Camrelizumab plus famitinib in previously chemo-immunotherapy treated patients with advanced NSCLC: results from an open-label multicenter phase 2 basket study.

BACKGROUND: The combination of immune checkpoint inhibitors and antiangiogenic agents has been effective in treating multiple cancers. This was further explored in an open-label, multicenter phase 2 basket study (NCT04346381), which evaluated the antitumor activity and safety of camrelizumab (an anti-PD-1 antibody) plus famitinib (a receptor tyrosine kinase inhibitor) in patients with advanced solid tumors. We herein report the findings from the cohort of advanced NSCLC patients who progressed after treatment with platinum-doublet chemotherapy and immunotherapy. METHODS: Eligible patients were enrolled and treated with camrelizumab (200 mg once every 3 weeks via intravenous infusion) and oral famitinib (20 mg once daily). The primary endpoint was the objective response rate (ORR). Secondary endpoints included the disease control rate (DCR), duration of response (DoR), progression-free survival (PFS), overall survival (OS), and safety. RESULTS: Forty patients were enrolled in this cohort, with a median follow-up duration of 11.5 months. Three patients (7.5%) achieved a partial response, and 29 patients (72.5%) achieved stable disease. The ORR and DCR with this combination regimen were 7.5% (95% CI, 1.6-20.4) and 80.0% (95% CI, 64.4-90.9), respectively. The median DoR was 12.1 months (95% CI, 10.3-not reached). The median PFS was 5.4 months (95% CI, 4.1-7.5), and the median OS was 12.1 months (95% CI, 9.1-16.7). The estimated 12-month OS rate was 51.5% (95% CI, 34.9-65.9). The most frequent grade 3 or higher treatment-related adverse events occurring in more than 5% of patients included hypertension (27.5%), palmar-plantar erythrodysesthesia syndrome (10%), decreased neutrophil count (10%), and proteinuria (7.5%). CONCLUSION: Camrelizumab plus famitinib demonstrated favorable benefits in PFS and OS, along with manageable safety profiles, in patients with advanced NSCLC who progressed after platinum-doublet chemotherapy and immunotherapy. This finding warrants further exploration.

First-line treatment with camrelizumab plus famitinib in advanced or metastatic NSCLC patients with PD-L1 TPS ≥1%: results from a multicenter, open-label, phase 2 trial.

BACKGROUND: The combination of immune-checkpoint inhibitors and antiangiogenic agents can synergistically modulate the tumor microenvironment and represents a promising treatment option. Here, we evaluated the efficacy and safety of camrelizumab plus famitinib (a receptor tyrosine kinase inhibitor) as a first-line treatment for advanced or metastatic NSCLC patients with a programmed death ligand-1 (PD-L1) tumor proportion score (TPS) of ≥1%, in an open-label, multicenter, phase 2 basket trial. METHODS: Eligible patients received camrelizumab (200 mg once every 3 weeks via intravenous infusion) plus oral famitinib at an initial dose of 20 mg once daily. The primary endpoint was the objective response rate (ORR), as assessed by the investigator per Response Evaluation Criteria in Solid Tumors V.1.1. Key secondary endpoints included disease control rate (DCR), duration of respons, progression-free survival (PFS), overall survival (OS), 12-month OS rate, and safety profile. RESULTS: Of the enrolled 41 patients, 21 (51.2%) had a PD-L1 TPS of 1-49%. As of the cut-off date on June 22, 2022, the combination regimen of camrelizumab and famitinib achieved an ORR of 53.7% (95% CI 37.4% to 69.3%) and a DCR of 92.7% (95% CI 80.1% to 98.5%). The median PFS was 16.6 months (95% CI 8.3 to not reached), and OS data were not yet mature, with an estimated 12-month OS rate of 76.8% (95% CI 60.0% to 87.3%). The most common treatment-related adverse events of grade 3 or higher included hypertension (22.0%), increased alanine aminotransferase (12.2%), decreased neutrophil count (9.8%), proteinuria (7.3%), decrease platelet count (7.3%), and hypokalemia (7.3%). One (2.4%) patient died from grade 5 hemoptysis, which was considered possibly related to the study treatment by the investigator. CONCLUSION: Camrelizumab plus famitinib demonstrated promising antitumor activity in advanced or metastatic NSCLC patients and had an acceptable safety profile. These findings suggest that this combination regimen could be an alternative therapeutic option and warrant further investigation. TRIAL REGISTRATION NUMBER: NCT04346381.

Ficonalkib (SY-3505) in Advanced ALK-Positive NSCLC: A Multicenter, Open-Label, Single-Arm, Phase 1/2 Study.

