Study of glycosidic changes in lung cancer: potential effectors in hematogenous metastasis

RESUMO: O cancro do pulmão (LC), uma das principais causas de mortalidade relacionada com cancro em Portugal, pode levar à formação de metástases hematogénicas. A adesão das células tumorais ao endotélio é considerada um dos passos fundamentais envolvidos na metástase. Em células sanguíneas, esta adesão é mediada por ligandos de E-selectina (E-SL), glicoproteínas ou glicolípidos decorados principalmente com sialyl-Lewis x (sLex) e sialyl-Lewis a (sLea). Tem sido descrito a expressão destes antigénios em LC, contudo o seu papel funcional em permitir a adesão das células de LC ao endotélio é ainda pouco compreendido. Foram analisadas amostras emparelhadas normais e tumorais de pacientes com cancro de pulmão de não-pequenas células (NSCLC) e três linhas celulares de LC. Immunoblotting assays com anti-sLex/sLea e molécula quimérica de E-selectina demonstraram que tecidos tumorais de LC sobreexpressam significativamente E-SL e resultados de citometria de fluxo demonstraram uma expressão elevada de E-SL nas linhas celulares. Para compreender o mecanismo da sobreexpressão de E-SL em tecidos tumorais e linhas celulares de LC, foi analisada a expressão de genes envolvidos na biossíntese de E-SL, nomeadamente FUT3, FUT4, FUT6, FUT7, ST3GAL3, ST3GAL4, ST3GAL6, β4GALT1, GCNT1 e GALNT3. Observou-se a sobreexpressão das fucosiltransferases FUT3, FUT6 e FUT7 em tecidos tumorais de LC e FUT3 em linhas celulares de LC, sendo que neste último, esta expressão é correlacionada com um aumento da adesão das células de LC às selectinas endoteliais. Foi observado que uma baixa expressão de FUT4 em tecidos tumorais está associada com estadios menos avançados de NSCLC. Foram analisadas ainda proteínas decoradas com sLex/sLea, tendo-se identificado como E-SL o antigénio carcinoembrionário em NSCLC. Em resumo, esta tese contribuiu para uma melhor compreensão das alterações glicosídicas e moléculas que podem influenciar a progressão tumoral do LC, podendo permitir identificar futuramente novos biomarcadores de diagnóstico/prognóstico e potenciais alvos terapêuticos para o NSCLC.--------------------------ABSTRACT: Lung cancer (LC), one of the major causes of mortality related to cancer in Portugal, may lead to hematogenous metastasis. Adhesion of cancer cells to endothelium is considered one of the crucial steps involved in metastasis. In blood cells, this adhesion is initiated by endothelial selectin ligands (E-SL) that are glycoproteins or glycolipids decorated mostly with sialyl-Lewis x (sLex) and sialyl-Lewis a (sLea). While LC has been described as expressing these sialyl Lewis antigens, its functional role in allowing LC adhesion to endothelium is still poorly understood. We analyzed paired tumor and normal tissues samples from non-small cell lung cancer (NSCLC) patients and three LC cell lines. Immunoblotting assays with anti-sLex/sLea and E-selectin chimera demonstrated that LC tumor tissues significantly overexpress E-SL and flow cytometry results indicated that E-SL are also abundantly expressed in LC cell lines. To understand the mechanism behind the overexpression of E-SL in LC tissues and cell lines, we analyzed the expression of genes involved in its biosynthesis, namely FUT3, FUT4, FUT6, FUT7, ST3GAL3, ST3GAL4, ST3GAL6, β4GALT1, GCNT1 and GALNT3. It was observed the overexpression of fucosyltransferases FUT3, FUT6 and FUT7 in LC tumor tissues and FUT3 in LC cell lines, being this last one correlated with an increased reactivity of the LC cells to endothelial selectins. It was described that low expression of FUT4 in tumor tissues is correlated with early stages of NSCLC. We also analyzed scaffolds proteins of sLex/sLea and it was identified the carcinoembryonic antigen as an E-SL in NSCLC. In summary, this thesis contributed to a better understanding of the glycosidic changes and molecules that can influence tumor progression of LC, allowing identifying in the future new diagnosis/prognosis biomarkers and potential therapeutic targets for NSCLC.

Stereotactic body radiation therapy in stage I inoperable lung cancer: from palliative to curative options.

