Transcriptional profiles of pulmonary artery endothelial cells in pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is characterized by endothelial cell (EC) dysfunction. There are no data from living patients to inform whether differential gene expression of pulmonary artery ECs (PAECs) can discern disease subtypes, progression and pathogenesis. We aimed to further validate our previously described method to propagate ECs from right heart catheter (RHC) balloon tips and to perform additional PAEC phenotyping. We performed bulk RNA sequencing of PAECs from RHC balloons. Using unsupervised dimensionality reduction and clustering we compared transcriptional signatures from PAH to controls and other forms of pulmonary hypertension. Select PAEC samples underwent single cell and population growth characterization and anoikis quantification. Fifty-four specimens were analyzed from 49 subjects. The transcriptome appeared stable over limited passages. Six genes involved in sex steroid signaling, metabolism, and oncogenesis were significantly upregulated in PAH subjects as compared to controls. Genes regulating BMP and Wnt signaling, oxidative stress and cellular metabolism were differentially expressed in PAH subjects. Changes in gene expression tracked with clinical events in PAH subjects with serial samples over time. Functional assays demonstrated enhanced replication competency and anoikis resistance. Our findings recapitulate fundamental biological processes of PAH and provide new evidence of a cancer-like phenotype in ECs from the central vasculature of PAH patients. This "cell biopsy" method may provide insight into patient and lung EC heterogeneity to advance precision medicine approaches in PAH.

Serotonin reduction in post-acute sequelae of viral infection.

Post-acute sequelae of COVID-19 (PASC, "Long COVID") pose a significant global health challenge. The pathophysiology is unknown, and no effective treatments have been found to date. Several hypotheses have been formulated to explain the etiology of PASC, including viral persistence, chronic inflammation, hypercoagulability, and autonomic dysfunction. Here, we propose a mechanism that links all four hypotheses in a single pathway and provides actionable insights for therapeutic interventions. We find that PASC are associated with serotonin reduction. Viral infection and type I interferon-driven inflammation reduce serotonin through three mechanisms: diminished intestinal absorption of the serotonin precursor tryptophan; platelet hyperactivation and thrombocytopenia, which impacts serotonin storage; and enhanced MAO-mediated serotonin turnover. Peripheral serotonin reduction, in turn, impedes the activity of the vagus nerve and thereby impairs hippocampal responses and memory. These findings provide a possible explanation for neurocognitive symptoms associated with viral persistence in Long COVID, which may extend to other post-viral syndromes.

Two cases of severe pulmonary toxicity from highly active mesothelin-directed CAR T cells.

Multiple clinical studies have treated mesothelin (MSLN)-positive solid tumors by administering MSLN-directed chimeric antigen receptor (CAR) T cells. Although these products are generally safe, efficacy is limited. Therefore, we generated and characterized a potent, fully human anti-MSLN CAR. In a phase 1 dose-escalation study of patients with solid tumors, we observed two cases of severe pulmonary toxicity following intravenous infusion of this product in the high-dose cohort (1-3 × 108 T cells per m2). Both patients demonstrated progressive hypoxemia within 48 h of infusion with clinical and laboratory findings consistent with cytokine release syndrome. One patient ultimately progressed to grade 5 respiratory failure. An autopsy revealed acute lung injury, extensive T cell infiltration, and accumulation of CAR T cells in the lungs. RNA and protein detection techniques confirmed low levels of MSLN expression by benign pulmonary epithelial cells in affected lung and lung samples obtained from other inflammatory or fibrotic conditions, indicating that pulmonary pneumocyte and not pleural expression of mesothelin may lead to dose-limiting toxicity. We suggest patient enrollment criteria and dosing regimens of MSLN-directed therapies consider the possibility of dynamic expression of mesothelin in benign lung with a special concern for patients with underlying inflammatory or fibrotic conditions.

Clinical Outcomes Associated With Pembrolizumab Monotherapy Among Adults With Diffuse Malignant Peritoneal Mesothelioma.