INTRODUCTION: Treatment options for second-generation (2nd-gen) ALK tyrosine kinase inhibitor (TKI)-resistant patients are limited. We evaluated the safety, pharmacokinetics, and efficacy of ficonalkib (SY-3505), a third-generation (3rd-gen) ALK TKI, in patients with advanced ALK-positive non-small cell lung cancer. METHODS: This first-in-human, phase 1/2 study (Chinese Clinical Trial Registry identifier: ChiCTR1900025619; ClinicalTrials.gov identifier: NCT05257512) had two parts. Phase 1 included a dose-escalation phase (25-800 mg quaque die [QD]) and a dose-expansion phase (500 mg QD or 600 mg QD). Phase 2 enrolled patients treated at recommended phase 2 dose. Primary end points were safety in phase 1 and objective response rate (ORR) in phase 2. RESULTS: Between April 21, 2020, and August 31, 2023, a total of 127 patients with advanced ALK-positive non-small cell lung cancer were enrolled, with 62 in phase 1. Ficonalkib was well absorbed and tolerated, with one dose-limited toxicity event occurring at 800 mg QD. Treatment-related adverse events occurred in 85.5% of patients, with 19.4% experienced greater than or equal to grade 3 events. The ORR was 38.3% (23 of 60, 95% confidence interval [CI]: 26.1%-51.8%) in phase 1, and 600 mg QD was established as recommended phase 2 dose. In phase 2, a total of 65 patients received ficonalkib at 600 mg QD. In total, 88 patients received ficonalkib at 600 mg QD in phase 1/2, and all had received prior 2nd-gen ALK TKI treatment. Furthermore, 90.9% of the patients experienced treatment-related adverse events and 14.8% experienced greater than or equal to grade 3 events. The ORR in efficacy-assessable patients who received ficonalkib at 600 mg QD was 47.5% (38 of 80, 95% CI: 36.2%-59.0%), with an intracranial ORR of 37.5% (12 of 32, 95% CI: 21.1%-56.3%) in these patients with measurable brain lesions at baseline. CONCLUSIONS: Ficonalkib (SY-3505) was well tolerated, with favorable safety profiles and promising efficacy in patients resistant to prior 2nd-gen ALK TKI.

A Phase 1b Study of Ivonescimab, a Programmed Cell Death Protein-1 and Vascular Endothelial Growth Factor Bispecific Antibody, as First- or Second-Line Therapy for Advanced or Metastatic Immunotherapy-Naive NSCLC.

INTRODUCTION: This study (HARMONi-5) aimed to evaluate the safety and efficacy of ivonescimab (a bispecific antibody against programmed cell death protein 1 and vascular endothelial growth factor) as first- or second-line monotherapy in patients with advanced immunotherapy-naive NSCLC. METHODS: Eligible patients received intravenous ivonescimab 10 mg/kg every 3 weeks (Q3W), 20 mg/kg every 2 weeks (Q2W), 20 mg/kg Q3W, or 30 mg/kg Q3W. The primary end points were safety and objective response rate (ORR) per Response Evaluation Criteria in Solid Tumors version 1.1. RESULTS: At data cutoff (October 5, 2022), 108 patients were enrolled and received ivonescimab. Programmed death ligand-1 tumor proportion score (TPS) was greater than or equal to 1% in 74 patients (68.5%), including 35 (32.4%) with TPS greater than or equal to 50%. The median follow-up was 10.4 months (range: 8.4-10.9 mo). For all patients, ORR and disease control rate were 39.8% and 86.1%, respectively. ORR by TPS was 14.7%, 51.4%, and 57.1% in patients with TPS less than 1%, greater than or equal to 1%, and greater than or equal to 50%, respectively. In the 67 programmed death ligand-1-positive patients receiving first-line ivonescimab, the ORR was 33.3%, 52.6%, 60.0%, and 75.0% at the doses of 10 mg/kg Q3W, 20 mg/kg Q2W, 20 mg/kg Q3W, and 30 mg/kg Q3W, respectively. Grade greater than or equal to 3 treatment-related adverse events (TRAEs) were observed in 24 patients (22.2%). TRAEs leading to treatment discontinuation occurred in one patient (0.9%). TRAEs leading to death occurred in three patients (2.8%) with squamous NSCLC. The occurrence of grade greater than or equal to 3 TRAEs and grade greater than or equal to 3 bleeding events in squamous versus nonsquamous NSCLC patients was 25.5% versus 18.9% and 0.0% versus 1.9%, respectively. CONCLUSIONS: Ivonescimab monotherapy was well tolerated and found to have a promising efficacy in patients with advanced or metastatic NSCLC. CLINICALTRIALS: gov identifier: NCT04900363.

The Evaluation of Prognostic Value and Immune Characteristics of Ferroptosis-Related Genes in Lung Squamous Cell Carcinoma.

