SMARCA4 deficiency and mutations are frequent in large cell lung carcinoma and are prognostically significant.

SMARCA4 mutation has emerged as a marker of poor prognosis in lung cancer and has potential predictive value in cancer treatment, but recommendations for which patients require its investigation are lacking. We comprehensively studied SMARCA4 alterations and the clinicopathological significance in a large cohort of immunohistochemically-subtyped non-small cell lung cancer (NSCLC). A total of 1416 patients was studied for the presence of SMARCA4 deficiency by immunohistochemistry (IHC). Thereafter, comprehensive sequencing of tumours was performed for 397 of these patients to study the mutational spectrum of SWI/SNF and SMARCA4 aberrations. IHC evidence of SMARCA4 deficiency was found in 2.9% of NSCLC. Of the sequenced tumours, 38.3% showed aberration in SWI/SNF complex, and 9.3% had SMARCA4 mutations. Strikingly, SMARCA4 aberrations were much more prevalent in large cell carcinoma (LCC) than other histological tumour subtypes. SMARCA4-deficient and SMARCA4-mutated tumours accounted for 40.5% and 51.4% of all LCC, respectively. Multivariable analyses confirmed SMARCA4 mutation was an independent prognostic factor in lung cancer. The immunophenotype of a subset of these tumours frequently showed TTF1 negativity and HepPAR1 positivity. SMARCA4 mutation or its deficiency was associated with positive smoking history and poor prognosis. It also demonstrated mutual exclusion with EGFR mutation. Taken together, the high incidence of SMARCA4 aberrations in LCC may indicate its diagnostic and prognostic value. Our study established the necessity of SMARCA4 IHC in the identification of SMARCA4-aberrant tumours, and this may be of particular importance in LCC and tumours without known driver events.

Safety and feasibility of transbronchial microwave ablation for subpleural lung nodules.

BACKGROUND: Transbronchial microwave ablation in treating lung nodules is gaining popularity. However, microwave ablation in subpleural lung nodules raised concerns about pleural-based complications due to the proximity between the pleura and the ablation zone. METHODS: Patients who underwent transbronchial microwave ablation between March 2019 and November 2022 were included in this analysis. The lung nodules were categorized into the subpleural group-less than 5 mm distance to the nearest pleural surface; the deep nodule group-larger or equal to 5 mm distance to the nearest pleural surface. A review of the safety profile of subpleural lung nodule ablation was conducted. RESULTS: Eighty-two lung nodules (n = 82) from 77 patients were treated. The mean nodule size was 14.2 ± 5.50 mm. The technical success rate was 100%. The mean procedural time was 133 min. No statistically significant differences were detected in the complication rate and the length of stay between the subpleural and deep nodule groups. Complications occured in 21 nodules (25.6%). No minor pneumothorax was reported. Total five cases of pneumothorax required drainage were observed (6.06% in subpleural nodules [n = 2] vs. 6.12% in deep nodules [n = 3], p = 0.991). Total seven cases of pleuritic chest pain were observed (12.1% in subpleural nodules [n = 4] vs. 6.12% in deep nodules [n = 3], p = 0.340). CONCLUSIONS: This single-center retrospective analysis found no significant difference in the safety outcomes between subpleural and nonsubpleural lung nodule ablation. The overall rate of complications was low in the cohort. This demonstrated that transbronchial microwave was feasible and safe for most lung nodules.

Protocol to study immunodynamics in the tumor microenvironment using a tyramide signal amplification-based immunofluorescent multiplex panel.

Immunodynamics in the tumor microenvironment can be precisely examined by using multiple antigen identification approaches. Here, we present a protocol for capturing expression levels of multiple target proteins in the same specimen at single-cell resolution using a tyramide signal amplification-based immunofluorescent multiplexing system. We describe steps for tumor tissue microarray preparation, multiplex immunohistochemistry staining, image acquisition, and quantification. This protocol can quantify immune cells in tissues from patients or experimental disease models at a protein level. For complete details on the use and execution of this protocol, please refer to Chung et al. (2023),1 Tang et al. (2022),2 and Tang et al. (2022).3.

Hematopoietic Transcription Factor RUNX1 is Essential for Promoting Macrophage-Myofibroblast Transition in Non-Small-Cell Lung Carcinoma.

