Liwen Xiong

Use of capture-based next-generation sequencing to detect ALK fusion in plasma cell-free DNA of patients with non-small-cell lung cancer.

Capture-based next-generation sequencing (NGS) is a potentially useful diagnostic method to measure tumor tissue DNA in blood as it can identify concordant mutations between cell-free DNA (cfDNA) and primary tumor DNA in lung cancer patients. In this study, the sensitivity, specificity and accuracy of capture-based NGS for detecting ALK fusion in plasma cfDNA was assessed. 24 patients with tissue ALK-positivity and 15 who did not harbor ALK fusion were enrolled. 13 ALK-positive samples were identified by capture-based NGS among the 24 samples with tissue ALK-positivity. In addition to EML4-ALK, 2 rare fusion types (FAM179A-ALK and COL25A1-ALK) were also identified. The overall sensitivity, specificity and accuracy for all cases were 54.2%, 100% and 71.8%, respectively. For patients without distant metastasis (M0-M1a) and patients with distant metastasis (M1b), the sensitivities were 28.6% and 64.7%, respectively. In the 15 patients who received crizotinib, the estimated median PFS was 9.93 months. Thus, captured-based NGS has acceptable sensitivity and excellent specificity for the detection of ALK fusion in plasma cfDNA, especially for patients with distant metastasis. This non-invasive method is clinically feasible for detecting ALK fusion in patients with advanced-stage NSCLC who cannot undergo traumatic examinations or have insufficient tissue samples for molecular tests.

A Classification Method Based on Principal Components of SELDI Spectra to Diagnose of Lung Adenocarcinoma

Purpose Lung cancer is the leading cause of cancer death worldwide, but techniques for effective early diagnosis are still lacking. Proteomics technology has been applied extensively to the study of the proteins involved in carcinogenesis. In this paper, a classification method was developed based on principal components of surface-enhanced laser desorption/ionization (SELDI) spectral data. This method was applied to SELDI spectral data from 71 lung adenocarcinoma patients and 24 healthy individuals. Unlike other peak-selection-based methods, this method takes each spectrum as a unity. The aim of this paper was to demonstrate that this unity-based classification method is more robust and powerful as a method of diagnosis than peak-selection-based methods. Results The results showed that this classification method, which is based on principal components, has outstanding performance with respect to distinguishing lung adenocarcinoma patients from normal individuals. Through leaving-one-out, 19-fold, 5-fold and 2-fold cross-validation studies, we found that this classification method based on principal components completely outperforms peak-selection-based methods, such as decision tree, classification and regression tree, support vector machine, and linear discriminant analysis. Conclusions and Clinical Relevance The classification method based on principal components of SELDI spectral data is a robust and powerful means of diagnosing lung adenocarcinoma. We assert that the high efficiency of this classification method renders it feasible for large-scale clinical use.

Prognostic impact of vascular endothelial growth factor-A and E-cadherin expression in completely resected pathologic stage I non-small cell lung cancer.

OBJECTIVE The purpose of this study was to evaluate the value of vascular endothelial growth factor-A and E-cadherin expression as well as other confirmed prognostic factors in predicting the clinical outcome after definitive surgery of pathologic stage I non-small cell lung cancer. METHODS One hundred and eighty-five consecutive and non-selected patients who underwent definitive surgery for stage I non-small cell lung cancer in our institute were included in this study. Formalin-fixed paraffin-embedded specimens were stained for vascular endothelial growth factor-A and E-cadherin and the correlation between the staining, its clinicopathological parameters and its prognostic power were analyzed statistically. RESULTS Of the 185 patients studied, 92 cases (49.7%) were strongly positive for vascular endothelial growth factor-A. Vascular endothelial growth factor-A expression was only related to visceral pleural involvement (P < 0.001). A total of 95 carcinomas (51.4%) were E-cadherin-negative tumors. E-cadherin expression correlated with histology (P < 0.001), tumor size (P = 0.001) and visceral pleural involvement (P < 0.001). In univariate analysis by log-rank test, gender, tumor size, lymphovascular invasion, visceral pleural involvement, vascular endothelial growth factor-A expression and E-cadherin expression were significant prognostic factors (P = 0.003, 0.042, 0.026, 0.035, 0.008 and 0.006, respectively). In multivariate analysis, gender, vascular endothelial growth factor-A and E-cadherin expression maintained its independent prognostic influence on overall survival (P = 0.013, <0.001 and 0.036, respectively). CONCLUSIONS Expression of vascular endothelial growth factor-A is related to visceral pleural involvement, and E-cadherin expression correlates with histology, tumor size and visceral pleural involvement. Multivariate analysis confirmed gender, vascular endothelial growth factor-A and E-cadherin expression were significant predictive factors for overall survival in completely resected pathologic stage I non-small cell lung cancer.

