identifying potential therapeutic targets.

A Call for Gene Expression Analysis in Whole Blood of Patients With Rheumatoid Arthritis (RA) as a Biomarker for RA-Associated Interstitial Lung Disease.

OBJECTIVE: Rheumatoid arthritis (RA)-associated interstitial lung disease (ILD) is one of the most common and prognostic organ manifestations of RA. Therefore, to allow effective treatment, it is of crucial importance to diagnose RA-ILD at the earliest possible stage. So far, the gold standard of early detection has been high-resolution computed tomography (HRCT) of the lungs. This procedure involves considerable radiation exposure for the patient and is therefore unsuitable as a routine screening measure for ethical reasons. Here, we propose the analysis of characteristic gene expression patterns as a biomarker to aid in the early detection and initiation of appropriate, possibly antifibrotic, therapy. METHODS: To investigate unique molecular patterns of RA-ILD, whole blood samples were taken from 12 female patients with RA-ILD (n = 7) or RA (n = 5). The RNA was extracted, sequenced by RNA-Seq, and analyzed for characteristic differences in the gene expression patterns between patients with RA-ILD and those with RA without ILD. RESULTS: The differential gene expression analysis revealed 9 significantly upregulated genes in RA-ILD compared to RA without ILD: arginase 1 (ARG1), thymidylate synthetase (TYMS), sortilin 1 (SORT1), marker of proliferation Ki-67 (MKI67), olfactomedin 4 (OLFM4), baculoviral inhibitor of apoptosis repeat containing 5 (BIRC5), membrane spanning 4-domains A4A (MS4A4A), C-type lectin domain family 12 member A (CLEC12A), and the long intergenic nonprotein coding RNA (LINC02967). CONCLUSION: All gene products of these genes (except for LINC02967) are known from the literature to be involved in the pathogenesis of fibrosis. Further, for some, a contribution to the development of pulmonary fibrosis has even been demonstrated in experimental studies. Therefore, the results presented here provide an encouraging perspective for using specific gene expression patterns as biomarkers for the early detection and differential diagnosis of RA-ILD as a routine screening test.

Proteomic Biomarkers of Quantitative Interstitial Abnormalities in COPDGene and CARDIA Lung Study.

Rationale: Quantitative interstitial abnormalities (QIAs) are early measures of lung injury automatically detected on chest computed tomography scans. QIAs are associated with impaired respiratory health and share features with advanced lung diseases, but their biological underpinnings are not well understood. Objectives: To identify novel protein biomarkers of QIAs using high-throughput plasma proteomic panels within two multicenter cohorts. Methods: We measured the plasma proteomics of 4,383 participants in an older, ever-smoker cohort (COPDGene [Genetic Epidemiology of Chronic Obstructive Pulmonary Disease]) and 2,925 participants in a younger population cohort (CARDIA [Coronary Artery Disease Risk in Young Adults]) using the SomaLogic SomaScan assays. We measured QIAs using a local density histogram method. We assessed the associations between proteomic biomarker concentrations and QIAs using multivariable linear regression models adjusted for age, sex, body mass index, smoking status, and study center (Benjamini-Hochberg false discovery rate-corrected P ⩽ 0.05). Measurements and Main Results: In total, 852 proteins were significantly associated with QIAs in COPDGene and 185 in CARDIA. Of the 144 proteins that overlapped between COPDGene and CARDIA, all but one shared directionalities and magnitudes. These proteins were enriched for 49 Gene Ontology pathways, including biological processes in inflammatory response, cell adhesion, immune response, ERK1/2 regulation, and signaling; cellular components in extracellular regions; and molecular functions including calcium ion and heparin binding. Conclusions: We identified the proteomic biomarkers of QIAs in an older, smoking population with a higher prevalence of pulmonary disease and in a younger, healthier community cohort. These proteomics features may be markers of early precursors of advanced lung diseases.

Functional Roles of CD26/DPP4 in Bleomycin-Induced Pulmonary Hypertension Associated with Interstitial Lung Disease.

Pulmonary hypertension (PH) with interstitial lung diseases (ILDs) often causes intractable conditions. CD26/Dipeptidyl peptidase-4 (DPP4) is expressed in lung constituent cells and may be related to the pathogenesis of various respiratory diseases. We aimed to clarify the functional roles of CD26/DPP4 in PH-ILD, paying particular attention to vascular smooth muscle cells (SMCs). Dpp4 knockout (Dpp4KO) and wild type (WT) mice were administered bleomycin (BLM) intraperitoneally to establish a PH-ILD model. The BLM-induced increase in the right ventricular systolic pressure and the right ventricular hypertrophy observed in WT mice were attenuated in Dpp4KO mice. The BLM-induced vascular muscularization in small pulmonary vessels in Dpp4KO mice was milder than that in WT mice. The viability of TGFß-stimulated human pulmonary artery SMCs (hPASMCs) was lowered due to the DPP4 knockdown with small interfering RNA. According to the results of the transcriptome analysis, upregulated genes in hPASMCs with TGFß treatment were related to pulmonary vascular SMC proliferation via the Notch, PI3K-Akt, and NFκB signaling pathways. Additionally, DPP4 knockdown in hPASMCs inhibited the pathways upregulated by TGFß treatment. These results suggest that genetic deficiency of Dpp4 protects against BLM-induced PH-ILD by alleviating vascular remodeling, potentially through the exertion of an antiproliferative effect via inhibition of the TGFß-related pathways in PASMCs.

Human pluripotent stem cell modeling of alveolar type 2 cell dysfunction caused by ABCA3 mutations.

Mutations in ATP-binding cassette A3 (ABCA3), a phospholipid transporter critical for surfactant homeostasis in pulmonary alveolar type II epithelial cells (AEC2s), are the most common genetic causes of childhood interstitial lung disease (chILD). Treatments for patients with pathological variants of ABCA3 mutations are limited, in part due to a lack of understanding of disease pathogenesis resulting from an inability to access primary AEC2s from affected children. Here, we report the generation of AEC2s from affected patient induced pluripotent stem cells (iPSCs) carrying homozygous versions of multiple ABCA3 mutations. We generated syngeneic CRISPR/Cas9 gene-corrected and uncorrected iPSCs and ABCA3-mutant knockin ABCA3:GFP fusion reporter lines for in vitro disease modeling. We observed an expected decreased capacity for surfactant secretion in ABCA3-mutant iPSC-derived AEC2s (iAEC2s), but we also found an unexpected epithelial-intrinsic aberrant phenotype in mutant iAEC2s, presenting as diminished progenitor potential, increased NFκB signaling, and the production of pro-inflammatory cytokines. The ABCA3:GFP fusion reporter permitted mutant-specific, quantifiable characterization of lamellar body size and ABCA3 protein trafficking, functional features that are perturbed depending on ABCA3 mutation type. Our disease model provides a platform for understanding ABCA3 mutation-mediated mechanisms of alveolar epithelial cell dysfunction that may trigger chILD pathogenesis.