Background The purpose of our study was to construct a prognostic model based on ferroptosis-related gene signature to improve the prognosis prediction of lung squamous carcinoma (LUSC). Methods The mRNA expression profiles and clinical data of LUSC patients were downloaded. LUSC-related essential differentially expressed genes were integrated for further analysis. Prognostic gene signatures were identified through random forest regression and univariate Cox regression analyses for constructing a prognostic model. Finally, in a preliminary experiment, we used the reverse transcription-quantitative polymerase chain reaction assay to verify the relationship between the expression of three prognostic gene features and ferroptosis. Results Fifty-six ferroptosis-related essential genes were identified by using integrated analysis. Among these, three prognostic gene signatures (HELLS, POLR2H, and POLE2) were identified, which were positively affected by LUSC prognosis but negatively affected by immune cell infiltration. Significant overexpression of immune checkpoint genes occurred in the high-risk group. In preliminary experiments, we confirmed that the occurrence of ferroptosis can reduce three prognostic gene signature expression. Conclusions The three ferroptosis-related genes could predict the LUSC prognostic risk of antitumor immunity.

Molecular Mechanism of Response and Adaptation of Antioxidant Enzyme System to Salt Stress in Leaves of Gymnocarpos przewalskii.

The antioxidant enzyme system is the main defense system responsible for maintaining cellular reactive oxygen species (ROS) homeostasis and normal plant growth and development after saline stress. In this study, we identified and characterized the members of the SOD, APX and CAT gene families of the antioxidant enzyme system in Gymnocarpos przewalskii, using plant physiology and molecular biology methods, and analyzed the pattern of enzyme activity in response to NaCl stress. It was found that seven, six and two genes of SOD, APX and CAT gene families, respectively, were expressed in the leaf tissue of G. przewalskii, in which most of the genes were significantly upregulated under NaCl stress, and the enzymatic activities were in accordance with the gene expression. Three positive selection sites in the GpCAT1 gene can increase the hydrophilicity of the GpCAT1 protein, increase the volume of the active site and increase the affinity for H2O2, thus improving the catalytic efficiency of GpCAT1. The results of the present study provide new insights for further investigations of the evolution and function of the SOD, APX and CAT gene families in G. przewalskii and their essential roles under salt stress, and the findings will be useful for revealing the molecular mechanism of salt tolerance and breeding of salt-tolerant plants.

A Novel Risk Score Model of Lactate Metabolism for Predicting over Survival and Immune Signature in Lung Adenocarcinoma.

BACKGROUND: The role of lactate acid in tumor progression was well proved. Recently, it was found that lactate acid accumulation induced an immunosuppressive microenvironment. However, these results were based on a single gene and it was unclear that lactate acid genes were associated with immunotherapy and able to predict overall survival. METHODS: Genes and survival data were acquired from TCGA, GEO and GENECARDS. PCA and TSNE were used to distinguish sample types according to lactate metabolism-associated gene expression. A Wilcox-test examined the expression differences between normal and tumor samples. The distribution in chromatin and mutant levels were displayed by Circo and MAfTools. The lactate metabolism-associated gene were divided into categories by consistent clustering and visualized by Cytoscape. Immune cell infiltration was evaluated by CIBERSORT and LM22 matrix. Enrichment analysis was performed by GSVA. We used the ConsensusClusterPlus package for consistent cluster analysis. A prognostic model was constructed by Univariate Cox regression and Lasso regression analysis. Clinical specimens were detected their expression of genes in model by IHC. RESULTS: Most lactate metabolism-associated gene were significantly differently expressed between normal and tumor samples. There was a strong correlation between the expression of lactate metabolism-associated gene and the abundance of immune cells. We divided them into two clusters (lactate.cluster A,B) with significantly different survival. The two clusters showed a difference in signal, immune cells, immune signatures, chemokines, and clinical features. We identified 162 differential genes from the two clusters, by which the samples were divided into three categories (gene.cluster A,B,C). They also showed a difference in OS and immune infiltration. Finally, a risk score model that was composed of six genes was constructed. There was significant difference in the survival between the high and low risk groups. ROC curves of 1, 3, 5, and 10 years verified the model had good predictive efficiency. Gene expression were correlated with ORR and PFS in patients who received anti-PD-1/L1. CONCLUSION: The lactate metabolism-associated genes in LUAD were significantly associated with OS and immune signatures. The risk scoring model that was constructed by us was able to well identify and predict OS and were related with anti-PD-1/L1 therapy outcome.

Interferon regulatory factor-1 regulates cisplatin-induced apoptosis and autophagy in A549 lung cancer cells.