γ-Glutamylcyclotransferase Knockdown Inhibits Growth of Lung Cancer Cells Through G0/G1 Phase Arrest.

Lung cancer as an aggressive type tumor is rapidly growing and has become the leading cause of cancer-related death worldwide. γ-Glutamylcyclotransferase (GGCT) has been shown as a diagnostic marker in various cancers. To reveal whether there is a correlation between GGCT and lung cancer, GGCT expression in human lung cancer cell lines was first determined by real-time quantitative PCR and western blot. GGCT is expressed in all tested lung cancer cell lines, A549, H1299, and H460. Then, a lentivirus-based system was applied to knock down GGCT in A549 cells, which were thus divided into Lv-shGGCT, Lv-shCon, and Con (noninfected) groups. Methylthiazol tetrazolium assay showed that the cell proliferation was decreased by over 50% in the Lv-shGGCT group compared with controls. The size and number of colonies were dramatically reduced in the GGCT knockdown group, as measured by colony formation assay. Moreover, A549 cells infected with Lv-shGGCT were arrested in the G0/G1 phase as assayed by flow cytometry. Furthermore, the expression levels of CDK4, CDK6, and cyclin D1 were decreased and the cleaved level of PARP was increased in GGCT knockdown cells. In conclusion, GGCT plays a critical role in lung cancer cell proliferation and may be a potential cancer therapeutic target.

Knockdown of eIF3d inhibits cell proliferation through G2/M phase arrest in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and remains the leading cause of cancer-related death worldwide. Eukaryotic translation initiation factor 3, subunit d (eIF3d) has been recognized recently in several human cancers. In this paper, we attempt to evaluate the functional role of eIF3d in NSCLC cells. Lentivirus-mediated RNA interference (RNAi) was applied to silence eIF3d in the human NSCLC cell lines A549 and 95D. Cell viability was measured by MTT. Cell colony-forming ability was measured by colony formation. Cell cycle progression was determined by propidium iodide staining and flow cytometry. Intracellular signaling molecules were detected using a PathScan® intracellular signaling array kit. In this study, we firstly proved that lentivirus-mediated RNAi specifically suppressed the expression of eIF3d both at the mRNA and protein levels in A549 and 95D cell lines. Further investigations revealed that eIF3d knockdown significantly inhibited cell proliferation and colony formation. Moreover, the cell cycle of A549 cells was arrested at G2/M phase after eIF3d knockdown. Furthermore, the activations of AKT, HSP27 and SAPK/JNK were suppressed by eIF3d knockdown. This study highlights the crucial role of eIF3d in promoting NSCLC cell proliferation, and provides a foundation for further study into the clinical potential of lentiviral-mediated delivery of eIF3d RNAi therapy for treatment of NSCLC.