Surgery has historically been the standard of care for operable stage I non-small cell lung cancer (NSCLC). However, nearly one-quarter of patients with stage I NSCLC will not undergo surgery because of medical comorbidity or other factors. Stereotactic ablative radiotherapy (SABR) is the new standard of care for these patients. SABR offers high local tumour control rates rivalling the historical results of surgery and is generally well tolerated by patients with both peripheral and centrally located tumours. This article reviews the history of SABR for stage I NSCLC, summarises the currently available data on efficacy and toxicity, and describes some of the currently controversial aspects of this treatment.

Perioperative in-stent thrombosis after lung resection performed within 3 months of coronary stenting

BACKGROUND: Incidence of perioperative in-stent thrombosis associated with myocardial infarction in patients undergoing major lung resection within 3 months of coronary stenting. METHODS: Retrospective multi-institutional trial including all patients undergoing major lung resection (lobectomy or pneumonectomy) within 3 months of coronary stenting with non-drug-eluting stents between 1999 and 2004. RESULTS: There were 32 patients (29 men and 3 women), with age ranging from 46 to 82 years. One, two or four coronary stents were deployed in 72%, 22% and 6% of the patients, respectively. The time intervals between stenting and lung surgery were <30 days, 30-60 days and 61-90 days in 22%, 53% and 25% of the patients, respectively. All patients had dual antiplatelet therapy after stenting. Perioperative medication consisted of heparin alone or heparin plus aspirin in 34% and 66% of the patients, respectively. Perioperative in-stent thrombosis with myocardial infarction occurred in three patients (9%) with fatal outcome in one (3%). Twenty patients underwent lung resection after 4 weeks of dual antiplatelet therapy as recommended by the ACC/AHA Guideline Update; however, two out of three perioperative in-stent thrombosis occurred in this group of patients. CONCLUSIONS: Major lung resection performed within 3 months of coronary stenting may be complicated by perioperative in-stent thrombosis despite 4 weeks of dual antiplatelet therapy after stenting as recommended by the ACC/AHA Guideline Update.

Modulation of MDR1 and MRP3 Gene Expression in Lung Cancer Cells after Paclitaxel and Carboplatin Exposure

Carboplatin-paclitaxel is a reference regimen in the treatment of locally advanced or disseminated non-small cell lung cancer (NSCLC). This paper discusses the multidrug resistance developed with this drug combination, which is one of the major obstacles to successful treatment. In order to understand and overcome the drug resistance pattern of NSCLC after carboplatin plus paclitaxel exposure, levels of mRNA expression of multidrug resistance 1 (MDR1) and multidrug resistance-associated protein 3 (MRP3) were investigated in primary NSCLC cell lines (A-549 and A-427) and a metastasis-derived NSCLC cell line (NODO). Our results showed that exposure of the three NSCLC lines to plasma concentrations of paclitaxel (5 μM) produced an increase in MDR1 expression, while MRP3 showed no alteration in expression. By contrast, the same cells exposed to carboplatin plasma concentrations (30 μM) showed overexpression of MRP3. In these cells, MDR1 showed no expression changes. Interestingly, the combination of both paclitaxel and carboplatin caused increased expression of the MDR1 drug resistance gene rather than the individual treatments. These results suggest that carboplatin and paclitaxel may induce drug resistance mediated by MDR1 and MRP3, which may be enhanced by the simultaneous use of both drugs.

Pattern of use of radiotherapy for lung cancer: a descriptive study.

BACKGROUND Lung cancer remains one of the most prevalent forms of cancer. Radiotherapy, with or without other therapeutic modalities, is an effective treatment. Our objective was to report on the use of radiotherapy for lung cancer, its variability in our region, and to compare our results with the previous study done in 2004 (VARA-I) in our region and with other published data. METHODS We reviewed the clinical records and radiotherapy treatment sheets of all patients undergoing radiotherapy for lung cancer during 2007 in the 12 public hospitals in Andalusia, an autonomous region of Spain. Data were gathered on hospital, patient type and histological type, radiotherapy treatment characteristics, and tumor stage. RESULTS 610 patients underwent initial radiotherapy. 37% of cases had stage III squamous cell lung cancer and were treated with radical therapy. 81% of patients with non-small and small cell lung cancer were treated with concomitant chemo-radiotherapy and the administered total dose was ≥60 Gy and ≥45 Gy respectively. The most common regimen for patients treated with palliative intent (44.6%) was 30 Gy. The total irradiation rate was 19.6% with significant differences among provinces (range, 8.5-25.6%; p<0.001). These differences were significantly correlated with the geographical distribution of radiation oncologists (r=0.78; p=0.02). Our results were similar to other published data and previous study VARA-I. CONCLUSIONS Our results shows no differences according to the other published data and data gathered in the study VARA-I. There is still wide variability in the application of radiotherapy for lung cancer in our setting that significantly correlates with the geographical distribution of radiation oncologists.