Importance: Diffuse malignant peritoneal mesothelioma (DMPM) represents a rare and clinically distinct entity among malignant mesotheliomas. Pembrolizumab has activity in diffuse pleural mesothelioma but limited data are available for DMPM; thus, DMPM-specific outcome data are needed. Objective: To evaluate outcomes after the initiation of pembrolizumab monotherapy in the treatment of adults with DMPM. Design, Setting, and Participants: This retrospective cohort study was conducted in 2 tertiary care academic cancer centers (University of Pennsylvania Hospital Abramson Cancer Center and Memorial Sloan Kettering Cancer Center). All patients with DMPM treated between January 1, 2015, and September 1, 2019, were retrospectively identified and followed until January 1, 2021. Statistical analysis was performed between September 2021 and February 2022. Exposures: Pembrolizumab (200 mg or 2 mg/kg every 21 days). Main Outcomes and Measures: Median progression-free survival (PFS) and median overall survival (OS) were assessed using Kaplan-Meier estimates. The best overall response was determined using RECIST (Response Evaluation Criteria in Solid Tumors) criteria, version 1.1. The association of disease characteristics with partial response was evaluated using the Fisher exact test. Results: This study included 24 patients with DMPM who received pembrolizumab monotherapy. Patients had a median age of 62 years (IQR, 52.4-70.6 years); 14 (58.3%) were women, 18 (75.0%) had epithelioid histology, and most (19 [79.2%]) were White. A total of 23 patients (95.8%) received systemic chemotherapy prior to pembrolizumab, and the median number of lines of prior therapy was 2 (range, 0-6 lines). Of the 17 patients who underwent programmed death ligand 1 (PD-L1) testing, 6 (35.3%) had positive tumor PD-L1 expression (range, 1.0%-80.0%). Of the 19 evaluable patients, 4 (21.0%) had a partial response (overall response rate, 21.1% [95% CI, 6.1%-46.6%]), 10 (52.6%) had stable disease, and 5 (26.3%) had progressive disease (5 of 24 patients [20.8%] were lost to follow-up). There was no association between a partial response and the presence of a BAP1 alteration, PD-L1 positivity, or nonepithelioid histology. With a median follow-up of 29.2 (95% CI, 19.3 to not available [NA]) months, the median PFS was 4.9 (95% CI, 2.8-13.3) months and the median OS was 20.9 (95% CI, 10.0 to NA) months from pembrolizumab initiation. Three patients (12.5%) experienced PFS of more than 2 years. Among patients with nonepithelioid vs epithelioid histology, there was a numeric advantage in median PFS (11.5 [95% CI, 2.8 to NA] vs 4.0 [95% CI, 2.8-8.8] months) and median OS (31.8 [95% CI, 8.3 to NA] vs 17.5 [95% CI, 10.0 to NA] months); however, this did not reach statistical significance. Conclusions and Relevance: The results of this retrospective dual-center cohort study of patients with DMPM suggest that pembrolizumab had clinical activity regardless of PD-L1 status or histology, although patients with nonepithelioid histology may have experienced additional clinical benefit. The partial response rate of 21.0% and median OS of 20.9 months in this cohort with 75.0% epithelioid histology warrants further investigation to identify those most likely to respond to immunotherapy.

Defining the spatial landscape of KRAS mutated congenital pulmonary airway malformations: a distinct entity with a spectrum of histopathologic features.

The potential pathogenetic mechanisms underlying the varied morphology of congenital pulmonary airway malformations (CPAMs) have not been molecularly determined, but a subset have been shown to contain clusters of mucinous cells (MCC). These clusters are believed to serve as precursors for potential invasive mucinous adenocarcinoma, and they are associated with KRAS codon 12 mutations. To assess the universality of KRAS mutations in MCCs, we sequenced exon 2 of KRAS in 61 MCCs from 18 patients, and we found a KRAS codon 12 mutation in all 61 MCCs. Furthermore, all MCCs from a single patient always had the same KRAS mutation, and the same KRAS mutation was also found in non-mucinous lesional tissue. Next generation sequencing of seven MCCs showed no other mutations or copy number variations. Sequencing of 46 additional CPAMs with MCCs revealed KRAS mutations in non-mucinous lesional tissue in all cases. RNA in situ hybridization confirmed widespread distribution of cells with mutant KRAS RNA, even extending outside of the bronchiolar type epithelium. We identified 25 additional CPAMs with overall histologic architecture similar to CPAMs with KRAS mutations but without identifiable MCCs, and we found KRAS mutations in 17 (68%). The histologic features of these KRAS mutated CPAMs included type 1 and type 3 morphology, as well as lesions with an intermediate histologic appearance, and analysis revealed a strong correlation between the specific amino acid substitution and histomorphology. These findings, together with previously published model organism data, suggests that the formation of type 1 and 3 CPAMs is driven by mosaic KRAS mutations arising in the lung epithelium early in development and places them within the growing field of mosaic RASopathies. The presence of widespread epithelial mutation explains late metastatic disease in incompletely resected patients and reinforces the recommendation for complete resection of these lesions.

