Integrated analysis of patients with KEAP1/NFE2L2/CUL3 mutations in lung adenocarcinomas.

OBJECTIVES: To explore the clinical features, molecular characteristics, and immune landscape of lung adenocarcinoma patients with KEAP1/NFE2L2/CUL3 mutations. METHODS: The multi-omics data from the GDC-TCGA LUAD project of The Cancer Genome Atlas (TCGA) database were downloaded from the Xena browser. The estimate of the immune infiltration was implemented by using the GSVA analysis and CIBERSORT. The status of KEAP1/NFE2L2/CUL3 mutation in 50 LUAD samples of our department was detected by using Sanger sequencing, following the relative expression level of differentially expressed genes (DEGs), miRNAs (DEmiRNAs), and lncRNAs (DElncRNAs) was validated by IHC and real-time quantitative polymerase chain reaction (RT-qPCR). RESULTS: The Kaplan-Meier and multivariable Cox regression analyses demonstrated that KEAP1/NFE2L2/CUL3 mutations had independent prognostic value for OS and PFS in LUAD patients. The differential analysis detected 207 upregulated genes (like GSR/UGT1A6) and 447 downregulated genes (such as PIGR). GO, KEGG, and GSEA analyses demonstrated that DEGs were enriched in glutamate metabolism and the immune response. The constructed ceRNA network shows the linkage of differential lncRNAs and mRNAs. Three hundred and nine somatic mutations were detected, alterations in immune infiltration DNA methylations and stemness scores were also founded between the two groups. Eight mutated LUAD patients were detected by Sanger DNA sequencing in 50 surgical patients. GSR and UGT1A6 were validated to express higher in the Mut group, whereas the expression of PIGR was restrained. Furthermore, the IHC staining conducted on paraffin-embedded tissue emphasizes the consistency of our result. CONCLUSION: This research implemented the comprehensive analysis of KEAP1/NFE2L2/CUL3 somatic mutations in the LUAD patients. Compared with the wild type of LUAD patients, the Mut group shows a large difference in clinical features, RNA sequence, DNA methylation, and immune infiltrations, indicating complex mechanism oncogenesis and also reveals potential therapeutic targets.

Examination of tracheal allografts after long-term survival in dogs.

OBJECTIVES: Little is known on the outcome of tracheal allografts after long-term survival. This study aimed to explore the changes in structure and composition by evaluating the status of the mucosa and cartilage of allografts with long-term survival in dogs. METHODS: Eight tracheal allografts that survived for ˃9 months were enrolled in our study. Epithelium, revascularization, monocyte infiltration and fibrosis were evaluated histologically. The fluorescent dye 4'-6-diamidino-2-phenylindole was used to evaluate the presence of chondrocyte nuclei. Glycosaminoglycan was detected using safranin-O staining and collagen II was evaluated using immunohistochemistry. RESULTS: The 8 animals survived from 277 to 783 days. Bronchoscopy demonstrated that 6 allografts showed no stenosis; 2 cases developed slight stenosis, but could maintain airway patency. Histological examination showed that the epithelium covered the surface of the allografts. In comparison to fresh tracheal controls, allografts demonstrated mild monocyte infiltration, evident revascularization and mild fibrosis in the mucosa or submucosa (all P < 0.05). There were a few viable chondrocytes scattered in the cartilage after long-term survival. Moreover, glycosaminoglycan and collagen II were significantly decreased in the allografts compared with fresh trachea (all P < 0.05). CONCLUSIONS: For tracheal allografts with long-term survival after transplantation, only a few viable chondrocytes were retained, and the extracellular matrix of the cartilage demonstrated degeneration. Despite this, the airway could maintain patency. Notably, the significance of monocyte infiltration in the mucosa or submucosa at different time points warrants further study.

Comparative expression analysis of PD-1, PD-L1, and CD8A in lung adenocarcinoma.

