Xiaoxuan Zheng

Wnt signaling regulates the stemness of lung cancer stem cells and its inhibitors exert anticancer effect on lung cancer SPC-A1 cells.

Abstract Wnt signaling plays an important role in regulating the activity of cancer stem cells (CSCs) in a variety of cancers. In this study, we explored the role of Wnt signaling in the lung cancer stem cells (LCSCs). LCSCs were obtained by sphere culture, for which human lung adenocarcinoma cell line SPC-A1 was treated with IGF, EGF and FGF-10. The stemness of LCSCs was confirmed by immunofluorescence, and pathway analysis was performed by functional genome screening and RT-PCR. The relationship between the identified signaling pathway and the expression of the stemness genes was explored by agonist/antagonist assay. Moreover, the effects of different signaling molecule inhibitors on sphere formation, cell viability and colony formation were also analyzed. The results showed that LCSCs were successfully generated as they expressed pluripotent stem cell markers Nanog and Oct 4, and lung distal epithelial markers CCSP and SP-C, by which the phenotype characterization of stem cells can be confirmed. The involvement of Wnt pathway in LCSCs was identified by functional genome screening and verified by RT-PCR. The expression of Wnt signaling components was closely related to the expression of the Nanog and Oct 4. Furthermore, targeting Wnt signaling pathway by using different signaling molecule inhibitors can exert anticancer effects. In conclusion, Wnt signaling pathway is involved in the stemness regulation of LCSCs and might be considered as a potential therapeutic target in lung adenocarcinoma.

Wnt blockers inhibit the proliferation of lung cancer stem cells

Background Previous study has confirmed that the occurrence of Wnt pathway activation is associated with risk of non-small-cell lung cancer recurrence. However, whether the pharmacologic blocking of the Wnt signaling pathway could provide therapeutic possibility remains unknown. The aim of the present study was to evaluate the therapeutic functions of the Wnt signaling pathway inhibitor pyrvinium pamoate (PP) on lung cancer stem cells (LCSCs) in vitro. Methods Colony formation and sphere culture were performed to enrich LCSCs from three lung cancer cell lines: PC9, SPC-A1, and A549. After confirming stemness by immunofluorescence, PP was employed for cell viability assay by comparison with three other kinds of Wnt signaling inhibitor: salinomycin, ICG-001, and silibinin. The effect of PP on LCSCs was further verified by colony formation assay and gene expression analysis. Results LCSCs were successfully generated by sphere culture from SPC-A1 and PC9 cells, but not A549 cells. Immunofluorescence assay showed that LCSCs could express pluripotent stem cell markers, including NANOG, Oct4, KLF5, and SOX2, and Wnt signaling pathway molecules β-catenin and MYC. Half-maximal inhibitory concentrations of PP on SPC-A1, PC9, and A549 were 10 nM, 0.44 nM, and 0.21 nM, respectively, which are much lower than those of salinomycin, ICG-001, and silibinin. Moreover, significantly decreased colony formation and downregulation of pluripotent stem cell signaling pathway were observed in lung cancer cells after treatment with PP. Conclusion Wnt signaling inhibitor PP can inhibit proliferation of LCSCs, and the Wnt signaling pathway could be considered a promising therapeutic or interventional target in lung adenocarcinoma.

Capture-Based Targeted Ultradeep Sequencing in Paired Tissue and Plasma Samples Demonstrates Differential Subclonal ctDNA-Releasing Capability in Advanced Lung Cancer.