Macrophage-myofibroblast transition (MMT) is a newly discovered pathway for mass production of pro-tumoral cancer-associated fibroblasts (CAFs) in non-small cell lung carcinoma (NSCLC) in a TGF-ß1/Smad3 dependent manner. Better understanding its regulatory signaling in tumor microenvironment (TME) may identify druggable target for the development of precision medicine. Here, by dissecting the transcriptome dynamics of tumor-associated macrophage at single-cell resolution, a crucial role of a hematopoietic transcription factor Runx1 in MMT formation is revealed. Surprisingly, integrative bioinformatic analysis uncovers Runx1 as a key regulator in the downstream of MMT-specific TGF-ß1/Smad3 signaling. Stromal Runx1 level positively correlates with the MMT-derived CAF abundance and mortality in NSCLC patients. Mechanistically, macrophage-specific Runx1 promotes the transcription of genes related to CAF signatures in MMT cells at genomic level. Importantly, macrophage-specific genetic deletion and systemic pharmacological inhibition of TGF-ß1/Smad3/Runx1 signaling effectively prevent MMT-driven CAF and tumor formation in vitro and in vivo, representing a potential therapeutic target for clinical NSCLC.

Smad3 is essential for polarization of tumor-associated neutrophils in non-small cell lung carcinoma.

Neutrophils are dynamic with their phenotype and function shaped by the microenvironment, such as the N1 antitumor and N2 pro-tumor states within the tumor microenvironment (TME), but its regulation remains undefined. Here we examine TGF-ß1/Smad3 signaling in tumor-associated neutrophils (TANs) in non-small cell lung carcinoma (NSCLC) patients. Smad3 activation in N2 TANs is negatively correlate with the N1 population and patient survival. In experimental lung carcinoma, TANs switch from a predominant N2 state in wild-type mice to an N1 state in Smad3-KO mice which associate with enhanced neutrophil infiltration and tumor regression. Neutrophil depletion abrogates the N1 anticancer phenotype in Smad3-KO mice, while adoptive transfer of Smad3-KO neutrophils reproduces this protective effect in wild-type mice. Single-cell analysis uncovers a TAN subset showing a mature N1 phenotype in Smad3-KO TME, whereas wild-type TANs mainly retain an immature N2 state due to Smad3. Mechanistically, TME-induced Smad3 target genes related to cell fate determination to preserve the N2 state of TAN. Importantly, genetic deletion and pharmaceutical inhibition of Smad3 enhance the anticancer capacity of neutrophils against NSCLC via promoting their N1 maturation. Thus, our work suggests that Smad3 signaling in neutrophils may represent a therapeutic target for cancer immunotherapy.

Use of Indocyanine Green Fluorescence Imaging in Thoracic and Esophageal Surgery.

BACKGROUND: Fluorescence imaging using indocyanine green in thoracic and esophageal surgery is gaining popularity because of the potential to facilitate surgical planning, to stage disease, and to reduce postoperative complications. To optimize use of fluorescence imaging in thoracic and esophageal surgery, an expert panel sought to establish a set of recommendations at a consensus meeting. METHODS: The panel included 12 experts in thoracic and upper gastrointestinal surgery from Asia-Pacific countries. Before meeting, 7 focus areas were defined: intersegmental plane identification for sublobar resections; pulmonary nodule localization; lung tumor detection; bullous lesion detection; lymphatic mapping of lung tumors; evaluation of gastric conduit perfusion; and lymphatic mapping in esophageal surgical procedures. A literature search of the PubMed database was conducted using keywords indocyanine green, fluorescence, thoracic, surgery, and esophagectomy. At the meeting, panelists addressed each focus area by discussing the most relevant evidence and their clinical experiences. Consensus statements were derived from the proceedings, followed by further discussions, revisions, finalization, and unanimous agreement. Each statement was assigned a level of evidence and a grade of recommendation. RESULTS: A total of 9 consensus recommendations were established. Identification of the intersegmental plane for sublobar resections, localization of pulmonary nodules, lymphatic mapping in lung tumors, and assessment of gastric conduit perfusion were applications of fluorescence imaging that have the most robust current evidence. CONCLUSIONS: Based on best available evidence and expert opinions, these consensus recommendations may facilitate thoracic and esophageal surgery using fluorescence imaging.

Single-cell RNA sequencing uncovers a neuron-like macrophage subset associated with cancer pain.