This study aimed to investigate the expression and function of interferon regulatory factor-1 (IRF-1) in non-small cell lung cancer (NSCLC). IRF-1 expression and its prognostic value were investigated through bioinformatic analysis. The protein expression levels of IRF-1, cleaved caspase 3, and LC3-I/II were analyzed by western blotting. A lentiviral vector was used to overexpress or knockdown IRF-1 in vitro. Mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) were analyzed by JC-1 and DCFH-DA staining, respectively. ATP, SOD, MDA, cell viability, LDH release, and caspase 3 activity were evaluated using commercial kits. Compared to the levels in normal tissues, IRF-1 expression was significantly lower in lung cancer tissues and was a prognostic factor for NSCLC. Cisplatin treatment-induced IRF-1 activation, ROS production, ATP depletion, SOD consumption, and MDA accumulation in A549 lung cancer cells. IRF-1 overexpression promoted mitochondrial depolarization, oxidative stress, and apoptotic cell death and inhibited autophagy in A549 cells, and these effects could be reversed by IRF-1 knockdown. These data suggest that IRF-1 regulates apoptosis, autophagy and oxidative stress, which might be served as a potential target for increasing chemotherapy sensitivity of lung cancer.

Neutrophil Extracellular Traps Facilitate A549 Cell Invasion and Migration in a Macrophage-Maintained Inflammatory Microenvironment.

INTRODUCTION: The biological functions of neutrophil extracellular traps (NETs) in tumorigenesis have drawn an increasing amount of attention. This study explored the relationship between NETs and the inflammatory microenvironment in lung cancer cell invasion and metastasis. METHODS: NETs were quantified using myeloperoxidase (MPO-DNA) and immunofluorescence staining. Cytokine levels were measured using ELISA kits. THP-1 and A549 cells were used for in vitro experiments. Transwell and Matrigel assays were used to assess the invasion and migration abilities of the cells. RESULTS: Neutrophil infiltration and NET formation were observed in the lung cancer tissues. Compared with healthy controls, the level of MPO-DNA complexes in lung cancer patients increased remarkably and was positively correlated with peripheral blood neutrophil counts, smoking status, and poor prognosis. Increased circulating NET levels were also positively correlated with the levels of inflammatory cytokines, including IL-1ß, IL-6, IL-18, and TNF-α. Neutrophils isolated from patients with lung cancer are more prone to NET release. NETs can promote the invasion and migration ability of THP-1 and A549 cell in coculture systems, while pretreatment with NET inhibitors can effectively reduce NET-induced invasion and metastasis. The ability of NETs to promote invasion and metastasis is partly dependent on macrophages. CONCLUSION: Taken together, our study demonstrated that NETs facilitate A549 cell invasion and migration in a macrophage-maintained inflammatory microenvironment.

Efficacy and Safety of Afatinib in the Treatment of Advanced Non-Small-Cell Lung Cancer with EGFR Mutations: A Meta-Analysis of Real-World Evidence.

INTRODUCTION: The purpose of this study was to explore the efficacy and safety of afatinib in advanced non-small-cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations based on real-world evidence. MATERIALS AND METHODS: Eligible real-world studies were identified from PubMed, Cochrane Library, and Embase. Cochrane guidelines were used to assess the quality of included studies. Cochran's Q test and I2 statistics were used for the heterogeneity analysis. RESULTS: Twenty-five studies were included in this meta-analysis; nine studies were included in the qualitative descriptive analysis. The summarized disease control rate (DCR) was 87.6% (81.5%, 92.7%), and the overall response rate (ORR) was 58.9% (48.8%, 68.7%). The pooled median progression-free survival (PFS) was 12.4 (10.3, 14.5) months, mean time to failure (TTF) was 15.4 (13.6, 17.2) months, and median overall survival (OS) was 31.6 (26.7, 36.5) months. The total incidences of adverse events (AEs) for skin rashes, diarrhea, paronychia, and mucositis were 71.4% (64.4%, 77.9%), 70.4% (60.1%, 79.8%), 52.1% (41.9, 62.3%), and 36.5% (29.5%, 43.8%), respectively. The incidences of severe adverse events (SAEs, Grade ≥3) for diarrhea, skin rashes, paronychia, and mucositis were 9.7% (6.8%, 13.1%), 5.8% (4.5%, 7.2%), 3.8% (2.0%, 6.2%), and 2.1% (1.0%, 3.6%), respectively. Differences in PFS and OS between the afatinib non-full-dose (<40 mg) and full-dose (>40 mg) groups were not significant (P > 0.05). However, the ORR in the full-dose group was 78.5% (66.7%, 88.4%), which was significantly higher than that in the non-full-dose group (67.8% [56.8%, 77.9%]). CONCLUSION: The efficacy and safety of afatinib has been confirmed by real-world evidence in advanced NSCLC with EGFR mutation, consistent with randomized controlled trial results. In real-world setting, tolerability-guided dose adjustment might not affect the afatinib efficacy.