Introduction Circulating tumor DNA (ctDNA), which represents an unbiased way to assess tumor genetic profile noninvasively, facilitates studying intratumor heterogeneity. Although intratumor heterogeneity has been elucidated substantially in a few cancer types, including NSCLC, how it influences the ability of tumor cells harboring different genetic abnormalities in releasing their DNA remains elusive. We designed a capture‐based panel targeting NSCLC to detect and quantify genetic alterations from plasma samples by using deep sequencing. By applying the panel to paired biopsy and plasma samples, we imputed and compared the ctDNA‐releasing efficiency in subclones harboring distinct genetic variants. Methods We collected 40 pairs of matched biopsy and plasma samples from patients with advanced lung cancer and applied capture‐based sequencing using our LungPlasma panel, which consists of critical exons and introns of 168 genes. We derived a normalized relative allelic fraction score (NRAFS) to reflect ctDNA‐releasing efficiency. Results By using mutations detected in biopsy samples as a reference, we achieved 87.2% by‐variant sensitivity, including for single‐nucleotide variants, insertions or deletions, and gene fusions. Furthermore, the by‐variant sensitivity for the seven most critical and actionable genes was 96.2%. The average NRAFS for subclones carrying mutations from seven actionable genes was 0.877; in contrast, the average NRAFS for other mutations was 0.658. Mutations from four genes involved in cell cycle pathways had a particularly low NRAFS (0.480) compared with the other two groups (p = 0.07). Conclusions We have demonstrated that subclones carrying driver mutations are more prone to release DNA. We have also demonstrated the quantitative ability of capture‐based sequencing, paving its way for routine utilization in clinical settings.

Learning curve of electromagnetic navigation bronchoscopy for diagnosing peripheral pulmonary nodules in a single institution

Background: Electromagnetic navigation bronchoscopy (ENB) is a novel technology that is designed to diagnose peripheral pulmonary lesions (PPLs). The purpose of the study was to explore the learning curve and evaluate the diagnostic yield and safety of ENB in diagnosing peripheral pulmonary nodules. Methods: A total of 40 patients discovered with peripheral pulmonary nodules suspicious for malignancy were chronologically enrolled into the study. Biopsy, brushing and flushing specimens were obtained through the extended working channel (EWC). Radial endobronchial ultrasound (R-EBUS) and fluoroscopy were used as means for localization and auxiliary implements in the procedure of ENB. Immunohistochemistry (IHC) and driver genes testing were performed on biopsy samples when it was necessary. All the patients performed chest radiographs to exclude pneumothorax after the procedure. Data of all patients were recorded prospectively and analyzed retrospectively. The learning curve was evaluated using cumulative sum (CUSUM) method. Results: The mean diameter of the 40 nodules was 21.1±5.3 mm. The average navigation time and total operation time were 8.1±3.2 and 24.6±4.1 min, respectively. The overall diagnostic yield was 82.5% (33/40). CUSUM analysis of learning curve based on the navigation time and total operation time both could be best modeled as polynomials and divided the learning curve into two phases at the point of case 14. The learning curve based on diagnostic yield did not demonstrate an obvious turning point. No complications occurred in the 40 procedures. Conclusions: ENB is a safe technology with a high diagnostic yield in diagnosing peripheral pulmonary nodules. The learning curve based on the procedure time could be stable after 14 procedures and there was no obvious learning curve based on the diagnostic yield for an experienced pulmonologist.

Application of Quantitative Autofluorescence Bronchoscopy Image Analysis Method in Identifying Bronchopulmonary Cancer.

Autofluorescence bronchoscopy shows good sensitivity and poor specificity in detecting dysplasia and cancer of the bronchus. Through quantitative analysis on the target area of autofluorescence bronchoscopy image, determine the optimal identification index and reference value for identifying different types of diseases and explore the value of autofluorescence bronchoscopy in diagnosis of lung cancer. Patients with 1 or more preinvasive bronchial lesions were enrolled and followed up by white-light bronchoscope and autofluorescence bronchoscopy. Color space quantitative image analysis was conducted on the lesion shown in the autofluorescence image using MATLAB image measurement software. A retrospective analysis was conducted on 218 cases with 1208 biopsies. One hundred seventy-three cases were diagnosed as positive, which included 151 true-positive cases and 22 false-positive cases. White-light bronchoscope associated with autofluorescence bronchoscopy was able to differentiate between benign and malignant lesion with a high sensitivity, specificity, positive predictive value, and negative predictive value (92.1%, 59.3%, 87.3%, and 71.1%, respectively). Taking 1.485 as the cutoff value of receiver operating characteristic of red-to-green value to differentiate benign and malignant diseases, the diagnostic sensitivity reached 82.3% and the specificity reached 80.5%. U values could differentiate invasive carcinoma and other groups well. Quantitative image analysis method of autofluorescence bronchoscopy provided effective scientific basis for the diagnosis of lung cancer and precancerous lesions.

