Pulmonary metastasectomy in colorectal cancer: a prospective study of demography and clinical characteristics of 543 patients in the Spanish colorectal metastasectomy registry (GECMP-CCR)

Objectives: To capture an accurate contemporary description of the practice of pulmonary metastasectomy for colorectal carcinoma in one national healthcare system. Design: A national registry set up in Spain by Grupo Español de Cirugía Metástasis Pulmonares de Carcinoma Colo-Rectal (GECMP-CCR). Setting: 32 Spanish thoracic units. Participants: All patients with one or more histologically proven lung metastasis removed by surgery between March 2008 and February 2010. Interventions: Pulmonary metastasectomy for one or more pulmonary nodules proven to be metastatic colorectal carcinoma. Primary and secondary outcome measures: The age and sex of the patients having this surgery were recorded with the number of metastases removed, the interval between the primary colorectal cancer operation and the pulmonary metastasectomy, and the carcinoembryonic antigen level. Also recorded were the practices with respect to mediastinal lymphadenopathy and coexisting liver metastases. Results: Data were available on 543 patients from 32 units (6-43/unit). They were aged 32-88 (mean 65) years, and 65% were men. In 55% of patients, there was a solitary metastasis. The median interval between the primary cancer resection and metastasectomy was 28 months and the serum carcinoembryonic antigen was low/normal in the majority. Liver metastatic disease was present in 29% of patients at some point prior to pulmonary metastasectomy. Mediastinal lymphadenectomy varied from 9% to 100% of patients. Conclusions: The data represent a prospective comprehensive national data collection on pulmonary metastasectomy. The practice is more conservative than the impression gained when members of the European Society of Thoracic Surgeons were surveyed in 2006/2007 but is more inclusive than would be recommended on the basis of recent outcome analyses. Furtheranalyses on the morbidity associated with this surgery and the correlation between imaging studies and pathological findings are being published separately by GECMP-CCR

Characterization, localization and comparison of c-Kit+ lung cells in never smokers and smokers with and without COPD

Background: c-Kit + lung stem cells have been described in the human healthy lung. Their potential relation with smoking and/or chronic obstructive pulmonary disease (COPD) is unknown. Methods: We characterized and compared c-Kit+ cells in lung tissue of 12 never smokers (NS), 15 smokers with normal spirometry (S) and 44 COPD patients who required lung resectional surgery. Flow cytometry (FACS) was used to characterize c-Kit+ cells in fresh lung tissue disaggregates, and immunofluorescence (IF) for further characterization and to determine their location in OCT- embedded lung tissue. Results: We identified 4 c-Kit+ cell populations, with similar proportions in NS, S and COPD: (1) By FACS, c-Kithigh/CD45 + cells (4.03 ± 2.97% (NS), 3.96 ± 5.30% (S), and 5.20 ± 3.44% (COPD)). By IF, these cells were tryptase+ (hence, mast cells) and located around the airways; (2) By IF, c-Kitlow/CD45+/triptase- (0.07 ± 0.06 (NS), 0.03 ± 0.02 (S), and 0.06 ± 0.07 (COPD) cells/field), which likely correspond to innate lymphoid cells; (3) By FACS, c-Kitlow/CD45-/CD34+ (0.95 ± 0.84% (NS), 1.14 ± 0.94% (S) and 0.95 ± 1.38% (COPD)). By IF these cells were c-Kitlow/CD45-/CD31+, suggesting an endothelial lineage, and were predominantly located in the alveolar wall; and, (4) by FACS, an infrequent c-Kitlow/CD45-/ CD34- population (0.09 ± 0.14% (NS), 0.08 ± 0.09% (S) and 0.08 ± 0.11% (COPD)) compatible with a putative lung stem cell population. Yet, IF failed to detect them and we could not isolate or grow them, thus questioning the existence of c-Kit+ lung stem-cells. Conclusions: The adult human lung contains a mixture of c-Kit+ cells, unlikely to be lung stem cells, which are independent of smoking status and/or presence of COPD

Multi-level immune response network in mild-moderate Chronic Obstructive Pulmonary Disease (COPD)