Identification and Prognosis of Patients With Interstitial Pneumonia With Autoimmune Features.

BACKGROUND/OBJECTIVE: Patients classified as interstitial pneumonia with autoimmune features (IPAF) have interstitial lung disease (ILD) and features of autoimmunity but do not fulfill criteria for connective tissue diseases (CTDs). Our goal was to identify patients classifiable as IPAF, CTD-ILD, and idiopathic pulmonary fibrosis (IPF) from a preexisting pulmonary cohort and evaluate the prognosis of patients with IPAF. METHODS: We reviewed the medical records of 456 patients from a single-center pulmonary ILD cohort whose diagnoses were previously established by a multidisciplinary panel that did not include rheumatologists. We reclassified patients as IPAF, CTD-ILD, or IPF. We compared transplant-free survival using Kaplan-Meier methods and identified prognostic factors using Cox models. RESULTS: We identified 60 patients with IPAF, 113 with CTD-ILD, and 126 with IPF. Transplant-free survival of IPAF was not statistically significantly different from that of CTD-ILD or IPF. Among IPAF patients, male sex (hazard ratio, 4.58 [1.77-11.87]) was independently associated with worse transplant-free survival. During follow-up, only 10% of IPAF patients were diagnosed with CTD-ILD, most commonly antisynthetase syndrome. CONCLUSION: Despite similar clinical characteristics, most patients with IPAF did not progress to CTD-ILD; those who did often developed antisynthetase syndrome, highlighting the critical importance of comprehensive myositis autoantibody testing in this population. As in other types of ILD, male sex may portend a worse prognosis in IPAF. The routine engagement of rheumatologists in the multidisciplinary evaluation of ILD will help ensure the accurate classification of these patients and help clarify prognostic factors.

Multiplexed detection of SARS-CoV-2 genomic and subgenomic RNA using in situ hybridization.

The widespread Coronavirus Disease 2019 (COVID-19) is caused by infection with the novel coronavirus SARS-CoV-2. Currently, we have a limited toolset available for visualizing SARS-CoV-2 in cells and tissues, particularly in tissues from patients who died from COVID-19. Generally, single-molecule RNA FISH techniques have shown mixed results in formalin fixed paraffin embedded tissues such as those preserved from human autopsies. Here, we present a platform for preparing autopsy tissue for visualizing SARS-CoV-2 RNA using RNA FISH with amplification by hybridization chain reaction (HCR). We developed probe sets that target different regions of SARS-CoV-2 (including ORF1a and N) as well as probe sets that specifically target SARS-CoV-2 subgenomic mRNAs. We validated these probe sets in cell culture and tissues (lung, lymph node, and placenta) from infected patients. Using this technology, we observe distinct subcellular localization patterns of the ORF1a and N regions, with the ORF1a concentrated around the nucleus and the N showing a diffuse distribution across the cytoplasm. In human lung tissue, we performed multiplexed RNA FISH HCR for SARS-CoV-2 and cell-type specific marker genes. We found viral RNA in cells containing the alveolar type 2 (AT2) cell marker gene (SFTPC) and the alveolar macrophage marker gene (MARCO), but did not identify viral RNA in cells containing the alveolar type 1 (AT1) cell marker gene (AGER). Moreover, we observed distinct subcellular localization patterns of viral RNA in AT2 cells and alveolar macrophages, consistent with phagocytosis of infected cells. In sum, we demonstrate the use of RNA FISH HCR for visualizing different RNA species from SARS-CoV-2 in cell lines and FFPE autopsy specimens. Furthermore, we multiplex this assay with probes for cellular genes to determine what cell-types are infected within the lung. We anticipate that this platform could be broadly useful for studying SARS-CoV-2 pathology in tissues as well as extended for other applications including investigating the viral life cycle, viral diagnostics, and drug screening.

Molecular imaging can identify the location to perform a frozen biopsy during intraoperative frozen section consultation.