Tumor innervation is a common phenomenon with unknown mechanism. Here, we discovered a direct mechanism of tumor-associated macrophage (TAM) for promoting de novo neurogenesis via a subset showing neuronal phenotypes and pain receptor expression associated with cancer-driven nocifensive behaviors. This subset is rich in lung adenocarcinoma associated with poorer prognosis. By elucidating the transcriptome dynamics of TAM with single-cell resolution, we discovered a phenomenon "macrophage to neuron-like cell transition" (MNT) for directly promoting tumoral neurogenesis, evidenced by macrophage depletion and fate-mapping study in lung carcinoma models. Encouragingly, we detected neuronal phenotypes and activities of the bone marrow-derived MNT cells (MNTs) in vitro. Adoptive transfer of MNTs into NOD/SCID mice markedly enhanced their cancer-associated nocifensive behaviors. We identified macrophage-specific Smad3 as a pivotal regulator for promoting MNT at the genomic level; its disruption effectively blocked the tumor innervation and cancer-dependent nocifensive behaviors in vivo. Thus, MNT may represent a precision therapeutic target for cancer pain.

Organoids in Lung Cancer Management.

Lung cancer is a complex milieu of genomically altered cancer cells, a diverse collection of differentiated cells and nonneoplastic stroma. Lung cancer organoids is a three-dimensional structure grown from patient cancer tissue that could mimic in vivo complex behavior and cellular architecture of the cancer. Furthermore, the genomic alterations of the primary lung tumor is captured ex vivo. Lung cancer organoids have become an important preclinical model for oncology studies in recent years. It could be used to model the development of lung cancer, investigate the process of tumorigenesis, and also study the signaling pathways. The organoids could also be a platform to perform drug screening and biomarker validation of lung cancer, providing a promising prediction of patient-specific drug response. In this review, we described how lung cancer organoids have opened new avenues for translating basic cancer research into clinical therapy and discussed the latest and future developments in organoid technology, which could be further applied in lung cancer organoids research.

Distinct Molecular Landscape of Epstein-Barr Virus Associated Pulmonary Lymphoepithelioma-Like Carcinoma Revealed by Genomic Sequencing.

Pulmonary lymphoepithelioma-like carcinoma (LELC) is a subtype of non-small cell lung cancer (NSCLC) characterized by marked lymphocytic infiltration and association with Epstein-Barr virus (EBV). The molecular basis underlying the disease remains unclear. We sought to study the molecular landscape by multiple approaches including whole genomic sequencing, capture-based targeted sequencing, fluorescent in situ hybridization and immunohistochemistry. Tumor cells from 57 EBV-positive pulmonary LELCs were isolated by careful microdissection prior to genomic sequencing. Integrated analysis revealed a distinct genomic landscape of low TP53 mutation rate (11%), low incidence of known drivers in the RTK/RAS/RAF (11%) and PI3K/AKT/mTOR pathways (7%), but enriched for loss-of-function mutations in multiple negative regulators of the NF-κB pathway. High level programmed cell death ligand-1 (PD-L1) expression was shown with 47% and 79% of the cases showing positive PD-L1 immunoreactivity at ≥50% and ≥1% tumor proportion score, respectively. Subsets of the patients with actionable fibroblast growth factor receptor 3 (FGFR3) aberrations (4%) and mismatch repair deficiency (4%) were potentially eligible for precision medicine. Pulmonary LELC showed a distinct genomic landscape, different from major NSCLC subtypes but resembled that of EBV-associated nasopharyngeal carcinoma. Our work facilitated the understanding of molecular basis underlying pulmonary LELC to explore potential therapeutic options.

EGFR mutation exists in squamous cell lung carcinoma.

Whether EGFR mutation occurs in lung squamous cell carcinoma (SCC) remains a controversial issue. Although numerous trials have shown positive response to tyrosine kinase inhibitors in SCC, these observations have not been well correlated with presence or absence of EGFR mutation. A complicating issue is that adenosquamous carcinoma, a mimic of SCC, frequently harbours EGFR mutations. We evaluated the EGFR mutation status of 191 cases initially diagnosed as SCC of lung origin in years 2000-2011, and performed a panel of markers including p40, p63, CK5/6, TTF-1, mucicarmine on the tissue microarray or tissue blocks from each case, to ascertain the squamous differentiation of each case. Four cases were found to have EGFR mutations, with three showing typical squamous morphological features and immunohistochemical profile on all available tumour blocks, and one reclassified as adenosquamous carcinoma. Mixed responses were noted for two of the patients with EGFR-mutated SCC treated with tyrosine kinase inhibitors. In conclusion, we report that a small subset of rigorously proven SCC harbours EGFR mutation. It also appears in our cohort that EGFR-mutated tumours, in the context of SCC, may have relatively poor response to tyrosine kinase inhibitors.

