Perilipin 2 expression is related to poor prognosis of patients with various malignant tumors. It exists on the surface of lipid droplets (LDs), which store lipids that can be used as an energy source during cancer progression. However, the underlying mechanism of lipid metabolism involving LDs in the progression of lung adenocarcinoma is unclear. This study investigated the role of perilipin 2 in the regulation of lipid metabolism in lung adenocarcinoma, as well as patient prognosis. In clinicopathologic analyses, high perilipin 2 expression in adenocarcinomas was significantly associated with poor differentiation, blood vessel and pleural invasion, advanced cancer stage, and large tumor size relative to perilipin 2-negative cases. Furthermore, patients with high perilipin 2 expression had significantly shorter recurrence-free survival times. LD accumulation was significantly reduced in A549 and PC-9 lung adenocarcinoma cells with clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) genome editing-mediated knockout of PLIN2 expression. Treatment of these cells with extracellular oleic acid induced accumulation of LDs, but the total amount of accumulated LDs was low in PLIN2 knockout cells compared with control cells. Cell proliferation and migration ability was also significantly reduced in PLIN2 knockout cells. Together, these results suggest that perilipin 2 mediates aggressive cancer progression in lung adenocarcinoma by regulating LD accumulation, and thus may represent a potential target for suppressing lung adenocarcinoma.

Background: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors are powerful molecular targeted therapeutic agents for lung cancer. We recently developed an original immunocytology and glass slide-based circulating tumor cell (CTC) detection platform for both CTC enumeration and EGFR mutation analysis with DNA extracted from CTCs. Methods: Using this platform, we conducted a pilot clinical study for CTC enumeration in peripheral blood (PB), pulmonary arterial blood (PA), and pulmonary venous blood (PV) from 33 patients with lung cancer (Stage I–III) who underwent surgery, followed by digital PCR-based EGFR mutation analysis of CTCs in PV from 12 patients. Results: The results showed that CTC levels were significantly higher in PV and PA than in PB (p < 0.05, p < 0.01. respectively), with a notably greater number of small and large CTC clusters (p < 0.01). Genetic analysis of EGFR mutations of CTCs from PV (n = 12) revealed six mutations, including three Exon19del and three L856R, in CTCs and eight EGFR mutations, including five Exon19del and three L856R, in lung tumor tissue. CTC mutation status matched that of tissue samples in nine patients, was unmatched in two patients, and controversial in one patient, indicating a sensitivity of 0.75 (6/8) and specificity of 1.0 (4/4) with some false-negative results for the mutation analysis of CTCs. Conclusions: This immunocytology-based CTC detection platform is a convenient method for detecting both CTC number and EGFR mutation status under microscopy, suggesting its potential as a liquid biopsy tool in the hospital for patients with lung cancer in some clinical settings.

Systemic arterial blood supply to a normal lung is a rare anatomical abnormality. Surgery is usually indicated because this abnormality leads to pulmonary hypertension. Herein, we report our experience and ideas for safe vessel dissection. Case 1 was a woman in her 50s. We performed a left lower lobectomy following percutaneous coil embolization. The aberrant artery with emboli was confirmed intraoperatively by cone-beam computed tomography (CBCT) to safely dissect under thoracoscopic surgery (TS). Case 2 was a man in his 40s. Following percutaneous endovascular plug occlusion, we performed a left partial resection using indocyanine green fluorescence navigation. Intraoperatively, CBCT imaging demonstrated the aberrant artery and exact position of the emboli. This combination technique of interventional radiology and TS with CBCT imaging was considered safe and more secure for the treatment of anomalous systemic arterial blood supply to a normal lung.