BACKGROUND: Intraoperative frozen section (FS) consultation is an important tool in surgical oncology that suffers from sampling error because the pathologist does not always know where to perform a biopsy of the surgical specimen. Intraoperative molecular imaging is a technology used in the OR to visualize lesions during surgery. We hypothesized that molecular imaging can address this pathology challenge in FS by visualizing the cancer cells in the specimen in the pathology suite. Here, we report the development and validation of a molecular-imaging capable cryostat called Smart-Cut. METHODS: A molecular imaging capable cryostat prototype was developed and tested using a murine model. Tumors grown in mice were targeted with a NIR contrast agent, indocyanine green (ICG), via tail vein injection. Tumors and adjacent normal tissue samples were frozen sectioned with Smart-Cut. Fluorescent sections and non-fluorescent sections were prepared for H&E and fluorescent microscopy. Fluorescent signal was quantified by tumor-to-background ratio (TBR). NIR fluorescence was tested in one patient enrolled in a clinical trial. RESULTS: The Smart-Cut prototype has a small footprint and fits well in the pathology suite. Fluorescence imaging with Smart-Cut identified cancerous tissue in the specimen in all 12 mice. No false positives or false negatives were seen, as confirmed by H&E. The mean TBR in Smart-Cut positive tissue sections was 6.8 (SD±3.8). In a clinical application in the pathology suite, NIR imaging identified two lesions in a pulmonary resection specimen, where traditional grossing only identified one. CONCLUSION: Molecular imaging can be integrated into the pathology suite via the Smart-Cut device, and can detect cancer in frozen tissue sections using molecular imaging in a murine model.

Alpha-defensins: risk factor for thrombosis in COVID-19 infection.

The inflammatory response to SARS/CoV-2 (COVID-19) infection may contribute to the risk of thromboembolic complications. α-Defensins, antimicrobial peptides released from activated neutrophils, are anti-fibrinolytic and prothrombotic in vitro and in mouse models. In this prospective study of 176 patients with COVID-19 infection, we found that plasma levels of α-defensins were elevated, tracked with disease progression/mortality or resolution and with plasma levels of interleukin-6 (IL-6) and D-dimers. Immunohistochemistry revealed intense deposition of α-defensins in lung vasculature and thrombi. IL-6 stimulated the release of α-defensins from neutrophils, thereby accelerating coagulation and inhibiting fibrinolysis in human blood, imitating the coagulation pattern in COVID-19 patients. The procoagulant effect of IL-6 was inhibited by colchicine, which blocks neutrophil degranulation. These studies describe a link between inflammation and the risk of thromboembolism, and they identify a potential new approach to mitigate this risk in patients with COVID-19 and potentially in other inflammatory prothrombotic conditions.

The concept of mesothelioma in situ, with consideration of its potential impact on cytology diagnosis.

Diffuse malignant mesothelioma (MM) is an incurable tumour of the serosal membranes, which is often caused by exposure to asbestos and commonly diagnosed at advanced stage. Malignant mesothelioma in situ (MMIS) is now included as diagnostic category by the World Health Organization (WHO). However, our international survey of 34 pulmonary pathologists with an interest in MM diagnosis highlights inconsistency regarding how the diagnosis is being made by experts, despite published guidelines. Whilst the WHO restricts the diagnosis to surgical samples, the very concept has implication for cytological diagnosis, which is already regarded as controversial in itself by some. MMIS is currently only applicable as precursor to MM with an epithelioid component, and raises the possibility for different molecular pathways for different histological MM subtypes. The clinical implications of MMIS at this stage are uncertain, but aggressive therapies are being initiated in some instances. Based on the results of the survey we here present a critical appraisal of the concept, its clinical and conceptual implications and provide practice suggestions for diagnosis. A low threshold for ancillary testing is suggested. The designations of 'malignant mesothelioma, cannot exclude MMIS' or 'atypical mesothelial proliferation with molecular indicators of malignancy, so-called MMIS' could be used on cytology samples, adding 'no evidence of invasion in sample provided' for surgical samples. Clinical and radiological correlation are integral to diagnosis and best done at multidisciplinary meetings. Finally, collaborative studies are required to improve our understanding of MMIS.

Subcellular Detection of SARS-CoV-2 RNA in Human Tissue Reveals Distinct Localization in Alveolar Type 2 Pneumocytes and Alveolar Macrophages.