Development and Validation of a Prognostic N6-Methyladenosine-Related Immune Gene Signature for Lung Adenocarcinoma.

PURPOSE: The prognostic value of an N6-methyladenosine (m6A) methylation-related immune gene signature for lung adenocarcinoma (LUAD) was investigated. PATIENTS AND METHODS: Gene expression and clinical phenotype data of LUAD patients were downloaded from The Cancer Genome Atlas database. A list of immune-related genes was retrieved from the InnateDB database. Correlation analysis, survival analysis, and univariate and multivariate Cox regression analyses were performed. After allocating patients into a high-risk or a low-risk group, the corresponding survival rates, immune microenvironment, expression of immune checkpoint genes, and modulation of Kyoto Encyclopedia of Genes and Genomes pathways were examined. Finally, the expression levels of prognostic biomarkers were assessed in the GSE126044 dataset. RESULTS: Seven m6A-related immune prognostic genes were identified. High expression of PSMD10P1, DIDO1, ABCA5, and CIITA was associated with high survival rates, while that of PRC1, ZWILCH, and ANLN was associated with low survival rates. The high- and low-risk groups showed significant differences in terms of the abundance of six tumor-infiltrating immune cell types and expression of 12 immune checkpoint genes. The risk group acted as an independent prognostic factor (hazard ratio = 0.398, 95% confidence interval = 0.217-0.729, P = 0.003). Finally, the developed nomogram could predict most efficiently the 1-, 2-, and 3-year survival probability of LUAD patients with a C-index of 0.833. CONCLUSION: A seven-gene risk signature, associated with the immune microenvironment in LUAD, showed independent prognostic value.

Multiple microarray analyses identify key genes associated with the development of Non-Small Cell Lung Cancer from Chronic Obstructive Pulmonary Disease.

Introduction: Chronic obstructive pulmonary disease (COPD) is an independent risk factor of non-small cell lung cancer (NSCLC). This study aimed to analyze the key genes and potential molecular mechanisms that are involved in the development from COPD to NSCLC. Methods: Expression profiles of COPD and NSCLC in GSE106899, GSE12472, and GSE12428 were downloaded from the Gene Expression Omnibus (GEO) database, followed by identification of the differentially expressed genes (DEGs) between COPD and NSCLC. Based on the identified DEGs, functional pathway enrichment and lung carcinogenesis-related networks analyses were performed and further visualized with Cytoscape software. Then, principal component analysis (PCA), cluster analysis, and support vector machines (SVM) verified the ability of the top modular genes to distinguish COPD from NSCLC. Additionally, the corrections between these key genes and clinical staging of NSCLC were studied using the UALCAN and HPA websites. Finally, a prognostic risk model was constructed based on multivariate Cox regression analysis. Kaplan-Meier survival curves of the top modular genes on the training and verification sets were generated. Results: A total of 2350, 1914, and 1850 DEGs were obtained from GSE106899, GSE12472, and GSE12428 datasets, respectively. Following analysis of protein-protein interaction networks, the identified modular gene signatures containing H2AFX, MCM2, MCM3, MCM7, POLD1, and RPA1 were identified as markers for discrimination between COPD and NSCLC. The modular gene signatures were mainly enriched in the processes of DNA replication, cell cycle, mismatch repair, and others. Besides, the expression levels of these genes were significantly higher in NSCLC than in COPD, which was further verified by the immunohistochemistry. In addition, the high expression levels of H2AFX, MCM2, MCM7, and POLD1 correlate with poor prognosis of lung adenocarcinoma (LUAD). The Cox regression prognostic risk model showed the similar results and the predictive ability of this model is independent of other clinical variables. Conclusions: This study revealed several key modules that closely relate to NSCLC with underlying disease COPD, which provide a deeper understanding of the potential mechanisms underlying the malignant development from COPD to NSCLC. This study provides valuable prognostic factors in high-risk lung cancer patients with COPD.

Comprehensive analysis of genomic alterations detected by next-generation sequencing-based tissue and circulating tumor DNA assays in Chinese patients with non-small cell lung cancer.