Several methods can be used to intraoperatively identify pulmonary lesion using radiation technology. However, little is known about patient radiation exposure during chest surgery. We aimed to measure patients’ radiation exposure from cone-beam computed tomography (CBCT) used in a hybrid operating room. This retrospective study included patients who underwent surgical treatment in a hybrid operating room between April 2019 and December 2023 at the Teikyo University Hospital. All data was obtained prospectively, but the study was approved by the IRB as a retrospective study because of repeated extensions of study period in order to collect more cases. Skin radiation exposure was measured using five wearable dosimeters per patient. The measurements were compared to cumulative Air Kerma. Furthermore, the radiation exposure dose on the surgical side, which cannot be measured, was estimated by computer simulation. Among 182 patients who underwent surgery in a hybrid operating room, radiation exposure measurements were conducted on 67 patients. The patients’ mean age was 60.7 years. The average number of CBCT scans was 2.1 (1–5) and the intraoperative identification rate was 100

Diaphragmatic eventration causes respiratory distress with the development of severe diaphragmatic compression of lung volume. While non-surgical treatment, such as physical therapy and pulmonary rehabilitation, is adequate for mild cases, surgical intervention is critical for severe diaphragmatic eventration. A 45-year-old man with respiratory fatigue was diagnosed with left diaphragmatic eventration and underwent surgery with diaphragmatic plication with double-row stapling under a video-assisted approach. Thoracoscopy with laparoscopic image display avoided visceral tissue involvement, and the double-stapling technique prevented diaphragmatic rupture and lowered the diaphragmatic level. The procedure improved the patient's respiratory function by reducing respiratory fatigue. This procedure is safe and effective for the thin and vulnerable diaphragmatic muscle.

PURPOSE:A prolonged air leak (PAL) is one of the common postoperative complications of pulmonary resection. The aim of this study was to evaluate the efficacy and safety of pleurodesis with sterile talc or OK-432 for postoperative air leak.METHODS:Patients with postoperative air leak who received chemical pleurodesis using sterile talc or OK-432 were retrospectively identified from medical records data. For pleurodesis with either agent, prior assessment and approval by the hospital safety department were carried out for each case, in addition to individual consent.RESULTS:Between February 2016 and June 2022, 39 patients had PALs and underwent chemical pleurodesis. Among them, 24 patients received pleurodesis with talc (Talc group) and 15 with OK-432 (OK-432 group). The leak resolved after less than two pleurodesis treatments in 22 patients (91.7%) in the Talc group compared with 14 patients (93.3%) in the OK-432 group. Pleurodesis significantly increased white blood cell counts, C-reactive protein concentration, and body temperature in the OK-432 group compared with that in the Talc group (p <0.001, p = 0.003, and p <0.001, respectively).CONCLUSIONS:Pleurodesis with talc may be an effective treatment option for postoperative air leak. Our findings suggest that talc was as effective as OK-432 and resulted in a milder systemic inflammatory response.

INTRODUCTION:Cancer-associated fibroblasts (CAFs) in the tumour microenvironment play a key role in tumour development, proliferation, invasion, and metastasis. The cytological features of spindle cells including CAFs-defined as stromal spindle cells (SSCs) adjacent to cancer cells-are frequently encountered in pulmonary adenocarcinomas. This study aimed to investigate the association between the presence of SSCs in cytological specimens and the clinicopathological features.METHODS:We evaluated 211 patients with pulmonary adenocarcinoma who underwent surgical resection. All participants had cytological specimens corresponding to the histological specimens available for review.RESULTS:Of the 211 cases examined, 89 were SSC-positive (SSC+ ) and 122 were SSC-negative (SSC- ). SSC+ cases were more frequently associated with higher pathological stage (P < 0.001), lymph node metastasis (P = 0.002), anaplastic lymphoma kinase (ALK) gene rearrangement (P = 0.04), high tumour grade (P < 0.001), solid and micropapillary predominant pattern (P = 0.02), and lymphatic vessel (P = 0.003), blood vessel (P < 0.001), and pleural invasion (P = 0.03) as compared to SSC- cases. Patients with SSC+ adenocarcinoma had a significantly shorter recurrence-free survival than those with SSC- adenocarcinoma (P = 0.009). Cytologically, necrotic background (P = 0.002), mucinous cancer cells (P = 0.02), pleomorphic cells (P < 0.001), and mutual cell inclusions (P = 0.01) were observed more frequently in SSC+ adenocarcinomas.CONCLUSIONS:The presence of SSCs could be an important cytological feature for predicting poor prognosis in lung adenocarcinomas.