The widespread coronavirus disease 2019 (COVID-19) is caused by infection with the novel coronavirus SARS-CoV-2. Currently, we have limited understanding of which cells become infected with SARS-CoV-2 in human tissues and where viral RNA localizes on the subcellular level. Here, we present a platform for preparing autopsy tissue for visualizing SARS-CoV-2 RNA using RNA fluorescence in situ hybridization (FISH) with amplification by hybridization chain reaction. We developed probe sets that target different regions of SARS-CoV-2 (including ORF1a and N), as well as probe sets that specifically target SARS-CoV-2 subgenomic mRNAs. We validated these probe sets in cell culture and tissues (lung, lymph node, and placenta) from infected patients. Using this technology, we observe distinct subcellular localization patterns of the ORF1a and N regions. In human lung tissue, we performed multiplexed RNA FISH HCR for SARS-CoV-2 and cell-type-specific marker genes. We found viral RNA in cells containing the alveolar type 2 (AT2) cell marker gene (SFTPC) and the alveolar macrophage marker gene (MARCO) but did not identify viral RNA in cells containing the alveolar type 1 (AT1) cell marker gene (AGER). Moreover, we observed distinct subcellular localization patterns of viral RNA in AT2 cells and alveolar macrophages. In sum, we demonstrate the use of RNA FISH HCR for visualizing different RNA species from SARS-CoV-2 in cell lines and FFPE (formalin fixation and paraffin embedding) autopsy specimens. We anticipate that this platform could be broadly useful for studying SARS-CoV-2 pathology in tissues, as well as extended for other applications, including investigating the viral life cycle, viral diagnostics, and drug screening. IMPORTANCE Here, we developed an in situ RNA detection assay for RNA generated by the SARS-CoV-2 virus. We found viral RNA in lung, lymph node, and placenta samples from pathology specimens from COVID patients. Using high-magnification microscopy, we can visualize the subcellular distribution of these RNA in single cells.

Mucinous Cell Clusters in Infantile Congenital Pulmonary Airway Malformations Mimic Adult Mucinous Adenocarcinoma But Are Not Associated With Poor Outcomes When Appropriately Resected.

Congenital pulmonary airway malformations (CPAMs) are abnormalities of the lung arising during development. At our institution the majority of type I infantile CPAMs contain mucinous cell clusters (MCCs). The overlapping histology of MCCs and adult in situ mucinous adenocarcinomas, as well as reports of metastatic mucinous adenocarcinoma arising in CPAMs resected later in childhood raise concerns about the malignant potential of these cells. However, after adequate surgical resection, malignant recurrence has not been reported in infants with CPAMs. Despite benign behavior, MCCs often have histologic features that, in an adult, would be consistent with a diagnosis of adenocarcinoma. Therefore, to assess the spectrum of features that may be seen in these presumed precursor lesions, we characterized the histology of 671 MCCs spread across 44 infantile CPAMs and compared them to 10 adult mucinous adenocarcinomas. MCCs in CPAMS were often numerous, widespread, and located outside of the large cysts. Mucinous and nonmucinous epithelium within CPAMs showed complex architecture, making application of adult adenocarcinoma architectural patterns difficult. The MCCs within CPAMs displayed nuclear features similar to adult mucinous adenocarcinomas. The proliferative index in infantile MCCs was higher than in adult mucinous adenocarcinomas but was also higher in uninvolved infantile lung tissue. This work illustrates that histologic features typically associated with adenocarcinoma frequently occur within CPAMs; however, this does not alter their benign behavior. Therefore, extreme caution should be used if adult lung cancer terminology is applied to avoid significant potential psychological and physical harms associated with the label of adenocarcinoma.

Function of Human Tumor-Infiltrating Lymphocytes in Early-Stage Non-Small Cell Lung Cancer.

Cancer progression is marked by dysfunctional tumor-infiltrating lymphocytes (TIL) with high inhibitory receptor (IR) expression. Because IR blockade has led to clinical responses in some patients with non-small cell lung cancer (NSCLC), we investigated how IRs influenced CD8+ TIL function from freshly digested early-stage NSCLC tissues using a killing assay and intracellular cytokine staining after in vitro T-cell restimulation. Early-stage lung cancer TIL function was heterogeneous with only about one third of patients showing decrements in cytokine production and lytic function. TIL hypofunction did not correlate with clinical factors, coexisting immune cells (macrophages, neutrophils, or CD4+ T regulatory cells), nor with PD-1, TIGIT, TIM-3, CD39, or CTLA-4 expression. Instead, we found that the presence of the integrin αeß7 (CD103), characteristic of tissue-resident memory cells (TRM), was positively associated with cytokine production, whereas expression of the transcription factor Eomesodermin (Eomes) was negatively associated with TIL function. These data suggest that the functionality of CD8+ TILs from early-stage NSCLCs may be influenced by competition between an antitumor CD103+ TRM program and an exhaustion program marked by Eomes expression. Understanding the mechanisms of T-cell function in the progression of lung cancer may have clinical implications for immunotherapy.