BACKGROUND: As a new strategy for advanced lung adenocarcinoma (LUAD), programmed cell death protein 1 (PD-1) pathway inhibitors have been used in clinic for several years. However, the roles of PD-1, programmed cell death-ligand 1 (PD-L1), and CD8A in LUAD are still unclear. In the study, we aimed to assess the correlation between the mRNA expression of these three factors and the clinical characteristics of LUAD, and to explore the influence of the PD-1/PD-L1/CD8A axis on the prognosis of LUAD. METHODS: The mRNA expression data and clinical characteristics of LUAD patients were retrieved from The Cancer Genome Atlas (TCGA). The optimal cutoff value for PD-1, PD-L1, and CD8A was determined by Cutoff Finder. The chi-square test was used to compare categorical variables. The prognostic effects of variables were analyzed using the Kaplan---Meier method and the Cox proportional hazards model. RESULTS: A total of 484 cases were enrolled in this study according to the selection process. The optimal cutoff values for identifying high/low mRNA expression were defined as 27.4 for PD-1, 29.41 for PD-L1, and 95.52 for CD8A. The high expression of PD-1 (P=0.015) and PD-L1 (P=0.027) was more frequent in women than in men. The high expression of PD-1 (P=0.003), PD-L1 (P=0.002), and CD8A (P=0.003) was associated with early T status, whereas CD8A showed a significantly higher expression in both the early stage (P=0.006) and early N stage groups (P=0.031). PD-1, PD-L1, and CD8A were significantly positively correlated among pairs (P<0.001). High expression of each of the three genes was associated with better prognosis (P=0.030 for PD-1, P=0.046 for PD-L1, P=0.019 for CD8A), although the relation did not reach statistical significance in the Cox regression hazards model. CONCLUSIONS: The study defined a group of cutoff values for PD-1, PD-L1, and CD8A to identify high and low mRNA expression in LUAD. The high expression of PD-1, PD-L1, and CD8A was associated with early T status, and CD8A showed significantly higher expression in both early stage and early N stage groups. Although the high expression of each of these three genes was associated with favorable overall survival (OS), they were not independent prognostic factors.

Evaluation of changes in cartilage viability in detergent-treated tracheal grafts for immunosuppressant-free allotransplantation in dogs.

OBJECTIVES: The first tissue-engineered clinical tracheal transplant prepared using the detergent-enzymatic method resulted in graft stenosis, possibly from detergent-enzymatic method-induced graft non-viability. We reported on the transplantation of de-epithelialized tracheal allografts while maintaining cartilage viability in dogs. No lethal stenosis occurred in allografts. Herein, on the basis of previous experimentation, we assessed cartilage viability in detergent-treated cartilages. METHODS: Six canine tracheal grafts were treated with detergent [1% t-octylphenoxypolyethoxyethanol (Triton X-100)] before transplantation. The histoarchitecture was evaluated, and the viable chondrocytes ratio was calculated. Glycosaminoglycan was detected using safranin-O staining. Collagen II was tested using immunohistochemistry. RESULTS: The epithelium was completely removed in 5 grafts. Compared with fresh tracheas, the viable chondrocyte ratio was significantly reduced in the de-epithelialized grafts (100 vs 54.70 ± 8.56%; P < 0.001). Image analysis revealed that the mean optical density of glycosaminoglycan (0.363 ± 0.027 vs 0.307 ± 0.012; P = 0.007) and collagen II (0.115 ± 0.013 vs 0.092 ± 0.011; P = 0.028) was decreased. The observation period ranged from 91 to 792 days. No stenosis occurred in 5 allografts; moderate stenosis developed in 1 allograft during the 4th week after surgery. The chondrocyte nuclei almost completely disappeared. Both glycosaminoglycan (0.307 ± 0.012 vs 0.164 ± 0.104; P = 0.044) and collagen II (0.092 ± 0.011 vs 0.068 ± 0.022; P = 0.022) were significantly degraded. CONCLUSIONS: This study demonstrated successful tracheal transplantation; about 50% of the viable chondrocytes were retained in the cartilage that could prevent development of a lethal stenosis in tracheal grafts.

Effects of cryopreservation on tracheal allograft antigenicity in dogs.