Management of secondary spontaneous pneumothorax caused by interstitial lung disease is challenging because of the limited treatment options. This report describes a complicated case of secondary refractory pneumothorax in a patient with chronic obstructive pulmonary disease and interstitial lung disease, which was treated successfully by bronchial occlusion followed by chemical pleurodesis. A 75-year-old man was referred to our institution with right tension pneumothorax caused by combined pulmonary fibrosis and emphysema, which was treated with drainage. Subsequently, surgery was performed to treat a persistent air leak after the site was identified. However, left pneumothorax occurred postoperatively, followed by right pneumothorax recurrence. The culprit bronchi on both sides were filled using Endobronchial Watanabe Spigots. Consequently, the air leaks decreased but did not disappear completely. Chemical pleurodesis with talc was effective for the right-sided air leak. On the left side, three chemical pleurodesis procedures with additional bronchial occlusion were required to stop the air leak. The patient was discharged without any adverse events. Our experience suggests that bronchial occlusion followed by chemical pleurodesis could be effective for refractory pneumothorax in nonsurgical patients with interstitial lung disease.

Supplementary Figures S1-S7 and figure legends

Background:Intraoperative identification of small pulmonary nodules has been an important technical issue. We aimed to develop a new localization method which is much safer and simple procedure compared with conventional methods.Methods:This was a retrospective study including patients with resected peripheral pulmonary nodules between November 2017 and April 2021 at Teikyo University School of Medicine, and Saitama Cardiovascular and Respiratory Center. All surgical procedure was wedge resection, and the tumor size was equal to or less than 20 mm which were detected by cone-beam computed tomography (CBCT; Philips Allura Xper FD 20, Philips). Some metal clips were put on several places of visceral pleura, where the target lesion was sandwiched by marking clips (sandwich marking technique). CBCT detected both the target lesion and metal clips, and video-assisted thoracoscopic surgery (VATS) was performed. Radiological and pathological findings were analyzed, and the correlation coefficient of tumor size was examined among pre-, intra-, and post-operative tumor sizes.Results:The average age of 90 patients was 65.2 years, and 47 were male (52.2%). All procedure was wedge resection including twelve bi-wedge resections, and one hundred nine peripheral pulmonary lesions were obtained by sandwich marking technique. The detection rate was 100%, and there was no marking-related complication.Conclusions:All small peripheral pulmonary lesions were successfully detected and resected by using CBCT with no marking-related complication. Sandwich marking technique was demonstrated to provide safe, reliable, and simple localization procedure for small peripheral pulmonary lesions.

Mediastinal M & uuml;llerian cysts are rare and are composed of ciliated M & uuml;llerian-type tubal epithelium. Preoperative diagnosis of this cyst is usually difficult owing to the local anatomy. We report a case of a M & uuml;llerian cyst in the mediastinum of a 25 -year -old female patient that was resected by robotassisted thoracoscopic surgery (RATS) without rupturing the cyst. The tumor was a monolocular cyst covered with a thin capsule, and pathological examination showed a thin -walled cyst lined by ciliated or nonciliated columnar cells resembling tubal epithelium. Immunohistochemical (IHC) analysis showed positive expression for paired box protein 8 (PAX8), Wilms' tumor 1 (WT -1), and estrogen receptors (ERs) and progesterone receptors (PRs). From the results of IHC analysis of control cases of mediastinal cystic tumors, four types of IHC analysis are quite helpful in obtaining a definitive diagnosis of M & uuml;llerian cyst. Although this cyst formation has been considered to be related to hormone imbalance, we suggest that hormone imbalance may be irrelevant to the etiology of M & uuml;llerian cyst, considering our case and two previous young cases. According to the fact that there is no significant difference between M & uuml;llerian cyst and non-M & uuml;llerian cyst in hematoxylin and eosin (HE) staining, a M & uuml;lleriancyst should always be considered in patients with a posterior mediastinal cyst, especially when it is adjacent to a thoracic vertebral body.

Pulmonary capillary hemangiomatosis (PCH) is a rare disease characterized by a proliferation of capillaries in the alveolar septa, bronchial and venous walls, pleura, and regional lymph nodes. However, the etiology of the disease remains unknown due to its rarity. Therefore, we present a case of a solitary PCH lesion without symptoms in a 38-year-old female patient. According to computed tomography, she was diagnosed with lung carcinoma, indicated by a tiny nodule with ground-glass opacity detected in her right upper lung. However, no other lesions were detected on systemic examination. Consequently, partial lung resection was conducted, since the lesion was suspected of lung adenocarcinoma. Pathologic results showed that the thick alveolar septa were caused by capillary growth without cellular atypia and hardly any infiltration of inflammatory cells. Finally, we diagnosed the pulmonary lesion as PCH, although solitary PCH has previously been reported in a few case reports. Therefore, further case studies are essential to clarify the causes of PCH.