Overexpression and biological function of TMEM48 in non-small cell lung carcinoma

Transmembrane protein 48 (TMEM48), localized to nuclear pore complexes (NPCs), has been reported crucial for NPC assembly. Alterations in NPC members have been reported in several malignancies. The present study was aimed to elucidate the expression and biological function of TMEM48 in non-small cell lung carcinoma (NSCLC). Here, TMEM48 expression level was higher in NSCLC tissues than that in the adjacent normal tissues. Moreover, higher TMEM48 expression was correlated with a more advanced tumor stage, lymph node metastasis, bigger tumor size tumor stage, and shorter survival time. Knockdown of TMEM48 in NSCLC cell lines, A549 and H1299, inhibited cell proliferation and significantly increased cells population in G1 phase. Gene set enrichment analysis (GSEA) showed that cell cycle pathway was correlative with the TMEM48 expression. Additionally, real-time PCR and western blot analysis revealed that several cell cycle and DNA replication genes, including Cyclin B1, CDK1, CDC6, PCNA, and RCF4, were reduced after TMEM48 knockdown. Additionally, inhibition of TMEM48 in NSCLC cells significantly stimulated cell apoptosis, while notably repressed cell adhesion, migration, invasion, and tumorigenicity in nude mice. Our data provide insight into the biological relevance of TMEM48 in NSCLC progression and highlight its usefulness as a prognostic factor and potential therapeutic target in NSCLC.

Pulmonary ground-glass nodules diagnosis: mean change rate of peak CT number as a discriminative factor of pathology during a follow-up

OBJECTIVE We aimed to analyse the peak CT number (PEAK) in CT number histogram of ground-glass nodules (GGN), meaning the most frequent density of pixels in the image of pulmonary nodule, based on three-dimensional (3D) reconstructive model pre-operatively, and the mean rate of PEAK change (V-PEAK) during a follow-up of GGN for differential diagnosis between pre-invasive adenocarcinoma (PIA) and invasive adenocarcinoma (IAC). METHODS CT number histogram of pixels in GGN was made automatically by 3D measurement software. Diameter, total volume, PEAK and V-PEAK were measured from CT data sets of different groups classified by pathology, subtype and number of GGN, respectively. RESULTS Among all 102 cases, 47 were PIA, including atypical adenomatous hyperplasia (n = 29) and adenocarcinoma in situ (n = 18), and 55 were IAC, including minimally IAC (MIA, n = 4). By Wilcoxon test, PEAK of IAC was significantly higher than that of PIA (p < 0.001). By receiver operating curve analysis, area under the curve (AUC) was 0.857 and threshold -820.50 Hounsfield units (HU) for differentiation between PIA and IAC. V-PEAK of IAC was unexpectedly remarkably smaller than that of PIA (p < 0.001) with AUC and threshold being 0.810 and -0.829 HU day(-1), respectively. CONCLUSION Pre-operative PEAK and V-PEAK, which interpret and evaluate the change of volume and density of pulmonary nodule simultaneously from both exterior and interior perspectives, can help to distinguish IAC from PIA. ADVANCES IN KNOWLEDGE This study provided researchers of GGN another perspective, taking both volume and density of nodules into consideration for pathological evaluation.

Radiologic Predictors for Clinical Stage IA Lung Adenocarcinoma with Ground Glass Components: A Multi-Center Study of Long-Term Outcomes