BACKGROUND: Prolonged cryopreservation (~8-10 months) has been shown to reduce the antigenicity of tracheal allografts due to subsequent denuding of epithelium. In the present study, tracheal epithelium was assessed after variable periods of cryopreservation. Immunosuppressant-free allotransplantation was then undertaken to evaluate the impact of cryopreservation on tracheal antigenicity in dogs. METHODS: Tracheal rings [7-8] were removed from mongrel adult dogs for cryopreservation (1-10 months, -85 °C) and grafting. Before transplantation, one ring was sectioned from each end for histologic examination. The residual five-ring segments of mediastinal trachea were then transplanted into recipient dogs after 1-7 months (group 1, n=9) or 8-10 months (group 2, n=6) of cryopreservation. Anastomotic sites and allografts were covered by omental pedicles. No immunosuppressants whatsoever were administered. RESULTS: In microscopic views, the ciliated tracheal epithelium of most grafts showed variable loss but was generally intact after cryopreservation, still demonstrating major histocompatibility complex (MHC)-II positivity. By postoperative bronchoscopy, allografts in both groups had largely developed lethal strictures. In group 1, eight dogs were sacrificed or died within 50 days post-transplantation, whereas survival times in group 2 were somewhat longer, with three dogs surviving for >60 days. Upon sacrifice, histologic preparations of grafted tissue in both groups were typically denuded of epithelium, with marked lymphocytic/monocytic submucosal infiltrates. Tracheal cartilage had been absorbed or destroyed. CONCLUSIONS: After cryopreservation, some degree of tracheal epithelium loss maybe expected, but complete denudation is not obligatory. Retained epithelial antigenicity is thus capable of triggering rejection, resulting in transplant failures. Although prolonging transplant survival to an extent, fatal rejection of tracheal allografts was not preventable by prior cryopreservation.

Collagen-conjugated tracheal prosthesis tested in dogs without omental wrapping and silicone stenting.

OBJECTIVES: Artificial tracheas fabricated from collagen-conjugated mesh appear to overcome fatal postoperative complications, namely anastomotic dehiscence and prosthesis dislocation. Such prostheses are incorporated by host tissue, provided they are wrapped in omentum (necessitating an additional abdominal procedure) and a silicone tube is used as a stent (to be extracted several weeks postoperatively). To mitigate related host impact (i.e. injury, pain and distress), we investigated the feasibility of implanting this type of tracheal prosthesis (∼5 cm in length) alone, without omental wrapping and use of a silicone stent. METHODS: Porous-type tracheal prostheses that were reinforced with a continuous polypropylene spiral and sealed by collagen sponge from porcine skin replaced segments of cervical trachea (∼5 cm long) in 10 dogs through the method of telescopic anastomosis. Omental wrapping and silicone stent placement were omitted. Postoperatively, bronchoscopic examination was performed periodically. When dogs died or were sacrificed, tracheal prostheses were harvested for haematoxylin and eosin staining and electron microscopic scanning of luminal surface conditions. RESULTS: With the exception of one death from an anaesthesia-related incident during fibre-optic bronchoscopy (postsurgical week 1), nine dogs survived uneventfully (until sacrifice), without prosthesis dislocation or anastomotic dehiscence. The longest observation period was 2 years and 8 months. Bronchoscopic examination revealed that no stenosis or local infection was evident in the prostheses of five dogs. Moderate (n = 2) and slight (n = 2) stenoses were observed in the other four animals. All four animals survived for a long time, without dyspnoea or stridor. Histological examination showed that partial inner surface of the artificial trachea was covered with the pseudostratified ciliated epithelium. Regeneration of ciliated epithelium was also confirmed by scanning electron microscopy. CONCLUSIONS: This pilot study revealed that implantation of a collagen-conjugated tracheal prosthesis (∼5 cm in length) is feasible without the need for omental wrapping and silicone stenting as ancillary measures. This approach considerably simplified the surgical procedures to minimize host intrusion, which indicated a possible clinical application.

Treatment with CA-074Me, a Cathepsin B inhibitor, reduces lung interstitial inflammation and fibrosis in a rat model of polymyositis.