Cancer pulmonaire: lobectomie par thoracoscopie [VATS lobectomy for early-stage primary lung cancer].

Lobectomy via video-assisted thoracoscopic surgery (VATS) is now considered as a valid alternative to conventional thoracotomy for early-stage primary lung cancer. Various studies have reported that VATS lobectomy is a safe technique associated with fewer postoperative complications and better post-operative recovery than open thoracotomy. Furthermore, studies suggest oncological equivalence between VATS and open lobectomy.

Micropapillary pattern in lung adenocarcinoma: aspect on 18F-fluorodeoxyglucose positron emission tomography/computed tomography imaging.

We diagnosed a non-small cell lung carcinoma in a 49-year-old female patient with the histopathological diagnosis of stage IIIB mixed bronchioloalveolar and papillary adenocarcinoma with extensive micropapillary feature, which was not visualized on the preoperative multimodality imaging with positron emission tomography (PET) and computed tomography (CT). The micropapillary component characterized by a unique growth pattern with particular morphological features can be observed in all subtypes of lung adenocarcinoma. Micropapillary component is increasingly recognized as a distinct entity associated with higher aggressiveness. Even the most modern multimodality PET/CT imaging technology may fail to adequately visualize this important component with highly relevant prognostic implications. Thus, the pathologist needs to consciously look for a micropapillary component in the surgical specimen or in preoperative biopsies or cytology. This may have potential future treatment implications, as adjuvant or neoadjuvant chemotherapy may be of relevance, even in the early stages of the disease.

The cooperative induction of hypoxia-inducible factor-1 alpha and STAT3 during hypoxia induced an impairment of tumor susceptibility to CTL-mediated cell lysis.

Hypoxia is an essential component of tumor microenvironment. In this study, we investigated the influence of hypoxia (1% PO(2)) on CTL-mediated tumor cell lysis. We demonstrate that exposure of target tumor cells to hypoxia has an inhibitory effect on the CTL clone (Heu171)-induced autologous target cell lysis. Such inhibition correlates with hypoxia-inducible factor-1alpha (HIF-1alpha) induction but is not associated with an alteration of CTL reactivity as revealed by granzyme B polarization or morphological change. Western blot analysis indicates that although hypoxia had no effect on p53 accumulation, it induced the phosphorylation of STAT3 in tumor cells by a mechanism at least in part involving vascular endothelial growth factor secretion. We additionally show that a simultaneous nuclear translocation of HIF-1alpha and phospho-STAT3 was observed. Interestingly, gene silencing of STAT3 by small interfering RNA resulted in HIF-1alpha inhibition and a significant restoration of target cell susceptibility to CTL-induced killing under hypoxic conditions by a mechanism involving at least in part down-regulation of AKT phosphorylation. Moreover, knockdown of HIF-1alpha resulted in the restoration of target cell lysis under hypoxic conditions. This was further supported by DNA microarray analysis where STAT3 inhibition resulted in a partly reversal of the hypoxia-induced gene expression profile. The present study demonstrates that the concomitant hypoxic induction of phospho-STAT3 and HIF-1alpha are functionally linked to the alteration of non-small cell lung carcinoma target susceptibility to CTL-mediated killing. Considering the eminent functions of STAT3 and HIF-1alpha in the tumor microenvironment, their targeting may represent novel strategies for immunotherapeutic intervention.

Lung Cancer That Harbors an HER2 Mutation: Epidemiologic Characteristics and Therapeutic Perspectives.