Background: Chronic Obstructive Pulmonary Disease (COPD) is associated with an abnormal pulmonary and systemic immune response to tobacco smoking. Yet, how do immune cells relate within and between these two biological compartments, how the pulmonary infiltrate influences the lung transcriptome, and what is the role of active smoking vs. presence of disease is unclear. Methods: To investigate these questions, we simultaneously collected lung tissue and blood from 65 individuals stratified by smoking habit and presence of the disease. The immune cell composition of both tissues was assessed by flow cytometry, whole lung transcriptome was determined with Affymetrix arrays, and we used Weighted Gene Co-expression Network Analysis (WGCNA) to integrate results. Results: Main results showed that: (1) current smoking and the presence of COPD were both independently associated with a reduction in the proportion of lung T cells and an increase of macrophages, specifically those expressing CD80 + CD163+; (2) changes in the proportion of infiltrating macrophages, smoking status or the level of airflow limitation were associated to different WGCNA modules, which were enriched in iron ion transport, extracellular matrix and cilium organization gene ontologies; and, (3) circulating white blood cells counts were correlated with lung macrophages and T cells. Conclusions: Mild-moderated COPD lung immune infiltrate is associated with the active smoking status and presence of disease; is associated with changes in whole lung tissue transcriptome and marginally reflected in blood

Exosome analysis in tumor-draining pulmonary vein identifies NSCLC patients with higher risk of relapse after curative surgery

Since tumor-draining pulmonary vein blood (PV) is enriched in tumor-secreted products, we hypothesized that it would also be enriched in tumor-derived exosomes, which would be important in the metastasis process. We characterized exosomes from PV of 61 resected non-small cell lung cancer (NSCLC) patients to evaluate its potential as relapse biomarkers. Exosomes were characterized using transmission electron microscopy, western blot and nanoparticle tracking analysis and we examined time to relapse (TTR) and overall survival (OS). Differences between PV and peripheral vein were found. PV was enriched in smaller exosomes than the paired peripheral vein (p = 0.01). Moreover, PV exosome size mode was able to identify relapsed patients (Area under the curve [AUC] = 0.781; 95%CI: 0.6641-0.8978), in whom exosome size was smaller (<112 nm; p < 0.001). The combination of PV exosome size and N (lymph node involvement) showed an AUC of 0.89 (95%CI: 0.80-0.97). Moreover, smaller PV exosome size was associated with shorter TTR (28.3 months vs. not reached, p < 0.001) and OS (43.9 months vs. not reached, p = 0.009). Multivariate analyses identified PV exosome size and stage as independent prognostic markers for TTR and OS. PV exosome size is a promising relapse biomarker after surgery that can add valuable information to clinical variables

YKT6 expression, exosome release, and survival in non-small cell lung cancer

BACKGROUND: Cancer-derived exosomes are involved in metastasis. YKT6 is a SNARE protein that participates in the regulation of exosome production and release, but its role in non-small cell lung cancer (NSCLC) has not been examined. MATERIALS AND METHODS: Ultracentrifugation-purified exosomes from the A549 cell line were studied by CRYO-TEM, nanoparticle tracking analysis and western blot (TSG101 marker). YKT6 was inhibited using a DsiRNA and selected pre-microRNAs. MicroRNAs targeting YKT6 were validated by Renilla/Luciferase assay and western blot. YKT6 expression and its prognostic impact were analyzed in 98 tissue specimens from resected NSCLC patients. RESULTS: Membranous nanosized vesicles (mode size: 128nm) with TSG101 protein were purified from A549 cells. YKT6 inhibition reduced exosome release by 80.9%. We validated miR-134 and miR-135b as miRNAs targeting YKT6, and transfection with the pre-miRNAs also produced a significant reduction in exosome release. The analysis of YKT6 in tumor samples showed that patients with high levels had shorter disease-free and overall survival. CONCLUSIONS: YKT6 is a key molecule in the regulation of exosome release in lung cancer cells and is in turn precisely regulated by miR-134 and miR-135b. Moreover, YKT6 levels impact prognosis of resected NSCLC patients

Extracellular Vesicle lincRNA-p21 Expression in Tumor-Draining Pulmonary Vein Defines Prognosis in NSCLC and Modulates Endothelial Cell Behavior