Cathepsin B (CB) is involved in the turnover of proteins and has various roles in maintaining the normal metabolism of cells. In our recent study, CB is increased in the muscles of polymyositis/dermatomyositis (PM/DM). However, the role of CB in interstitial lung disease (ILD) has not been reported. ILD is a frequent complication of PM/DM, which is the leading cause of death in PM/DM. It carries high morbidity and mortality in connective tissue diseases, characterized by an overproduction of inflammatory cytokines and induced fibrosis, resulting in respiratory failure. The etiology and pathogenesis of ILD remain incompletely understood. This study investigated whether treatment with CA-074Me, a specific inhibitor of CB, attenuates ILD in PM. CB expression, inflammation, and fibrosis were analyzed in the lung tissues from patients with PM/DM. The animal model of PM was induced in guinea pigs with Coxsackie virus B1 (CVB1). CA-074Me was given 24 h after CVB1 injection for 7 consecutive days. At the end of the experiment, the animals were killed and lung tissues were collected for the following analysis. Inflammation, fibrosis and apoptosis cells, and cytokines were assessed by histological examinations and immunohistochemical analyses, western blot analysis and transferase-mediated dUTP nick-end labeling assay. In patients with PM/DM, the protein levels of CB were significantly elevated in lung tissues compared with healthy controls, which correlated with increases in inflammation and fibrosis. Similarly, the expression of CB, inflammation and fibrosis, CD8(+) T cell, CD68(+) cell, tumor necrosis factor-alpha, transforming growth factor-beta1 infiltrations, and apoptotic cell death were significantly increased in lung tissues of the guinea-pig model of CVB1-induced PM. These changes were attenuated by the administration of CA-074Me. In conclusion, this study demonstrates that PM/DM increases CB expression in lung tissues and inhibition of CB reduces ILD in a guinea-pig model of CVB1-induced PM. This finding suggests that CB may be a potential therapeutic target for ILD.

Identification of reference miRNAs in human tumors by TCGA miRNA-seq data.

Although the accuracy of detecting the expression of miRNAs by quantitative real-time polymerase chain reaction (qRT-PCR) is highly dependent on reliable reference miRNAs, many commonly used reference miRNAs are not stably expressed and as such are not suitable for quantification and normalization of qRT-PCR data. To solve this problem, we analyzed the global expression profiles of thousands of samples in 14 types of common human tumors released by The Cancer Genome Atlas (TCGA), and identified the most stably and highly expressed miRNAs as candidate reference miRNAs in each type of tumor. We found that miR-361-5p and let-7i-5p were the most recommended candidate reference miRNAs in nine and eight types of tumors, respectively, followed by let-7a-5p, mir-28-5p and miR-99b-5p. Our results are of important value to those researchers focused on miRNA; however, these candidate reference miRNAs still need to be validated prior to their use in qRT-PCR studies.

Ratio of γ-H2AX level in lymphocytes to that in granulocytes detected using flow cytometry as a potential biodosimeter for radiation exposure.

This study aims to assess utilisation of the ratio of γ-H2AX in lymphocytes to that in granulocytes (RL/G of γ-H2AX) in blood as a rapid method for population triage and dose estimation during large-scale radiation emergencies. Blood samples from healthy volunteers exposed to 0-10 Gy of (60)Co irradiation were collected. The samples were cultured for 0-24 h and then analysed using flow cytometry to measure the levels of γ-H2AX in lymphocytes and granulocytes. The basal RL/G levels of γ-H2AX in healthy human blood, the response of RL/G of γ-H2AX to ionising radiation and its relationship with doses, time intervals after exposure and individual differences were also analysed. The level of γ-H2AX in lymphocytes increased in a dose-dependent manner after irradiation, whereas the level in granulocytes was not affected. A linear dose-effect relationship with low inter-experimental and inter-individual variations was observed. The RL/G of γ-H2AX may be used as a biomarker for population triage and dose estimation during large-scale radiation emergencies if blood samples can be collected within 24 h.

Selection of candidate radiation biomarkers in the serum of rats exposed to gamma-rays by GC/TOFMS-based metabolomics.

In the study, gas chromatography/time-of-flight mass spectrometry (GC/TOFMS) techniques coupled with principal components analysis (PCA) were used to investigate metabolite perturbations in the serum of the rats exposed to 0.75, 3 or 8 Gy gamma rays. Male standard deviation rats were gamma-irradiated at doses of 0.75, 3 and 8 Gy (1.9 Gy min(-1)) or sham-irradiated. Serum samples were collected over the first 24 h under the exposure to irradiation in order to analyse the samples by GC/TOFMS. And multivariate data were analysed by PCA. The composition of metabolites in serum yielded distinct metabolomic phenotypes for 0.75, 3 and 8 Gy at 24 h after irradiation. Nine serum metabolites were significantly altered as a result of radiation exposure. Up-regulated metabolites included inositol, serine, lysine, glycine, threonine and glycerol; down-regulated metabolites included isocitrate, gluconic acid and stearic acid. The nine metabolites were significantly altered after ionising radiation for they may be the potential biomarkers for the diagnosis of radiation injury.