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Ipilimumab improves clinical outcomes when combined with nivolumab in metastatic non-small cell lung cancer (NSCLC), but its efficacy and impact on the immune microenvironment in operable NSCLC remain unclear. We report the results of the phase 2 randomized NEOSTAR trial (NCT03158129) of neoadjuvant nivolumab or nivolumab + ipilimumab followed by surgery in 44 patients with operable NSCLC, using major pathologic response (MPR) as the primary endpoint. The MPR rate for each treatment arm was tested against historical controls of neoadjuvant chemotherapy. The nivolumab + ipilimumab arm met the prespecified primary endpoint threshold of 6 MPRs in 21 patients, achieving a 38% MPR rate (8/21). We observed a 22% MPR rate (5/23) in the nivolumab arm. In 37 patients resected on trial, nivolumab and nivolumab + ipilimumab produced MPR rates of 24% (5/21) and 50% (8/16), respectively. Compared with nivolumab, nivolumab + ipilimumab resulted in higher pathologic complete response rates (10% versus 38%), less viable tumor (median 50% versus 9%), and greater frequencies of effector, tissue-resident memory and effector memory T cells. Increased abundance of gut Ruminococcus and Akkermansia spp. was associated with MPR to dual therapy. Our data indicate that neoadjuvant nivolumab + ipilimumab-based therapy enhances pathologic responses, tumor immune infiltrates and immunologic memory, and merits further investigation in operable NSCLC.

Abstract Little is known of the geospatial architecture of individual cell populations in lung adenocarcinoma (LUAD) evolution. Here, we perform single-cell RNA sequencing of 186,916 cells from five early-stage LUADs and 14 multiregion normal lung tissues of defined spatial proximities from the tumors. We show that cellular lineages, states, and transcriptomic features geospatially evolve across normal regions to LUADs. LUADs also exhibit pronounced intratumor cell heterogeneity within single sites and transcriptional lineage-plasticity programs. T regulatory cell phenotypes are increased in normal tissues with proximity to LUAD, in contrast to diminished signatures and fractions of cytotoxic CD8+ T cells, antigen-presenting macrophages, and inflammatory dendritic cells. We further find that the LUAD ligand–receptor interactome harbors increased expression of epithelial CD24, which mediates protumor phenotypes. These data provide a spatial atlas of LUAD evolution, and a resource for identification of targets for its treatment. Significance: The geospatial ecosystem of the peripheral lung and early-stage LUAD is not known. Our multiregion single-cell sequencing analyses unravel cell populations, states, and phenotypes in the spatial and ecologic evolution of LUAD from the lung that comprise high-potential targets for early interception. This article is highlighted in the In This Issue feature, p. 2355

The mechanism by which anti-cancer immunity shapes early carcinogenesis of lung adenocarcinoma (ADC) is unknown. In this study, we characterize the immune contexture of invasive lung ADC and its precursors by transcriptomic immune profiling, T cell receptor (TCR) sequencing and multiplex immunofluorescence (mIF). Our results demonstrate that anti-tumor immunity evolved as a continuum from lung preneoplasia, to preinvasive ADC, minimally-invasive ADC and frankly invasive lung ADC with a gradually less effective and more intensively regulated immune response including down-regulation of immune-activation pathways, up-regulation of immunosuppressive pathways, lower infiltration of cytotoxic T cells (CTLs) and anti-tumor helper T cells (Th), higher infiltration of regulatory T cells (Tregs), decreased T cell clonality, and lower frequencies of top T cell clones in later-stages. Driver mutations, chromosomal copy number aberrations (CNAs) and aberrant DNA methylation may collectively impinge host immune responses and facilitate immune evasion, promoting the outgrowth of fit subclones in preneoplasia into dominant clones in invasive ADC. The evolution of immune landscape in lung adenocarcinoma (ADC) is largely unknown. Here the authors use a cohort of resected invasive lung ADC and its precursors and show a gradual increase of immunosuppression and decrease of anti-tumor response associated with specific genomic and epigenetic features.