Hypoxia-induced upregulation of lincRNA-p21 in tumor tissue was previously shown by our group to be related to poor prognosis in resected non-small cell lung cancer (NSCLC) patients. In the present study, we have evaluated the presence of lincRNA-p21 in extracellular vesicles (EVs) from NSCLC patients and assessed its potential as a prognostic biomarker. High EV lincRNA-p21 levels in blood from the tumor-draining vein were associated with shorter time to relapse and shorter overall survival. Moreover, the multivariate analysis identified high lincRNA-p21 levels as an independent prognostic marker. In addition, lincRNA-p21 was overexpressed in H23 and HCC44 NSCLC cell lines and their derived EVs under hypoxic conditions. Functional assays using human umbilical vein endothelial cells (HUVECs) showed that tumor-derived EVs enriched in lincRNA-p21 affected endothelial cells by promoting tube formation and enhancing tumor cell adhesion to endothelial cells. Additionally, the analysis of selected EV microRNAs related to angiogenesis and metastasis showed that the microRNAs correlated with EV lincRNA-p21 levels in both patients and cell lines. Finally, EV co-culture with HUVEC cells increased the expression of microRNAs and genes related to endothelial cell activation. In conclusion, EV lincRNA-p21 acts as a novel prognosis marker in resected NSCLC patients, promoting angiogenesis and metastasis

Clinical significance of long non-coding RNA HOTTIP in early-stage non-small-cell lung cancer

Background HOTTIP, a long non-coding RNA located in the HOXA cluster, plays a role in the patterning of tissues with mesodermal components, including the lung. Overexpression of HOXA genes, including HOTTIP, has been associated with a more aggressive phenotype in several cancers. However, the prognostic impact of HOTTIP has not yet been explored in non-small-cell lung cancer (NSCLC). We have correlated HOTTIP expression with time to relapse (TTR) and overall survival (OS) in early-stage NSCLC patients. Methods Ninety-nine early-stage NSCLC patients who underwent surgical resection in our center from June 2007 to November 2013 were included in the study. Mean age was 66; 77.8% were males; 73.7% had stage I disease; and 55.5% had adenocarcinoma. A validation data set comprised stage I-II patients from The Cancer Genome Atlas (TCGA) Research Network. Results HOTTIP was expressed in all tumor samples and was overexpressed in squamous cell carcinoma (p = 0.007) and in smokers (p = 0.018). Patients with high levels of HOTTIP had shorter TTR (78.3 vs 58 months; p = 0.048) and shorter OS (81.2 vs 61 months; p = 0.023) than those with low levels. In the multivariate analysis, HOTTIP emerged as an independent prognostic marker for TTR (OR: 2.05, 95%CI: 1-4.2; p = 0.05), and for OS (OR: 2.31, 95%CI: 1.04-5.1; p = 0.04). HOTTIP was validated as a prognostic marker for OS in the TCGA adenocarcinoma cohort (p = 0.025). Moreover, we identified a 1203-mRNA and a 61-miRNA signature that correlated with HOTTIP expression. Conclusions The lncRNA HOTTIP can be considered a prognostic biomarker in early-stage NSCLC

miR-141 and miR-200c as markers of overall survival in early stage non-small cell lung cancer adenocarcinoma

Several treatments in non-small cell lung cancer (NSCLC) are histology-dependent, and the need for histology-related markers is increasing. MicroRNAs (miRNAs) are promising molecular markers in multiple cancers and show differences in expression depending on histological subtype. The miRNA family miR-200 has been associated with the regulation of epithelial-mesenchymal (EMT)/mesenchymal-epithelial transition (MET). EMT involves profound phenotypic changes that include the loss of cell-cell adhesion, the loss of cell polarity, and the acquisition of migratory and invasive properties that facilitates metastasis. A dual role for the miR-200 family in the prognosis of several tumors has been related to tumor cell origin. However, the prognostic role and function of miR-200 family in early-stage NSCLC adenocarcinoma and squamous cell carcinoma (SCC) have not been well established. Methods: miRNA expression was determined using TaqMan assays in 155 tumors from resected NSCLC patients. Functional studies were conducted in three NSCLC cell lines: H23, A-549 and HCC-44. Results: High miR-200c expression was associated with shorter overall survival (OS) in the entire cohort (p = 0.024). High miR-200c (p = 0.0004) and miR-141 (p = 0.009) expression correlated with shorter OS in adenocarcinoma - but not in SCC. In the multivariate analysis, a risk score based on miR-141 and miR-200c expression emerged as an independent prognostic factor for OS in the entire cohort (OR, 2.787; p = 0.033) and in adenocarcinoma patients (OR, 10.649; p = 0.002). Functional analyses showed that miR-200c, was related to mesenchymal-epithelial transition (MET) and affected cell migration and E-cadherin levels, while overexpression of miR-141 reduced KLF6 protein levels and produced an increase of secretion of VEGFA in vitro (H23, p = 0.04; A-549, p = 0.03; HCC-44, p = 0.02) and was associated with higher blood microvessel density in patient tumor samples (p<0.001). Conclusion: High miR-141 and miR-200c expression are associated with shorter OS in NSCLC patients with adenocarcinoma through MET and angiogenesis

