Rationale: The contributions of diverse cell populations in the human lung to pulmonary fibrosis pathogenesis are poorly understood. Single-cell RNA sequencing can reveal changes within individual cell populations during pulmonary fibrosis that are important for disease pathogenesis. Objectives: To determine whether single-cell RNA sequencing can reveal disease-related heterogeneity within alveolar macrophages, epithelial cells, or other cell types in lung tissue from subjects with pulmonary fibrosis compared with control subjects. Methods: We performed single-cell RNA sequencing on lung tissue obtained from eight transplant donors and eight recipients with pulmonary fibrosis and on one bronchoscopic cryobiospy sample from a patient with idiopathic pulmonary fibrosis. We validated these data using in situ RNA hybridization, immunohistochemistry, and bulk RNA-sequencing on flow-sorted cells from 22 additional subjects. Measurements and Main Results: We identified a distinct, novel population of profibrotic alveolar macrophages exclusively in patients with fibrosis. Within epithelial cells, the expression of genes involved in Wnt secretion and response was restricted to nonoverlapping cells. We identified rare cell populations including airway stem cells and senescent cells emerging during pulmonary fibrosis. We developed a web-based tool to explore these data. Conclusions: We generated a single-cell atlas of pulmonary fibrosis. Using this atlas, we demonstrated heterogeneity within alveolar macrophages and epithelial cells from subjects with pulmonary fibrosis. These results support the feasibility of discovery-based approaches using next-generation sequencing technologies to identify signaling pathways for targeting in the development of personalized therapies for patients with pulmonary fibrosis.

Purpose: This phase 1 dose escalation study evaluated the safety and feasibility of single-dose intrapleural IFN-β gene transfer using an adenoviral vector (Ad.IFN-β) in patients with malignant pleural mesothelioma (MPM) and metastatic pleural effusions (MPE). Experimental Design: Ad.IFN-β was administered through an indwelling pleural catheter in doses ranging from 9 × 1011 to 3 × 1012 viral particles (vp) in two cohorts of patients with MPM (7 patients) and MPE (3 patients). Subjects were evaluated for (a) toxicity, (b) gene transfer, (c) humoral, cellular, and cytokine-mediated immune responses, and (d) tumor responses via 18-fluorodeoxyglucose-positron emission tomography scans and chest computed tomography scans. Results: Intrapleural Ad.IFN-β was generally well tolerated with transient lymphopenia as the most common side effect. The maximally tolerated dose achieved was 9 × 1011 vp secondary to idiosyncratic dose-limiting toxicities (hypoxia and liver function abnormalities) in two patients treated at 3 × 1012 vp. The presence of the vector did not elicit a marked cellular infiltrate in the pleural space. Intrapleural levels of cytokines were highly variable at baseline and after response to gene transfer. Gene transfer was documented in 7 of the 10 patients by demonstration of IFN-β message or protein. Antitumor immune responses were elicited in 7 of the 10 patients and included the detection of cytotoxic T cells (1 patient), activation of circulating natural killer cells (2 patients), and humoral responses to known (Simian virus 40 large T antigen and mesothelin) and unknown tumor antigens (7 patients). Four of 10 patients showed meaningful clinical responses defined as disease stability and/or regression on 18-fluorodeoxyglucose-positron emission tomography and computed tomography scans at day 60 after vector infusion. Conclusions: Intrapleural instillation of Ad.IFN-β is a potentially useful approach for the generation of antitumor immune responses in MPM and MPE patients and should be investigated further for overall clinical efficacy.