Background How neoadjuvant chemo-immunotherapy modulates tumor immune composition and response is not completely understood. We interrogate immunomodulation of neoadjuvant platinum-based chemotherapy (C), nivolumab (N), and N-plus-C (NC) and their connections to therapeutic efficacy in resected non-small cell lung cancer (NSCLC) by integrating immunomic data from the ImmunogenomiC PrOfiling of NSCLC (ICON) study and NEOSTAR trial cohorts. Methods In NEOSTAR (NCT03158129), patients with stage I-IIIA (single N2) resectable NSCLC (AJCC7th) received N (3 mg/kg IV, D1,15,29); patients with stage IB(≥4cm)-IIIA (single N2) resectable NSCLC received NC (N 360 mg IV plus C, D1,22,43 for 3 cycles, every 3 weeks) before surgery; major pathologic response (MPR) was the primary endpoint. In ICON, patients with stage IB(≥4cm)-IIIA resectable NSCLC received C before surgery. Surgically resected tumor samples underwent immune profiling via flow cytometry (n=16,13,9 for C,N,NC), immunohistochemistry (IHC;n=0,18,14), and multiplexed immunofluorescence (mIF;n=28,16,10). Treatment-associated immunomodulation and associations with therapeutic efficacy were analyzed using: 1) a shared nearest neighbors-based network we developed linking measurements across datasets; 2) MetaCyto, a specialized cytometry analysis method for identifying cell subsets by clustering. Results We holistically explored the immunomic data by integration across cohorts. Through hierarchical regression of the integrated data, we determined the overall effect of a given treatment controlling for the presence or absence of the other treatment. We examined C's effects across all cohorts controlling for N. Across all patients, regardless of MPR, C is associated with immunosuppression, increasing PD1+ T cell (CD45+CD3+) populations: regulatory (CD4+CD25+FOXP3+), helper (CD4+), and effector (CD8+) (effect size(ES):1.48,1.61,1.26;q<0.05). C also decreases proliferative (Ki67+) populations: helper and effector T cells as well as NK (CD45+CD3-CD56+) cells (ES:-1.27,-1.43;-1.36;q<0.05). In patients without MPR (i.e., non-responding patients), immunosuppression appears heightened by increased Ki67+ regulatory T cells (ES:1.86;q<0.05). Conversely, we examined N's effects across all cohorts controlling for C. Across all patients, regardless of MPR, N is associated with immune activation, increasing ICOS+ T cell populations: regulatory, helper, and effector (ES:1.29,1.29,1.47;q<0.05). Comparing N and NC reveals that adding C may drive exhaustion by increasing TIM3+ regulatory, helper and effector T cells (ES:1.16,1.17,1.23;q<0.05), an effect more pronounced in non-responding patients (ES:1.31,1.33,1.35;q<0.05). Conclusions We report the first integrated examination of the immunomodulatory effect of neoadjuvant C and N. C is associated with immunosuppression while N with immune activation; together, N appears to lessen C's suppressive effects. Incorporation of transcriptomics into this integrated network of flow cytometry, mIF, and IHC immune profiling data is ongoing to augment translational insights for neoadjuvant chemo/immunotherapies.