RGB and HSV quantitative analysis of autofluorescence bronchoscopy used for characterization and identification of bronchopulmonary cancer

Autofluorescence bronchoscopy (AFB) shows good sensitivity in detecting dysplasia and bronchopulmonary cancer. However, the poor specificity of AFB would lead to excessive biopsy. The aim of the study is to establish a more effective quantitative method (optimal identification index and reference value) for characterizing the AFB images within the region of interest and discuss AFBs significance in the diagnosis of central‐type lung cancer. A total of 218 suspected lung cancer patients were enrolled in this study. A quantitative analysis based on color space (red, green, blue[RGB] and HSV system) was conducted and the result was compared with the final diagnosis obtained by the pathology of biopsy. Cases were divided into different groups according to the pathological diagnosis of normal bronchial mucosa, inflammation, low‐grade preinvasive (LGD), high‐grade preinvasive (HGD), and invasive cancer. Quantitative analyses in multi‐color spaces for the lesions showed by AFB images were conducted by software MATLAB. Finally, there is statistical significance among the different groups in some parameter in RGB and HSV system. So, both RGB and HSV quantitative analysis of autofluorescence bronchoscopy are useful to define benign and malignant diseases, which can objectively guide the bronchoscopist in selecting sites for biopsy with good pathologic correlation.

Reliability of using circulating tumor cells for detecting epidermal growth factor receptor mutation status in advanced non-small-cell lung cancer patients: a meta-analysis and systematic review

Purpose To evaluate the clinical value of circulating tumor cells as a surrogate to detect epidermal growth factor receptor mutation in advanced non-small-cell lung cancer (NSCLC) patients. Methods We searched the electronic databases, and all articles meeting predetermined selection criteria were included in this study. The pooled sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio were calculated. The evaluation indexes of the diagnostic performance were the summary receiver operating characteristic curve and area under the summary receiver operating characteristic curve. Results Eight eligible publications with 255 advanced NSCLC patients were included in this meta-analysis. Taking tumor tissues as reference, the pooled sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio of circulating tumor cells for detecting the epidermal growth factor receptor mutation status were found to be 0.82 (95% confidence interval [CI]: 0.50–0.95), 0.95 (95% CI: 0.24–1.00), 16.81 (95% CI: 0.33–848.62), 0.19 (95% CI: 0.06–0.64), and 86.81 (95% CI: 1.22–6,154.15), respectively. The area under the summary receiver operating characteristic curve was 0.92 (95% CI: 0.89–0.94). The subgroup analysis showed that the factors of blood volume, histological type, EGFR-tyrosine kinase inhibitor therapy, and circulating tumor cell and tissue test methods for EGFR accounted for the significant difference of the pooled specificity. No significant difference was found between the pooled sensitivity of the subgroup. Conclusion Our meta-analysis confirmed that circulating tumor cells are a good surrogate for detecting epidermal growth factor receptor mutation when tumor tissue is unavailable in advanced NSCLC patients, but more precise techniques are needed to improve their clinical efficiency.

Comparison of genetic profiles among primary lung tumor, metastatic lymph nodes and circulating tumor DNA in treatment-naïve advanced non-squamous non-small cell lung cancer patients

OBJECTIVES Genetic profiles of primary and metastatic lung tumor have been investigated by previous studies. However, whether they can be replaced by each other to guide treatment remains controversial. Moreover, it is unclear that whether genetic profiles of plasma can reflect genetic divergence between primary and metastatic lesions. MATERIALS AND METHODS In this prospective study, we collected 35 pairs of matched primary tumor tissue, metastatic lymph nodes and plasma from treatment-naïve patients with advanced non-squamous non-small cell lung cancer (NSCLC) and applied to capture-based sequencing using a panel consisting 56 NSCLC-related genes to interrogate the heterogeneity and similarity among the 3 sites. RESULTS We observed 62.0% (67/108) by-variant concordance rate among primary tumor, metastatic lymph nodes and plasma as well as 76.4% (81/106) by-variant concordance rate between primary tumor and metastatic lymph nodes. When the analysis restricted to driver genes, we achieved 60.9% (28/46) and 77.3% (34/44) concordance, respectively. Furthermore, there is no statistically significant difference in progression-free survival (PFS) of 17 patients who used matched targeted therapy between patients having 100% concordance rate between primary tumor and metastatic lymph nodes and patients having partially matched mutational profiles. CONCLUSION Collectively, our study revealed a similar genetic profile shared between primary tumor and metastatic lymph nodes. The limited discordance observed can be partially reflected by plasma. Sequencing results obtained from either site can be utilized for providing treatment guidance.