PURPOSE HER2 mutations are identified in approximately 2%of non-small-cell lung cancers (NSCLC). There are few data available that describe the clinical course of patients with HER2-mutated NSCLC. PATIENTS AND METHODS We retrospectively identified 65 NSCLC, diagnosed with a HER2 in-frame insertion in exon 20. We collected clinicopathologic characteristics, patients' outcomes, and treatments. Results HER2 mutation was identified in 65 (1.7%) of 3,800 patients tested and was almost an exclusive driver, except for one single case with a concomitant KRAS mutation. Our population presented with a median age of 60 years (range, 31 to 86 years), a high proportion of women (45 women v 20 men; 69%), and a high proportion of never-smokers (n= 34; 52.3%). All tumors were adenocarcinomas and 50% were stage IV at diagnosis. For these latter cases, 22 anti-human epidermal growth factor receptor 2 (HER2) treatments were administered after conventional chemotherapy in 16 patients. Subsequently, four patients experienced progressive disease, seven experienced disease stabilizations, and 11 experienced partial responses (overall response rate, 50%; disease control rate [DCR], 82%). Specifically, we observed a DCR of 93% for trastuzumab-based therapies (n = 15) and a DCR of 100% for afatinib (n = 3) but no response to other HER2-targeted drugs (n = 3). Progression-free survival for patients with HER2 therapies was 5.1 months. Median survival was of 89.6 and 22.9 months for early-stage and stage IV patients, respectively. CONCLUSION This study, the largest to date dedicated to HER2-mutated NSCLC, reinforces the importance of screening for HER2 mutations in lung adenocarcinomas and suggests the potential efficacy of HER2-targeted drugs in this population.

Video-assisted thoracoscopic resection of a small pulmonary nodule after computed tomography-guided localization with a hook-wire system. Experience in 45 consecutive patients.

BACKGROUND: This study is a single-institution validation of video-assisted thoracoscopic (VATS) resection of a small solitary pulmonary nodule (SPN) previously localized by a CT-guided hook-wire system in a consecutive series of 45 patients. METHODS: The records of all patients undergoing VATS resection for SPN preoperatively localized by CT-guided a hook-wire system from January 2002 to December 2004 were assessed with respect to failure to localize the lesion by the hook-wire system, conversion thoracotomy rate, duration of operation, postoperative complications, and histology of SPN. RESULTS: Forty-five patients underwent 49 VATS resections, with simultaneous bilateral SPN resection performed in 4. Preoperative CT-guided hook-wire localization failed in two patients (4%). Conversion thoracotomy was necessary in two patients (4%) because it was not possible to resect the lesion by a VATS approach. The average operative time was 50 min. Postoperative complications occurred in 3 patients (6%), one hemothorax and two pneumonia. The mean hospital stay was 5 days (range: 2-18 days). Histological assessment revealed inflammatory disease in 17 patients (38%), metastasis in 17 (38%), non-small-cell lung cancer (NSCLC) in 4 (9%), lymphoma in 3 (6%), interstitial fibrosis in 2 (4%), histiocytoma in one (2%), and hamartoma in one (2%). CONCLUSIONS: Histological analysis of resected SPN revealed unexpected malignant disease in more than 50% of the patients indicating that histological clarification of SPN seems warranted. Video-assisted thoracoscopic resection of SPN previously localized by a CT-guided hook-wire system is related to a low conversion thoracotomy rate, a short operation time, and few postoperative complications, and it is well suited for the clarification of SPN.

Traitements antiangiogéniques des cancers métastatiques du côlon et du rectum, du sein et du poumon: bénéfices et risques [Anti-angiogenic therapies for metastatic colorectal, breast and lung cancer: benefits and risks]

Anti-angiogenic therapies have recently enriched the therapeutic armentarium against the most common cancers. Among these, bevacizumab, a monoclonal antibody against vascular endothelial growth factor, is currently used most frequently. While the addition of bevacizumab to chemotherapy improves overall survival in first and second line treatment of metastatic colorectal cancer, its effect in metastatic breast cancer is limited to improvements in tumor response and progression-free-survival. In non-small-cell lung cancer, the positive results of a first American phase III study have not been confirmed by a second European study and are subject to controversies. A summary of the data concerning anti-angiogenic therapies in these three cancers is presented including safety information.

Impact of induction therapy on airway complications after sleeve lobectomy for lung cancer.

BACKGROUND: Sleeve lobectomy is a valid alternative to pneumonectomy for the treatment of centrally located operable non-small cell lung cancer (NSCLC), but concern has been evoked regarding a potentially increased risk of bronchial anastomosis complications after induction therapy. This study examined the impact of induction therapy on airway healing after sleeve lobectomy for NSCLC. METHODS: Bronchial anastomosis complications were recorded with respect to the induction regimen applied (neoadjuvant chemotherapy vs chemoradiotherapy) in a consecutive series of patients with sleeve lobectomy for NSCLC. RESULTS: Ninety-nine patients underwent sleeve resection, 28 of them after induction therapy. Twelve patients received chemotherapy alone, and 16 patients had radiochemotherapy. There were no significant differences in postoperative 90-day mortality (3.6% vs 2.8%) and morbidity (54% vs 49%) for patients with and without induction therapy. Bronchial anastomosis complications occurred in 3 patients (10.8%) with neoadjuvant therapy and in 2 (2.8%) without (p = 0.3). In the induction therapy group, two bronchial stenoses occurred after radiochemotherapy and one bronchopleural fistula after chemotherapy alone. In patients without induction therapy, one bronchial stenosis and one bronchopleural fistula were observed. All bronchial stenoses were successfully treated by dilatation, and both bronchopleural fistulas occurring after right lower lobectomy were successfully treated by reoperation and completion sleeve bilobectomy with preservation of the upper lobe. CONCLUSIONS: Sleeve lobectomy for NSCLC can be safely performed after induction chemotherapy and radiochemotherapy with mortality and incidence of airway complications similar to that observed in nonpretreated patients. The treatment of airway complications does not differ for patients with and without induction therapy.