Abstract Introduction: Despite the recent approval of immune checkpoints inhibitors (ICI) as a treatment option in the extensive-stage small cell lung cancer (SCLC) setting, survival has not significantly changed in the last decades. Recent scientific efforts have led to the identification of 4 major subtypes defined by expression of three transcription factors: ASCL1 (SCLC-A), NEUROD1 (SCLC-N), POU2F3 (SCLC-P) with the fourth subtype characterized by increased expression of immune genes (Inflamed subtype - SCLC-I). Transcriptomic results, along with recent immunotherapy trials, suggest that modulation of tumor immune microenvironment (TIME) could potential be critical to achieving clinical responses in a subset of patients, hence a comprehensive study of the TIME in SCLC is imperative. Here we report the feasibility of a multiplex immunofluorescence (mIF) methodology to analyze the TIME in SCLC. Methods: FFPE sections from surgically resected SCLC (N=4, one representative case across all SCLC subtypes) were identified from the ICON Project at UT MD Anderson Cancer Center. We used mIF to identify and quantify immune markers grouped into two 6-antibody panels: Panel 1: cytokeratin (CK, AE1/AE3), CD3, CD8, PD-1, PD-L1 and CD68; Panel 2: CK, CD20, granzyme B, FOXP3, CD45RO, and CD57. Finally, genomic (WES), transcriptomic (RNA sequencing) and proteomic (RPPA) data from these cases were integrated with the mIF data. Results: SCLC molecular subtypes (SCLC-A, N, P, I) were classified using transcriptomic and proteomic data. Analysis of TIME unveils a higher immune cell infiltration within SCLC-I subtype compared with the other cases representing, immune “cold” SCLC subtypes. SCLC-I subtype showed a 2-13 folder higher (range) immune cell density than SCLC-A, N and P subtypes (measured as a median of cell density). Specifically, T cells (CD3+) (695 and 242 cells/mm2, for SCLC-I and the median for the other subtypes respectively), T cytotoxic cells (CD3+ CD8+) (206 and 105), activated T cells (CD3+ CD8+ granzyme+) (20 and 2), antigen experienced ‘like' T cells (CD3+ PD-1+) (17 and 0), memory T cells (CD3+ CD45RO+) (328 and 91) and macrophages (CD68+) (773 and 57). PD-L1 expression in malignant cells did not show significant differences within the 4 SCLC subtypes. However, PD-L1 expression in macrophages was significantly higher in the SCLC-I subtype, suggesting an increase of IFN-gamma in the TIME. Conclusions: TIME study show the use of mIF in SCLC tumors to be feasible, and could potentially provide key information towards the identification of SCLC patients that could benefit from ICI. For the first time we complemented transcriptomic data from SCLC tumors with mIF analysis unveiling the complex interplay of the host immune response and malignant cells. Our preliminary results warrant further studies to explore the role of TIME in immunotherapeutic response in SCLC. Citation Format: Pedro Rocha, C. Allison Stewart, Edwin Parra, Luisa M. Solis, Carl M. Gay, Robert J. Cardnell, Naohiro Uraoka, Alejandro Francisco-Cruz, Hitoshi Dejima, Yuanxin Xi, Lixia Diao, Jing Wang, Marcelo V. Negrao, Jianjun Zhang, Ignacio Wistuba, Don L. Gibbons, Lauren A. Byers. Multiplex immunofluorescence (mIF) reveals differences in tumor immune microenvironment between molecularly-defined subsets of small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2758.

SUMMARYHow anti-cancer immunity shapes early carcinogenesis of lung adenocarcinoma (ADC) is unknown. We characterized immune contexture of invasive lung ADC and its precursors by transcriptomic immune profiling, T cell receptor (TCR) sequencing and multiplex immunofluorescence. Our results demonstrated that anti-tumor immunity evolved as a continuum from lung preneoplasia, to preinvasive ADC, minimally-invasive ADC and frankly invasive lung ADC with a gradually less effective and more intensely regulated immune response including down-regulation of immune-activation pathways, up-regulation of immunosuppressive pathways, higher infiltration of CD4+ T cells, lower infiltration of CD8+ T cells, decreased T cell clonality, and lower frequencies of top T cell clones in later stages. Driver mutations, HLA loss, chromosomal copy number aberrations and DNA methylation changes may collectively impinge host immune responses and facilitate immune evasion as a potential mechanism underlying outgrowth of the most fit subclones in preneoplasia into dominant clones in invasive ADC.SIGNIFICANCEThere has been a drastic increase in the detection of lung nodules, many of which are lung ADC precursors. The management of these lung nodules is controversial. We discovered that immune activation and evasion have started at preneoplastic stage and lung ADC precursors may exhibit an overall better-preserved anti-tumor immune contexture suggesting therapeutic strategies reprograming the immune microenvironment in patients with lung ADC precursors prior to further immunosuppression in invasive lung cancers may be beneficial. These findings have served as the critical scientific rationale for our immunoprevention clinical trial IMPRINT-Lung (NCT03634241) recruiting individuals diagnosed with lung nodules at high risk developing invasive lung cancers.