Toll-like receptor 5 agonism protects mice from radiation pneumonitis and pulmonary fibrosis.

Radiation pneumonitis and pulmonary fibrosis are the main complications with radiotherapy for thoracic neoplasms, directly limiting the efficient dose in clinical application and currently there are few medicines that effectively function as radioprotectants. However, a TLR5 agonist, CBLB502, was confirmed to have protective efficacy against hematopoietic and gastrointestinal radiation syndromes in mice and primates. This study points to a new direction for protection against thoracic radiation-induced pulmonary syndromes and skin injury by CBLB502. We utilized the TUNEL assay, pathological analysis and immunohistochemistry to obtain evidence that CBLB502 could alleviate the occurrence of radiation pneumonitis and pulmonary fibrosis as well as radiation- induced skin injury. It may thus play a promising role in facilitating clinical radiotherapy of thoracic neoplasms.

Toll-like receptor 5 agonist inhibition of growth of A549 lung cancer cells in vivo in a Myd88 dependent manner.

The purpose of this study was to examine the effect of a Toll-like receptor 5 (TLR5) agonist, CBLB502, on the growth and radiosensitivity of A549 lung cancer cells in vivo. Expression of myeloid differentiation factor 88 (MyD88) or TLR5 was stably knocked down in human lung cancer cells (A549) using lentivirus expressing short hairpin RNA targeting human MyD88 or TLR5. Lack of MyD88 or TLR5 expression enhanced tumor growth in mouse xenografts of A549 lung cancer cells. CBLB502 inhibited the growth of A549 lung cancer cells, not A549-MyD88-KD cells in vivo in the murine xenograft model. Our results showed that the inhibition of A549 by CBLB502 in vivo was realized through regulating the expression of neutrophil recruiting cytokines and neutrophil infiltration. Finally, we found that activation of TLR5 signaling did not affect the radiosensitivity of tumors in vivo.

Histological examination of cryopreserved rat tracheal grafts.

It has been reported that tracheal tissue treated by cryopreservation can be used for tracheal replacement in the absence of immunosuppressants. However, the mechanism of reduced antigenicity is unclear. We investigated this issue in cryopreserved rat trachea using detailed histologic evaluation. Rat tracheal segments were preserved in a cryopreservative solution at -85 degrees C. The epithelium of tracheal segments (n = 6 in each group) was subjected to light microscopic and scanning electron microscopic examination before freezing and after cryopreservation for 1 week, 1 month, 3 months, and 6 months. The expression of major histocompatibility complex II (MHC-II) was determined using frozen sections. Ultrastructure of dendritic cells (DCs) was observed by transmission electron microscopy. Tracheal epithelium was partially intact even after 6 months in cryopreservation, although cellularity decreased with time. MHC-II antigen expression was detected even at 6 months, although expression was lower than that measured on fresh tissue. Tracheal tissue DCs displayed dilatations of perinuclear cisterna and degeneration of vacuoles. Density of the mitochondrial matrix was increased. These results suggest that damage to the epithelium and DCs during cryopreservation and concomitant loss of MHC-II expression might explain the reduction of antigenicity.

[Impact of micrometastasis in pathologically negative lymph node on staging and prognosis of non-small cell lung cancers].

OBJECTIVE: To study the influence of micrometastasis in lymph node on staging and prognosis of non-small-cell lung cancer (NSCLC). METHODS: In 39 NSCLC patients, micrometastasis in pathologically negative lymph nodes were tested through immunohistochemical cytokeratin (CK) analysis and the relationship between CK(+) and staging, survival were analyzed. RESULTS: In these 39 patients, the survival of CK(+) and CK(-) patients were 32 months and 48 months respectively (P = 0.0178). Multivariate analysis of Cox regression model showed: clinical stage (P = 0.0288) and relapse or metastasis (P = 0.0053) affected the prognosis while micrometastasis in lymphnodes (P = 0.7740) did not. CONCLUSION: The detection of micrometastasis in the lymphnodes may serve as a supplement to the present staging system for lung cancer. Even though the prognosis of patients with micrometastasis being poorer than those without, micrometastasis in the lymph nodes should not be regarded as an independent prognostic factor.