While tumor genotyping is the standard treatment for patients with non-small cell lung cancer (NSCLC), spatial and temporal tumor heterogeneity and insufficient specimens can lead to limitations in the use of tissue-based sequencing. Circulating tumor DNA (ctDNA) fully encompasses tumor-specific sequence alterations and offers an alternative to tissue sample biopsies. However, few studies have evaluated whether the frequency of multiple genomic alterations observed following ctDNA sequencing is similar to that observed following tissue sequencing in NSCLC. Therefore, in the present study, targeted next-generation sequencing (NGS) was performed on tissue and plasma ctDNA samples in 99 patients with NSCLC. Overall, the frequencies of genetic alterations detected in ctDNA were positively correlated with those detected via tissue profiling (r=0.812; P=0.022). Genomic data revealed significant mutual exclusivity between alterations in epidermal growth factor receptor (EGFR) and tumor protein 53 (TP53; P=0.020), and between alterations in EGFR and KRAS (P=0.008), as well as potential mutual exclusivity between alterations in EGFR and Erb-B2 receptor tyrosine kinase 2 (P=0.059). Furthermore, the EGFR mutant allele frequency (MAF) was positively correlated with the TP53 MAF in individual tumors (r=0.773; P=0.005), and there was a marked difference in the EGFR MAF between patients with and without the TP53 mutation (P=0.001). Levels of the tumor serum marker CA242 in patients with ctDNA-detectable mutations were higher compared with those in patients without ctDNA-detectable mutations. The data from the present study highlight the importance of tissue and plasma ctDNA screening by NGS to guide personalized therapy and promote the clinical management of patients with NSCLC.

Clinical application of plasma mitochondrial DNA content in patients with lung cancer.

Alterations of mitochondrial DNA (mtDNA) have been identified in several types of solid tumor. However, to the best of our knowledge, the clinical significance of plasma mtDNA content in lung cancer remains unknown. Thus, the current study explored the diagnostic and prognostic value of plasma mtDNA quantification in patients with lung cancer. Plasma mtDNA copy numbers of patients with lung cancer (n=128) and healthy individuals (n=107) were quantified by quantitative polymerase chain reaction. Plasma mtDNA copy numbers in patients and healthy controls were 0.89×104 and 1.37×104 copies/µl, respectively (P<0.0001). Furthermore, lower plasma mtDNA content was associated with tumor size, lymph node metastases, distant metastases and serum carcinoembryonic antigen levels (P<0.05), but was not associated with pathological type, age, sex or main driver gene mutation status (P>0.05). Plasma mtDNA facilitated the detection of lung cancer at a threshold of 1.19×104 copies/µl with a sensitivity of 71.1% and specificity of 70.1%, as determined by receiver operating characteristic curve analysis. Advanced stage (III and IV) patients with a lower mtDNA copy number (cutoff: 1.02×104 copies/µl) tended to exhibit poorer prognosis (P<0.05). These results indicated that plasma mtDNA content is a promising and complementary candidate with tissue mtDNA for diagnosis and prognostic prediction for lung cancer.

Inhibition of autophagy promotes cisplatin-induced apoptotic cell death through Atg5 and Beclin 1 in A549 human lung cancer cells.

Recent studies have indicated that autophagy contributes to tumorigenesis and participates in acquired chemotherapeutic resistance. The present study aimed to determine the function and underlying mechanism of cisplatin­induced autophagy in A549 human lung cancer cells. Autophagy was measured by LC3B­I/II conversion, LC3B puncta and autophagosomes formation. Apoptotic cell death was measured by caspase­3 activity, caspase­3 cleavage and LDH release. The transcriptional and expressional level of autophagy related proteins were measured by reverse transcription-quantitative polymerase chain reaction and western blot analysis. Beclin 1 and Atg 5 siRNA transfection was used to explore the function of cisplatin­induced autophagy. The results demonstrated that cisplatin induces apoptotic cell death in A549 cells and triggers an autophagic response, as indicated by increased microtubule­associated protein 1 light chain 3ß (LC3B)­I/II conversion, increased LC3B puncta and autophagosome formation. Mechanisms underlying cisplatin­induced autophagic responses were also investigated. Cisplatin induced autophagy by upregulating the mRNA and protein expression levels of autophagy protein (Atg)5 and Beclin 1, whereas the mRNA and protein expression levels of serine/threonine­protein kinase ULK1, Atg3, Atg7, Atg12, and sequestosome­1 were not markedly upregulated. In addition, knockdown of Atg5 and Beclin 1 by small interfering RNA transfection impaired cisplatin­induced activation of autophagic responses, increased caspase­3 cleavage and inhibited cell viability. These findings suggested that disruption of autophagy via the inhibition of Atg5 and Beclin 1 may promote cisplatin­induced apoptotic cell death in A549 human lung cancer cells. In conclusion, the present study demonstrated that targeting autophagy may be used in the future for the treatment of lung cancer.

[Expression of TLR5 in different types of non-small cell lung cancer cell lines and its activation mechanism].