Optimized exosome isolation protocol for cell culture supernatant and human plasma.

Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research.

Estimation of lung cancer diagnosis and treatment costs based on a patient-level analysis in Catalonia (Spain)

Background: Assessing of the costs of treating disease is necessary to demonstrate cost-effectiveness and to estimate the budget impact of new interventions and therapeutic innovations. However, there are few comprehensive studies on resource use and costs associated with lung cancer patients in clinical practice in Spain or internationally. The aim of this paper was to assess the hospital cost associated with lung cancer diagnosis and treatment by histology, type of cost and stage at diagnosis in the Spanish National Health Service. Methods: A retrospective, descriptive analysis on resource use and a direct medical cost analysis were performed. Resource utilisation data were collected by means of patient files from nine teaching hospitals. From a hospital budget impact perspective, the aggregate and mean costs per patient were calculated over the first three years following diagnosis or up to death. Both aggregate and mean costs per patient were analysed by histology, stage at diagnosis and cost type. Results: A total of 232 cases of lung cancer were analysed, of which 74.1% corresponded to non-small cell lung cancer (NSCLC) and 11.2% to small cell lung cancer (SCLC); 14.7% had no cytohistologic confirmation. The mean cost per patient in NSCLC ranged from 13,218 Euros in Stage III to 16,120 Euros in Stage II. The main cost components were chemotherapy (29.5%) and surgery (22.8%). Advanced disease stages were associated with a decrease in the relative weight of surgical and inpatient care costs but an increase in chemotherapy costs. In SCLC patients, the mean cost per patient was 15,418 Euros for limited disease and 12,482 Euros for extensive disease. The main cost components were chemotherapy (36.1%) and other inpatient costs (28.7%). In both groups, the Kruskall-Wallis test did not show statistically significant differences in mean cost per patient between stages. Conclusions: This study provides the costs of lung cancer treatment based on patient file reviews, with chemotherapy and surgery accounting for the major components of costs. This cost analysis is a baseline study that will provide a useful source of information for future studies on cost-effectiveness and on the budget impact of different therapeutic innovations in Spain.

Cytotoxicity of Marchantia convoluta leaf extracts to human liver and lung cancer cells

The cytotoxicity of three extracts (petroleum ether, ethyl acetate and n-butanol) from a plant used in folk medicine, Marchantia convoluta, to human non-small cell lung carcinoma (H1299) and liver carcinoma (HepG2) cell lines was tested. After 72-h incubation of lung and liver cancer cell cultures with varying concentrations of extracts (15 to 200 µg/mL), cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and reported in terms of cell viability. The extracts that showed a significant cytotoxicity were subjected to gas chromatography-mass spectrometry analysis to identify the components. The ethyl acetate, but not the petroleum ether or n-butanol extract, had a significant cytotoxicity against lung and liver carcinoma cells with IC50 values of 100 and 30 µg/mL, respectively. A high concentration of ethyl acetate extract (100 µg/mL) rapidly reduced the number of H1299 cells. At lower concentrations of ethyl acetate extract (15, 30, and 40 µg/mL), the numbers of HepG2 cells started to decrease markedly. Gas chromatography-mass spectrometry analysis of the ethyl acetate extract revealed the presence of several compounds such as phytol (23.42%), 1,2,4-tripropylbenzene (13.09%), 9-cedranone (12.75%), ledene oxide (7.22%), caryophyllene (1.82%), and caryophyllene oxide (1.15%). HPLC analysis result showed that there were no flavonoids in ethyl acetate extract, but flavonoids are abundant in n-butanol extract. Further studies are needed regarding the identification, toxicity, and mechanism of action of active compounds.