Antigen presentation of the Oct4 and Sox2 peptides by CD154-activated B lymphocytes enhances the killing effect of cytotoxic T lymphocytes on tumor stem-like cells derived from cisplatin-resistant lung cancer cells

The present study investigated whether antigen presentation of the Oct4 and Sox2 peptides by CD154-activated B lymphocytes can enhance the killing effect of CD8+ cytotoxic T lymphocytes (CTLs) on lung stem-like cancer cells (SLCCs). The CTLs were generated using an accelerated co-cultured dendritic cells (DC) (acDC) assay by incubating human peripheral blood mononuclear cells (PBMCs) from non-small-cell lung cancer patients with antigen peptides of Oct4 and Sox2 in the presence of several DC-activating agents. CD154+ NIH3T3 cells prepared by CD154 lentiviral transfection were used as feeder layer to activate primary B cells (CD19+) obtained from PBMCs. Activated B cells were co-cultured with CTLs to present antigen peptides of Oct4 and Sox2. CTLs co-cultured with activated B cells were evaluated for the levels of secreted inflammatory cytokines using ELISA. In addition, the killing effect of the CTLs on SLCCs derived from cisplatin-resistant strain of human lung cancer cell line PC9 was evaluated by flow cytometry using CFSE labeling of the target cells. After the acDC assay, the PBMCs exhibited a significant (p<0.01) increase in the population of CD8+/CD3+ cells, indicating successful preparation of CTLs. The primary B cells cultured on the CD154+ NIH3T3 feeder layer resulted in significant (p<0.01) increase in the proportions of population expressing CD80, CD86, or HLA-A, indicating successful activation of the B cells. The co-culture of CTLs with CD154-activated B cells presenting the Oct4 and Sox2 peptides caused significant increase in the levels of secretory inflammatory cytokines and exhibited enhanced killing of the SLCCs derived from cisplatin-resistant PC9 cells. Antigen presentation of the Oct4 and Sox2 peptides by CD154-activated B cells can enhance the killing effect of CTLs towards lung SLCCs.

Can peripheral blood be used as surrogate in detecting epidermal growth factor receptor mutation status in advanced non-small cell lung cancer patients? A meta-analysis

Background Apply peripheral blood as a surrogate for detecting epidermal growth factor receptor mutation status in tumor, also called liquid biopsy, has been reported to be a feasible method in patients with advanced non-small lung cancer. But the diagnostic yield varies in different studies. Methods A meta-analysis was carried out to evaluate the sensitivity and specificity of peripheral blood in detection epidermal growth factor receptor mutation status in advanced non-small lung cancer patients. Publications up to October 2016 were searched using PubMed, Embase and Web of Science databases. Sensitivity, specificity and other parameters were pooled using the bivariate mixed-effects regression model. Results Fifteen studies meeting the inclusion criteria were included. The pooled sensitivity, specificity, positive likelihood ratio, negative likelihood ratio and diagnostic odds ratio were 0.69 (95% CI: 0.59~0.78), 0.97 (95% CI: 0.94~0.99), 23.1 (95% CI: 11.6~46.1), 0.32 (95% CI: 0.23~0.44), 73 (95% CI: 33~159), respectively. The summary receiver operating characteristic curve was 0.93 (95% CI: 0.91–0.95). Discussion Detecting epidermal growth factor receptor mutation in peripheral blood is a reliable and non-invasive method in patients with advanced non-small lung cancer. More sensitive detection methods are required to increase the sensitivity of liquid biopsy of ctDNA.