BACKGROUND: It has been proven that toll-like receptor 5 (TLR5) plaied an important role in the development of tumor. In our previous study, we found that the expression of TLR5 was remarkably higher in non-small cell lung cancer (NSCLC) tissues than that in normal tissues, but the activation of TLR5 signaling pathway in NSCLC was still unknown. The aim of this study is to investigate the expression of TLR5 in different types of NSCLC cell lines, and analyze the activity of the signaling pathway after stimulated by its specific exogenous ligand flagellin. METHODS: The TLR5 protein was detected by immunofluorescence and Western blot in three kinds of NSCLC cell lines, and the TLR5 mRNA was detected by RT-PCR. Select the cell line of TLR5 highest expression as the research object, and select the suitable concentration of flagellin. NF-κB luciferase activity was detected to validate the TLR5 activation pathway through inhibitory signaling pathways by 0 µg/mL, 0.01 µg/mL, 0.1 µg/mL, 1 µg/mL, 10 µg/mL TLR5 antibody. The chosen cell line was transfected by TLR5 shRNA plasmid, and p-IKBα, IKBα, p-ERK1/2, ERK1/2 and p-JNK of untrasfected and transfected cells were detected in the activity of TLR5 signaling pathway by Western blot at 0 min, 10 min, 30 min and 60 min, respectively. RESULTS: The expression of TLR5 was the highest in the lung adenocarcinoma cell line SPC-A-1 by immunofluorescence, mainly expressed on the cell membrane. NF-κB luciferase activity of SPC-A-1 cells was the highest, and the activity was increased in a dose-dependent manner. 0.1 µg/mL flagellin could significantly increase the NF-κB luciferase activity (P<0.05), while its activity could be inhibited by the TLR5 antibody in a negative correlation. Treated by 0.1 µg/mL flagellin, compared with that of 0 min group, the levels of p-IKBα, p-ERK1/2, p-JNK of SPC-A-1 cells increased significantly after 10 min, reached the peak at 30 min, and declined at 60 min (P<0.05). Compared with that of 10 min and 60 min group, the levels of p-IKBα, p-ERK1/2, p-JNK significantly increased at 30 min (P<0.05). While the levels of IKBα, ERK1/2 at 0 min, 10 min, 30 min and 60 min had no significant changes (P>0.05). SPC-A-1 cells transfected TLR5-shRNA were also stimulated by flagellin (0.1 µg/mL). At 0 min, 10 min, 30 min and 60 min, p-IKBα and p-JNK proteins could not be detected, and the levels of IKBα and ERK1/2 had no significant changes (P>0.05), but the levels of p-ERK1/2 significantly increased as time went on (P<0.05). CONCLUSIONS: Exogenous ligand flagellin can activate TLR5 protein in NSCLC cell lines and initiate downstream signaling pathways. It may be relative to the development of NSCLC.

High expression of Toll-like receptor 5 correlates with better prognosis in non-small-cell lung cancer: an anti-tumor effect of TLR5 signaling in non-small cell lung cancer.

PURPOSE: Toll-like receptor 5 (TLR5) is a pattern recognition receptor and plays key roles in inflammatory responses against pathogen infection. Recent evidence suggests that TLR5 is expressed in a wide variety of tumors and exhibits either pro-tumor or anti-tumor activities. In this study, we explored expression of TLR5 in the context of non-small-cell lung cancer (NSCLC) and evaluated the effects of TLR5 signaling in NSCLC cells. METHODS: The lung carcinoma samples were collected from 113 patients diagnosed as NSCLC at Tumor Hospital of Xiangya School of Medicine from January 2005 to December 2008. Immunohistochemistry was performed for TLR5 and the protein expression score was quantified using an established scoring system. Kaplan-Meier survival curves were generated for disease-free survival (DFS) and overall survival (OS) in all patients. TLR5 expression levels were correlated with DFS and OS using univariate and multivariate Cox regression analysis. Expression and subcellular localization of TLR5 were analyzed in NSCLC cell lines including SPC-A1, A549, H1975, and H1299 cells. Activation of TLR5 signaling pathway by flagellin was characterized by western blotting. Effects of flagellin on NSCLC cell proliferation, anchorage-independent growth, migration, and invasion were analyzed by BrdU incorporation assay, soft agar colony formation assay, wound-healing migration assay, and transwell invasion assay, respectively. RESULTS: High expression of TLR5 was significantly associated with better prognosis in patients with NSCLC. We further demonstrated that activation of TLR5 by flagellin in NSCLC cells inhibited cell proliferation, migration, and invasion in vitro. CONCLUSION: TLR5 may serve as a potential therapeutic target in NSCLC patients.

Comparison of graft healing in anterior cruciate ligament reconstruction with and without a preserved remnant in rabbits.