Role of the epithelial-mesenchymal transition-related circular RNA, circ-10720, in non-small-cell lung cancer

Background: Circular RNAs (circRNAs) are non-coding RNAs with a circular structure that have recently emerged as important regulators of tumorogenesis. Recently, several circRNAS, including circ-10720 have been related to epithelial-mesenchymal transition (EMT) process. In the present study, we have analyzed the role of circ-10720 in non-small-cell lung cancer (NSCLC) and studied its prognostic relevance in resected stage I-IIIa NSCLC patients. Methods: Circ-10720 expression was analyzed using a custom TaqMan assay in four NSCLC cell lines (HCC44, A549, H23 and H1299) and in the normal immortalized lung cell line BEAS2B. Silencing of circ10720 was performed using two custom siRNAs which were transfected using lipofectamine 2000. Protein levels were evaluated by Western blot and immunofluorescence. Wound healing and invasion assays were performed to evaluate the impact the circRNA on cell motility. Apoptosis was analyzed by evaluation of Caspase 3-7 activity and proliferation by MTS assay. Moreover, the expression levels of the circRNA were studied in 119 resected NSCLC patients. The expression in tumor tissue was correlated with the main clinicopathological characteristics and with time to relapse (TTR). Results: Circ-10720 was overexpressed in HCC44 and A549 and underexpressed in H23 and H1299 NSCLC cell lines in comparison to BEAS2B normal immortalized lung cell line. CircRNA knockdown in the two circ-10720 overexpressing cell lines was associated with a decrease of Vimentin (VIM) and an increase of E-cadherin (CDH1) protein levels, loss of mesenchymal phenotype, and a significant reduction of migration and invasion capacity. After silencing circ-10720, the apoptosis rate increased and the proliferation was significantly reduced. Furthermore, circ-10720 was upregulated in tumor vs. normal tissue from 119 resected NSCLC patients. In the group of patients not receiving adjuvant treatment, those with high levels of circ-10720 had a shorter TTR than those with low levels and emerged as an independent prognostic value in the multivariate analysis. In tumor tissue, circ-10720 levels positively correlated with the EMT gene Twist1 levels. Conclusions: Circ-10720 regulates EMT, apoptosis and proliferation and acts as a biomarker of relapse in NSCLC

Characterization of the MicroRNA Cargo of Extracellular Vesicles Isolated from a Pulmonary Tumor-Draining Vein Identifies miR-203a-3p as a Relapse Biomarker for Resected Non-Small Cell Lung Cancer.

In resected non-small cell lung cancer (NSCLC), post-surgical recurrence occurs in around 40% of patients, highlighting the necessity to identify relapse biomarkers. An analysis of the extracellular vesicle (EV) cargo from a pulmonary tumor-draining vein (TDV) can grant biomarker identification. We studied the pulmonary TDV EV-miRNAome to identify relapse biomarkers in a two-phase study (screening and validation). In the screening phase, a 17-miRNA relapse signature was identified in 18 selected patients by small RNAseq. The most expressed miRNA from the signature (EV-miR-203a-3p) was chosen for further validation. Pulmonary TDV EV-miR-203a-3p was studied by qRT-PCR in a validation cohort of 70 patients, where it was found to be upregulated in relapsed patients (p = 0.0194) and in patients with cancer spread to nearby lymph nodes (N+ patients) (p = 0.0396). The ROC curve analysis showed that TDV EV-miR-203a-3p was able to predict relapses with a sensitivity of 88% (AUC: 0.67; p = 0.022). Moreover, patients with high TDV EV-miR-203a-3p had a shorter time to relapse than patients with low levels (43.6 vs. 97.6 months; p = 0.00703). The multivariate analysis showed that EV-miR-203a-3p was an independent, predictive and prognostic post-surgical relapse biomarker. In conclusion, pulmonary TDV EV-miR-203a-3p is a promising new relapse biomarker for resected NSCLC patients