We previously showed that a single intrapleural dose of an adenoviral vector expressing interferon-beta (Ad.IFN-beta) in patients with malignant pleural mesothelioma (MPM) or malignant pleural effusions (MPE) resulted in gene transfer, humoral antitumor immune responses, and anecdotal clinical responses manifested by modified Response Evaluation Criteria in Solid Tumors (RECIST) disease stability in 3 of 10 patients at 2 months and an additional patient with significant metabolic response on positron emission tomography (PET) imaging. This phase I trial was conducted to determine whether using two doses of Ad.IFN-beta vector would be superior. Ten patients with MPM and seven with MPE received two doses of Ad.IFN-beta through an indwelling pleural catheter. Repeated doses were generally well tolerated. High levels of IFN-beta were detected in pleural fluid after the first dose; however, only minimal levels were seen after the second dose of vector. Lack of expression correlated with the rapid induction of neutralizing Ad antibodies (Nabs). Antibody responses against tumor antigens were induced in most patients. At 2 months, modified RECIST responses were as follows: one partial response, two stable disease, nine progressive disease, and two nonmeasurable disease. One patient died after 1 month. By PET scanning, 2 patients had mixed responses and 11 had stable disease. There were seven patients with survival times longer than 18 months. This approach was safe, induced immune responses and disease stability. However, rapid development of Nabs prevented effective gene transfer after the second dose, even with a dose interval as short as 7 days.

Diagnostic yield and number of lymph nodes sampled using deep sedation is superior to moderate sedation in patients undergoing EBUS-TBNA. Prospective studies accounting for other factors including patient selection and cost are needed.

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New therapeutic strategies are needed for malignant pleural mesothelioma (MPM). We conducted a single-center, open-label, nonrandomized, pilot and feasibility trial using two intrapleural doses of an adenoviral vector encoding human IFN-a (Ad.IFN-a2b). Nine subjects were enrolled at two dose levels. The first three subjects had very high pleural and systemic IFN-a concentrations resulting in severe "flu-like" symptoms necessitating dose de-escalation. The next six patients had reduced (but still significant) pleural and serum IFN-a levels, but with tolerable symptoms. Repeated vector administration appeared to prolong IFN-a expression levels. Antitumor humoral immune responses against mesothelioma cell lines were seen in seven of the eight subjects evaluated. No clinical responses were seen in the four subjects with advanced disease. However, evidence of disease stability or tumor regression was seen in the remaining five patients, including one dramatic example of partial tumor regression at sites not in contiguity with vector infusion. These data show that Ad.IFN-a2b has potential therapeutic benefit in MPM and that it generates anti-tumor immune responses that may induce anatomic and/or metabolic reductions in distant tumor. Clinical trial registered with www.clinicaltrials.gov (NCT 01212367).

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<b><i>Background:</i></b> Bronchoscopy for the diagnosis of peripheral pulmonary lesions continues to present clinical challenges, despite increasing experience using newer guided techniques. Robotic bronchoscopic platforms have been developed to potentially improve diagnostic yields. Previous studies in cadaver models have demonstrated increased reach into the lung periphery using robotic systems compared to similarly sized conventional bronchoscopes, although the clinical impact of additional reach is unclear. <b><i>Objectives:</i></b> This study was performed to evaluate the performance of a robotic bronchoscopic system’s ability to reach and access artificial tumor targets simulating peripheral nodules in human cadaveric lungs. <b><i>Methods:</i></b> Artificial tumor targets sized 10–30 mm in axial diameter were implanted into 8 human cadavers. CT scans were performed prior to procedures and all cadavers were intubated and mechanically ventilated. Electromagnetic navigation, radial probe endobronchial ultrasound, and fluoroscopy were used for all procedures. Robotic-assisted bronchoscopy was performed on each cadaver by an individual bronchoscopist to localize and biopsy peripheral lesions. <b><i>Results:</i></b> Sixty-seven nodules were evaluated in 8 cadavers. The mean nodule size was 20.4 mm. The overall diagnostic yield was 65/67 (97%) and there was no statistical difference in diagnostic yield for lesions &#x3c;20 mm compared with lesions measuring 21–30 mm, the presence of a concentric or eccentric radial ultrasound image, or relative distance from the pleura. <b><i>Conclusions:</i></b> The robotic bronchoscopic system was successful at biopsying 97% of peripheral pulmonary lesions 10–30 mm in size in human cadavers. These findings support further exploration of this technology in prospective clinical trials in live human subjects.