BACKGROUND: The remnant of the native anterior cruciate ligament (ACL) might contribute to the biological integration of the graft in ACL reconstruction. The aim of this study was to explore whether the preserved remnant enhanced graft healing in ACL reconstruction. METHODS: Forty New Zealand rabbits underwent bilateral anterior cruciate ligament reconstructions. One knee was treated with a 2-mm remnant preserved on the tibial side (remnant-preservation, RP group) while the contralateral knee underwent a complete removal of the remnants by cauterization (remnant-resection, RR group) in each animal. Gross observations combined with microangiography, histological evaluation, and uniaxial load testing were performed after 4, 8, and 12weeks. RESULTS: The vascular density on the graft surface was statistically higher in the RP group as compared to that of the RR group at 4 (P=0.002) and 8weeks (P=0.020). Additionally, the accelerated intra-articular and intra-tunnel graft integration were histologically observed in the RP group. Histological scores in the RP group were statistically higher than the RR group at 4weeks (P=0.028 for the intra-articular healing and P=0.046 for the intra-tunnel healing) and 8weeks (P=0.031 for the intra-articular healing and P=0.014 for the intra-tunnel healing). The ultimate failure load (P=0.017), yield load (P=0.025), and stiffness (P=0.004) were statistically higher in the RP group as compared to those of the RR group, with corresponding significant differences in the failure mode (P=0.020) between the two groups at 8weeks. CONCLUSIONS: The preserved remnant enhanced ACL graft healing with improved biomechanical properties in the rabbit model. LEVEL OF EVIDENCE: Level II.

[Case-control studies on therapeutic effects of arthroscopic reconstruction of anterior cruciate ligament with preservation of remnant through outside-in and transtibial tunel].

OBJECTIVE: To compare the properties and clinical outcomes of arthroscopic reconstruction of anterior cruciate ligament (ACL) with preservation of remnant through outside-in and transtibial tunel. METHODS: From June 2005 to January 2012, 145 patients were treated with arthroscopic reconstruction of ACL with preservation of remnant. Among the patients, 88 patients were treated with outside-in techniques (outside-in group), including 55 males and 33 females, ranging in age from 18 to 52 years, with a mean of (29.22 +/- 7.31) years; 57 patients were treated with transtibial technique (transtibial group), including 35 males and 22 females, ranging in age from 18 to 51 years, with a mean of (29.28 +/- 8.07) years. The Lysholm, VAS and IKDC scores were compared between two groups before operation, after operation and at the latest follow-up time. RESULTS: The average operation time was (76.94 +/- 10.83) min in the outside-in group, and (70.35 +/- 10.11) min in the transbibial group, there was a significant difference between two groups. There was no significant difference of hydrops articuli scores at the early stage between the two groups (P = 0.065). At follow-up from 18 to 60 months, there were great improvements in the knee stabilities in each group compared with the preoperative data respectively. The Lysholm score improved from 54.75 +/- 10.58 preoperatively to 95.80 +/- 5.16 at the follow-up in the outside-in group; and improved from 52.51 +/- 11.38 preoperatively to 94.86 +/- 5.50 at follow-up in the transtibial group. Additionally, IKDC grades also improved in both groups. However, no significant differences were seen in stabilities shown by pivot shift test, Lachman test and anterior drawer test. And there also no significant differences of Lysholm scores and IKDC grades between two groups after operation. CONCLUSION: The outside-in technique has advantages to create an anatomical femoral tunnel easily with minimal intra-articular interference, and disadvantages of complicated manipulation. The transtibial technique is easy to operate and gain time. Using either of responding technique according to the actual situation, satisfactory outcome could be archived.

Knockdown of TRB3 induces apoptosis in human lung adenocarcinoma cells through regulation of Notch 1 expression.

The upregulation of tribbles homolog 3 (TRB3), a pseudokinase in mammals, has been observed in several types of malignant cancer, including thyroid, ovarian, liver and colorectal cancer. However, the pathological role and the regulatory mechanism of TRB3 in cancer remain unknown. In the current study, we demonstrated that the expression of TRB3 was upregulated in non-small cell lung cancer (NSCLC), correlating with tumor metastasis, disease recurrence and poor survival in patients. Knocking down TRB3 in aggressive lung cancer cell lines was demonstrated to significantly inhibit their malignant behaviors, including in vitro invasion and cell proliferation, as well as in vivo metastasis and tumor growth. The correlation between TRB3 and Notch 1 expression revealed that Notch 1 was downregulated by the knockdown of TRB3 in the lung adenocarcinoma cell lines. These results have provided insights into the correlation between TRB3 expression and lung cancer progression, and thus may have potential for the prognosis and therapy of lung cancer.