Nuclear grade and necrosis predict prognosis in malignant epithelioid pleural mesothelioma: a multi-institutional study.

A recently described nuclear grading system predicted survival in patients with epithelioid malignant pleural mesothelioma. The current study was undertaken to validate the grading system and to identify additional prognostic factors. We analyzed cases of epithelioid malignant pleural mesothelioma from 17 institutions across the globe from 1998 to 2014. Nuclear grade was computed combining nuclear atypia and mitotic count into a grade of I-III using the published system. Nuclear grade was assessed by one pathologist for three institutions, the remaining were scored independently. The presence or absence of necrosis and predominant growth pattern were also evaluated. Two additional scoring systems were evaluated, one combining nuclear grade and necrosis and the other mitotic count and necrosis. Median overall survival was the primary endpoint. A total of 776 cases were identified including 301 (39%) nuclear grade I tumors, 354 (45%) grade II tumors and 121 (16%) grade III tumors. The overall survival was 16 months, and correlated independently with age (P=0.006), sex (0.015), necrosis (0.030), mitotic count (0.001), nuclear atypia (0.009), nuclear grade (<0.0001), and mitosis and necrosis score (<0.0001). The addition of necrosis to nuclear grade further stratified overall survival, allowing classification of epithelioid malignant pleural mesothelioma into four distinct prognostic groups: nuclear grade I tumors without necrosis (29 months), nuclear grade I tumors with necrosis and grade II tumors without necrosis (16 months), nuclear grade II tumors with necrosis (10 months) and nuclear grade III tumors (8 months). The mitosis-necrosis score stratified patients by survival, but not as well as the combination of necrosis and nuclear grade. This study confirms that nuclear grade predicts survival in epithelioid malignant pleural mesothelioma, identifies necrosis as factor that further stratifies overall survival, and validates the grading system across multiple institutions and among both biopsy and resection specimens. An alternative scoring system, the mitosis-necrosis score is also proposed.

Exposure to Ambient Particulate Matter Is Associated With Accelerated Functional Decline in Idiopathic Pulmonary Fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF), a progressive disease with an unknown pathogenesis, may be due in part to an abnormal response to injurious stimuli by alveolar epithelial cells. Air pollution and particulate inhalation of matter evoke a wide variety of pulmonary and systemic inflammatory diseases. We therefore hypothesized that increased average ambient particulate matter (PM) concentrations would be associated with an accelerated rate of decline in FVC in IPF. METHODS: We identified a cohort of subjects seen at a single university referral center from 2007 to 2013. Average concentrations of particulate matter < 10 and < 2.5 µg/m3 (PM10 and PM2.5, respectively) were assigned to each patient based on geocoded residential addresses. A linear multivariable mixed-effects model determined the association between the rate of decline in FVC and average PM concentration, controlling for baseline FVC at first measurement and other covariates. RESULTS: One hundred thirty-five subjects were included in the final analysis after exclusion of subjects missing repeated spirometry measurements and those for whom exposure data were not available. There was a significant association between PM10 levels and the rate of decline in FVC during the study period, with each µg/m3 increase in PM10 corresponding with an additional 46 cc/y decline in FVC (P = .008). CONCLUSIONS: Ambient air pollution, as measured by average PM10 concentration, is associated with an increase in the rate of decline of FVC in IPF, suggesting a potential mechanistic role for air pollution in the progression of disease.

Microbial Lineages in Sarcoidosis. A Metagenomic Analysis Tailored for Low-Microbial Content Samples.