Objective This study was to define preoperative predictors from radiologic findings for the pathologic risk groups based on long-term surgical outcomes, in the aim to help guide individualized patient management. Methods We retrospectively reviewed 321 consecutive patients with clinical stage IA lung adenocarcinoma with ground glass component on computed tomography (CT) scanning. Pathologic diagnosis for resection specimens was based on the 2011 IASLC/ATS/ERS classification of lung adenocarcinoma. Patients were classified into different pathologic risk grading groups based on their lymph node status, local regional recurrence and overall survival. Radiologic characteristics of the pulmonary nodules were re-evaluated by reconstructed three-dimension CT (3D-CT). Univariate and multivariate analysis identifies independent radiologic predictors from tumor diameter, total volume (TV), average CT value (AVG), and solid-to-tumor (S/T) ratio. Receiver operating characteristic curves (ROC) studies were carried out to determine the cutoff value(s) for the predictor(s). Univariate cox regression model was used to determine the clinical significance of the above findings. Results A total of 321 patients with clinical stage IA lung adenocarcinoma with ground glass components were included in our study. Patients were classified into two pathologic low- and high- risk groups based on their distinguished surgical outcomes. A total of 134 patients fell into the low-risk group. Univariate and multivariate analyses identified AVG (HR: 32.210, 95% CI: 3.020–79.689, P<0.001) and S/T ratio (HR: 12.212, 95% CI: 5.441–27.408, P<0.001) as independent predictors for pathologic risk grading. ROC curves studies suggested the optimal cut-off values for AVG and S/T ratio were-198 (area under the curve [AUC] 0.921), 2.9 (AUC 0.996) and 54% (AUC 0.907), respectively. The tumor diameter and TV were excluded for the low AUCs (0.778 and 0.767). Both the cutoff values of AVG and S/T ratio were correlated with pathologic risk classification (p<0.001). Univariate Cox regression model identified clinical risk classification (RR: 3.011, 95%CI: 0.796–7.882, P = 0.095) as a good predictor for recurrence-free survival (RFS) in patients with clinical stage IA lung adenocarcinoma. Statistical significance of 5-year OS and RFS was noted among clinical low-, moderate- and high-risk groups (log-rank, p = 0.024 and 0.010). Conclusions The AVG and the S/T ratio by reconstructed 3D-CT are important preoperative radiologic predictors for pathologic risk grading. The two cutoff values of AVG and S/T ratio are recommended in decision-making for patients with clinical stage IA lung adenocarcinoma with ground glass components.

Role of high expression levels of STK39 in the growth, migration and invasion of non-small cell type lung cancer cells.

Non-small cell type lung cancer (NSCLC) is the most common malignancy and the leading cause of cancer related mortality. In this study, serine/threonine kinase 39 (STK39) was identified as an up-regulated gene in NSCLC tissues by next-generation RNA sequencing. Although STK39 gene polymorphisms may be prognostic of overall survival in patients with early stage NSCLC, the roles of STK39 in NSCLC cancer are poorly understood. In the current study, Genome Set Enrichment Analysis (GSEA) on the RNA-seq data of NSCLC specimens indicated that cancer-related process and pathways, including metastasis, cell cycle, apoptosis and p38 pathway, were significantly correlated with STK39 expression. STK39 expression was significantly increased in NSCLC cases and its protein expression was positively correlated with the poor tumor stage, large tumor size, advanced lymphnode metastasis and poor prognosis. Down-regulation of STK39 in NSCLC cells significantly decreased cell proliferation by blocking of cell cycle and inducing apoptosis. We also found that STK39 knockdown in NSCLC cells remarkably repressed cell migration and invasion. On the contrary, overexpression of STK39 in NSCLC cells had inverse effects on cell behaviors. Taken together, STK39 acts as a tumor oncogene in NSCLC and can be a potential biomarker of carcinogenesis.

Ubiquitin-specific protease 39 is overexpressed in human lung cancer and promotes tumor cell proliferation in vitro

Lung cancer is the most frequent cancer in the world. Previous studies have shown that ubiquitin-specific protease 39 (USP39) is upregulated in several cancers and associated with tumor malignant characters. However, the effects of USP39 in lung cancer have not been well understood. In the present study, we found USP39 was generally expressed higher in human lung cancer tissues than in normal tissues by Oncomine database mining, qRT-PCR, and western blot assay. Knockdown of USP39 expression markedly reduced the proliferative and colony-forming ability of lung cancer cell lines 95D and A549. Flow cytometric analysis showed that USP39 knockdown induced cell cycle arrest at G2/M phase and enhanced cell apoptosis in 95D cells. Moreover, depletion of USP39 blocked activation of Akt, mTOR, p53, and PARP signaling pathways. Taken together, our study indicates that USP39 may be functionally involved in lung cancer growth and act as a potential molecular target for human lung cancer diagnosis and treatment.