Ten-Year Trends of Clinicopathologic Features and Surgical Treatment of Lung Cancer in China.

BACKGROUND: Lung cancer has changed significantly during the past 2 decades in its epidemiology and treatment. This retrospective analysis used data from 7 major areas of China over 10 years to evaluate clinicopathologic and surgical treatment trends of lung cancer in China during the past decade. METHODS: Data from 7184 patients with primary lung cancer who were treated between 2005 and 2014 in 8 provinces of China were retrospectively collected. Their clinicopathologic features and surgical treatment information were recorded. Simple linear regression models and the Cochrane-Armitage trend test were used to assess temporal trends. RESULTS: The proportion of female patients (from 57.4% to 59.6%; P < .001) and nonsmoking patients (from 37.1% to 48.9%; P < .001) and of patients with a family history of malignant tumors (from 7.0% to 11.5%; P < .001) increased significantly. The percentage of adenocarcinomas increased significantly (from 36.4% to 53.5%; P < .001), with a decrease in squamous cell carcinomas (from 45.4% to 34.4%; P < .001). After 2008, the application of minimally invasive surgery significantly increased in China (from 2.4% in 2008 to 34.4% in 2014; P < .001), with a decline in the rate of conversion to open operation (from 14.3% in 2008 to 4.8% in 2014; P = .146) and an increase in the proportion of systematic mediastinal lymph node dissection (from 50.0% in 2008 to 84.1% in 2014; P = .001). CONCLUSIONS: This study investigated recent 10-year trends in the clinicopathologic features and surgical treatment of lung cancer in China and found significant important changes. These findings provide valuable information and evidence for the future control of the disease in China.

A comparison of EGFR mutation status in tissue and plasma cell-free DNA detected by ADx-ARMS in advanced lung adenocarcinoma patients.

BACKGROUND: Previous studies have shown that there are different methods used to detect the epidermal growth factor receptor (EGFR) mutation status in plasma cell-free DNA (cfDNA) for advanced lung adenocarcinoma patients including the ADx-Amplification Refractory Mutation System (ADx-ARMS). We explored the performance of the ADx-ARMS in detecting the EGFR mutations in cfDNA. METHODS: This prospective cohort study enrolled patients who presented with advanced (stage IIIb/IV) lung adenocarcinoma. EGFR mutations in plasma cfDNA and tumor tissues by ADx-ARMS were detected. Next-generation sequencing (NGS) in plasma was performed in patients with inconsistent gene region mutations in the plasma and matched tissue samples. We calculated the clinical parameters of the ADx-ARMS for EGFR mutation status in the plasma of cfDNA, using the tumor tissues as the standard for measurement. The objective response rate (ORR) and progression-free survival (PFS) were also calculated for patients receiving first-generation EGFR-tyrosine kinase inhibitors (TKIs) therapy. RESULTS: In total, 203 patients were included in the final analysis. Mutations were discovered in 58.6% (119/203) of the tumor tissues and 31.0% (63/203) were detected EGFR mutations in both tumor tissues and matched plasma. The sensitivity and the specificity setting for detecting the EGFR mutations in the plasma using the ADx-ARMS were configured to 52.9% and 98.8%. An ORR of 64.8% was observed among the 71 patients who were identified as being EGFR-positive in their tumor tissues, who had received treatments using Gefitinib or Icotinib. Next, the ORR was observed to be 69.0% among the 42 patients with an EGFR mutation in their plasma. The median PFS of the patients with an EGFR mutation in tumor tissues and plasma were 10.0 vs. 11.0 months (P=0.175). The median PFS of the patients with an EGFR wild-type in the plasma was 8.7 months, which was significantly shorter than the EGFR mutant-type in plasma (P=0.001). CONCLUSIONS: Using ADx-ARMS as an approach with high specificity but moderate sensitivity to detect the EGFR mutations in plasma cfDNA and EGFR mutation status in plasma cfDNA using the ADx-ARMS can predict the tumor response for EGFR-TKIs.

Accuracy of a 3-Dimensionally Printed Navigational Template for Localizing Small Pulmonary Nodules: A Noninferiority Randomized Clinical Trial.