RATIONALE: The etiology of sarcoidosis is unknown, but microbial agents are suspected as triggers. OBJECTIVES: We sought to identify bacterial, fungal, or viral lineages in specimens from patients with sarcoidosis enriched relative to control subjects using metagenomic DNA sequencing. Because DNA from environmental contamination contributes disproportionately to samples with low authentic microbial content, we developed improved methods for filtering environmental contamination. METHODS: We analyzed specimens from subjects with sarcoidosis (n = 93), control subjects without sarcoidosis (n = 72), and various environmental controls (n = 150). Sarcoidosis specimens consisted of two independent sets of formalin-fixed, paraffin-embedded lymph node biopsies, BAL, Kveim reagent, and fresh granulomatous spleen from a patient with sarcoidosis. All specimens were analyzed by bacterial 16S and fungal internal transcribed spacer ribosomal RNA gene sequencing. In addition, BAL was analyzed by shotgun sequencing of fractions enriched for viral particles, and Kveim and spleen were subjected to whole-genome shotgun sequencing. MEASUREMENTS AND MAIN RESULTS: In one tissue set, fungi in the Cladosporiaceae family were enriched in sarcoidosis compared with nonsarcoidosis tissues; in the other tissue set, we detected enrichment of several bacterial lineages in sarcoidosis but not Cladosporiaceae. BAL showed limited enrichment of Aspergillus fungi. Several microbial lineages were detected in Kveim and spleen, including Cladosporium. No microbial lineage was enriched in more than one sample type after correction for multiple comparisons. CONCLUSIONS: Metagenomic sequencing revealed enrichment of microbes in single types of sarcoidosis samples but limited concordance across sample types. Statistical analysis accounting for environmental contamination was essential to avoiding false positives.

High Complication Rate after Introduction of Transbronchial Cryobiopsy into Clinical Practice at an Academic Medical Center.

RATIONALE: Transbronchial cryobiopsy is an emerging technique for obtaining biopsies of lung parenchyma. Despite limited evidence of its safety and efficacy in direct comparison with other available biopsy procedures, pulmonologists are integrating this technique into clinical practice with the hope of avoiding the risks of surgical lung biopsy. OBJECTIVES: To report the rate of severe complications and diagnostic outcomes immediately after introduction of transbronchial cryobiopsy into the clinical practice of a single-center, high-volume, interventional pulmonary group at a large academic medical center in the United States. METHODS: We conducted a retrospective review of a case series. RESULTS: Twenty-five consecutive patients underwent transbronchial cryobiopsy for a variety of indications over a period of 14 weeks. In the absence of a strict protocol, a variety of techniques were employed by four attending interventional pulmonologists and one advanced interventional pulmonology fellow to plan and complete the procedures. Three patients (12%) experienced serious hemorrhage immediately after biopsy, including one patient who survived a life-threatening bleed. Two procedures were complicated by an iatrogenic pneumothorax. One patient experienced hypercapnic respiratory failure shortly after the procedure. A definitive diagnosis was made with 14 cryobiopsies (56%). Another five biopsies (20%) contributed to a presumptive diagnosis achieved by multidisciplinary consensus. CONCLUSIONS: Transbronchial cryobiopsy may have diagnostic and safety limitations that are not yet well appreciated, given the state of the published medical literature. Major questions remain regarding the safest procedural protocol to be used when performing transbronchial cryobiopsy. Thorough planning and a high degree of caution are encouraged on first introduction of this technique into a clinical practice.

Interstitial Lung Disease in the Elderly.

BACKGROUND: Despite the relationship between idiopathic pulmonary fibrosis (IPF) and advancing age, little is known about the epidemiology of interstitial lung disease (ILD) in the elderly. We describe the diagnoses, clinical characteristics, and outcomes of patients who were elderly at the time of ILD diagnosis. METHODS: Among subjects from a prospective cohort study of ILD, elderly was defined as age ≥ 70 years. Diagnoses were derived from a multidisciplinary review. Differences between elderly and nonelderly groups were determined using the χ2 test and analysis of variance. RESULTS: Of the 327 subjects enrolled, 80 (24%) were elderly. The majority of elderly subjects were white men. The most common diagnoses were unclassifiable ILD (45%), IPF (34%), connective tissue disease (CTD)-ILD (11%), and hypersensitivity pneumonitis (8%). Most elderly subjects (74%) with unclassifiable ILD had an imaging pattern inconsistent with usual interstitial pneumonia (UIP). There were no significant differences in pulmonary function or 3-year mortality between nonelderly and elderly subjects combined or in a subgroup analysis of those with IPF. CONCLUSIONS: Although IPF was the single most common diagnosis, the majority of elderly subjects had non-IPF ILD. Our findings highlight the need for every patient with new-onset ILD, regardless of age, to be surveyed for exposures and findings of CTD. Unclassifiable ILD was common among the elderly, but for most, the radiographic pattern was inconsistent with UIP. Although the effect of ILD may be more pronounced in the elderly due to reduced global functionality, ILD was not more severe or aggressive in this group.