Importance: Localization of small lung nodules are challenging because of the difficulty of nodule recognition during video-assisted thoracoscopic surgery. Using 3-dimensional (3-D) printing technology, a navigational template was recently created to assist percutaneous lung nodule localization; however, the efficacy and safety of this template have not yet been evaluated. Objective: To assess the noninferiority of the efficacy and safety of a 3-D-printed navigational template guide for localizing small peripheral lung nodules. Design, Setting, and Participants: This noninferiority randomized clinical trial conducted between October 2016 and October 2017 at Shanghai Pulmonary Hospital, Shanghai, China, compared the safety and precision of lung nodule localization using a template-guided approach vs the conventional computed tomography (CT)-guided approach. In total, 213 surgical candidates with small peripheral lung nodules (<2 cm) were recruited to undergo either CT- or template-guided lung nodule localization. An intention-to-treat analysis was conducted. Interventions: Percutaneous lung nodule localization. Main Outcomes and Measures: The primary outcome was the accuracy of lung nodule localization (localizer deviation), and secondary outcomes were procedural duration, radiation dosage, and complication rate. Results: Of the 200 patients randomized at a ratio of 1:1 to the template- and CT-guided groups, most were women (147 vs 53), body mass index ranged from 15.4 to 37.3, the mean (SD) nodule size was 9.7 (2.9) mm, and the mean distance between the outer edge of target nodule and the pleura was 7.8 (range, 0.0-43.9) mm. In total, 190 patients underwent either CT- or template-guided lung nodule localization and subsequent surgery. Among these patients, localizer deviation did not significantly differ between the template- and CT-guided groups (mean [SD], 8.7 [6.9] vs 9.6 [5.8] mm; P = .36). The mean (SD) procedural durations were 7.4 (3.2) minutes for the template-guided group and 9.5 (3.6) minutes for the CT-guided group (P < .001). The mean (SD) radiation dose was 229 (65) mGy × cm in the template-guided group and 313 (84) mGy × cm in CT-guided group (P < .001). Conclusions and Relevance: The use of the 3-D-printed navigational template for localization of small peripheral lung nodules showed efficacy and safety that were not substantially worse than those for the CT-guided approach while significantly simplifying the localization procedure and decreasing patient radiation exposure. Trial Registration: ClinicalTrials.gov identifier: NCT02952261.

The Society for Translational Medicine: clinical practice guidelines for mechanical ventilation management for patients undergoing lobectomy.

Patients undergoing lobectomy are at significantly increased risk of lung injury. One-lung ventilation is the most commonly used technique to maintain ventilation and oxygenation during the operation. It is a challenge to choose an appropriate mechanical ventilation strategy to minimize the lung injury and other adverse clinical outcomes. In order to understand the available evidence, a systematic review was conducted including the following topics: (I) protective ventilation (PV); (II) mode of mechanical ventilation [e.g., volume controlled (VCV) versus pressure controlled (PCV)]; (III) use of therapeutic hypercapnia; (IV) use of alveolar recruitment (open-lung) strategy; (V) pre-and post-operative application of positive end expiratory pressure (PEEP); (VI) Inspired Oxygen concentration; (VII) Non-intubated thoracoscopic lobectomy; and (VIII) adjuvant pharmacologic options. The recommendations of class II are non-intubated thoracoscopic lobectomy may be an alternative to conventional one-lung ventilation in selected patients. The recommendations of class IIa are: (I) Therapeutic hypercapnia to maintain a partial pressure of carbon dioxide at 50-70 mmHg is reasonable for patients undergoing pulmonary lobectomy with one-lung ventilation; (II) PV with a tidal volume of 6 mL/kg and PEEP of 5 cmH2O are reasonable methods, based on current evidence; (III) alveolar recruitment [open lung ventilation (OLV)] may be beneficial in patients undergoing lobectomy with one-lung ventilation; (IV) PCV is recommended over VCV for patients undergoing lung resection; (V) pre- and post-operative CPAP can improve short-term oxygenation in patients undergoing lobectomy with one-lung ventilation; (VI) controlled mechanical ventilation with I:E ratio of 1:1 is reasonable in patients undergoing one-lung ventilation; (VII) use of lowest inspired oxygen concentration to maintain satisfactory arterial oxygen saturation is reasonable based on physiologic principles; (VIII) Adjuvant drugs such as nebulized budesonide, intravenous sivelestat and ulinastatin are reasonable and can be used to attenuate inflammatory response.