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Liquid biopsy analyses are already incorporated in the routine clinical practice in many hospitals and oncology departments worldwide, improving the selection of treatments and monitoring of lung cancer patients. Although they have not yet reached its full potential, liquid biopsy-based tests will soon be as widespread as “standard” biopsies and imaging techniques, offering invaluable diagnostic, prognostic, and predictive information. This review summarizes the techniques available for the isolation and analysis of circulating free DNA and RNA, exosomes, tumor-educated platelets, and circulating tumor cells from the blood of cancer patients, presents the methodological challenges associated with each of these materials, and discusses the clinical applications of liquid biopsy testing in lung cancer.

Summary Background The tyrosine kinase inhibitor erlotinib improves the outcomes of patients with advanced non-small-cell lung carcinoma (NSCLC) harbouring epidermal growth factor receptor ( EGFR ) mutations. The coexistence of the T790M resistance mutation with another EGFR mutation in treatment-naive patients has been associated with a shorter progression-free survival to EGFR inhibition than in the absence of the T790M mutation. To test this hypothesis clinically, we developed a proof-of-concept study, in which patients with EGFR -mutant NSCLC were treated with the combination of erlotinib and bevacizumab, stratified by the presence of the pretreatment T790M mutation. Methods BELIEF was an international, multicentre, single-arm, phase 2 trial done at 29 centres in eight European countries. Eligible patients were aged 18 years or older and had treatment-naive, pathologically confirmed stage IIIB or stage IV lung adenocarcinoma with a confirmed, activating EGFR mutation (exon 19 deletion or L858R mutation). Patients received oral erlotinib 150 mg per day and intravenous bevacizumab 15 mg/kg every 21 days and were tested centrally for the pretreatment T790M resistance mutation with a peptide nucleic acid probe-based real-time PCR. The primary endpoint was progression-free survival. The primary efficacy analysis was done in the intention-to-treat population and was stratified into two parallel substudies according to the centrally confirmed pretreatment T790M mutation status of enrolled patients (T790M positive or negative). The safety analysis was done in all patients that have received at least one dose of trial treatment. This trial was registered with ClinicalTrials.gov, number NCT01562028. Findings Between June 11, 2012, and Oct 28, 2014, 109 patients were enrolled and included in the efficacy analysis. 37 patients were T790M mutation positive and 72 negative. The overall median progression-free survival was 13·2 months (95% CI 10·3–15·5), with a 12 month progression-free survival of 55% (95% CI 45–64). The primary endpoint was met only in substudy one (T790M-positive patients). In the T790M-positive group, median progression-free survival was 16·0 months (12·7 to not estimable), with a 12 month progression-free survival of 68% (50–81), whereas in the T790M-negative group, median progression-free survival was 10·5 months (9·4–14·2), with a 12 month progression-free survival of 48% (36–59). Of 106 patients included in the safety analysis, five had grade 4 adverse events (one acute coronary syndrome, one biliary tract infection, one other neoplasms, and two colonic perforations) and one died due to sepsis. Interpretation The BELIEF trial provides further evidence of benefit for the combined use of erlotinib and bevacizumab in patients with NSCLC harbouring activating EGFR mutations. Funding European Thoracic Oncology Platform, Roche.

Lung cancer is the leading cause of cancer-associated deaths worldwide. Surgery is the standard treatment for early-stage non-small cell lung cancer (NSCLC). However, 30% to 80% of these patients will die within 5 yearS of diagnosis. Circulating cell-free DNA (cfDNA) harbors pathologic characteristics of the original tumor, such as gene mutations or epigenetic alterations. Analysis of cfDNA has revolutionized the clinical care of advanced lung cancer patients undergoing targeted therapies. However, the low concentration of cfDNA in the blood of early-stage NSCLC patients has hampered its use for management of early disease. Continuing development of more specific and sensitive techniques for detection and analysis of cfDNA will soon enable its leverage in early stage and, perhaps, even screening settings. Therefore, cfDNA analysis may become a tool used for routine NSCLC diagnosis and for monitoring tumor burden, as well as for identifying hidden residual disease. In this review, we will focus on the current evidence of cfDNA in patients with early-stage NSCLC, new and upcoming approaches to identify circulating-tumor biomarkers, their clinical applications and future directions.

In lung cancer, as well as in other malignancies, the so-called “liquid biopsy” is quickly moving from research into clinical practice. Although it has not yet reached its full potential, the “liquid biopsy” is no longer a promise but a reality that is allowing a better monitoring and treatment selection of cancer patients in many hospitals and oncology departments. We can already envision a day when “liquid biopsy” tests will be as common and useful as imaging techniques and “standard” biopsies, offering invaluable prognostic and predictive information. The objective of this special issue of Translational Lung Cancer Research ( TLCR ) is to offer an overview of this exciting area, with a particular emphasis on the clinical utility of the so-called “liquid biopsy” and the diversity of applications, methodologies and information that can be derived from it.

Introduction Smoking is the cause of multiple diseases. Effective treatment is critical, and when there is, and fails, to detect posible contributing factors. Objectives Analyze the characteristics of people who fail in anti-smoking treatment. Material and methods Retrospective study of the patients evaluated in smoking unit between 2009-2011. Patients who had treatment failure were detected and were compared with those who had any time of abstinence. Chi square, Fisher statistic and student t was used. We defined failure as that one who has not been able to leave smoking even 24 hours. Results N=901 patients. 83 failed. Conclusions 1. Male gender influences in therapeutic failure. 2. Age, number of cigarettes, ECM and SBP are also associated. 3. Among respiratory diseases, suffering COPD and SAHS is more likely to fail. 4. Arrythmias, dyslipidemia, IBD and neoplasias also have a higher frecuency of treatment failure. 5. Neither psychiatric illness nor the use of other drugs or alcohol were associated with a higher frecuency of failure.

Introduction Smoking is a systemic disease. It is the the most important isolated cause of premature morbidity and mortality preventable and avoidable in developed countries. Multiple diseases and pathological conditions are directly caused or have been related to snuff exposure. Objectives We decided to see if with the experience the success rate, defined as a year of continuous abstinence, of our patients had improved. Material and methods Retrospective study using the database of our unit over years 2008 and 2012. Chi-square test was used to compare the success and relapse in one year and another, and student t test and chi-square were used to compare if the main characteristic of the samples were comparable.The study was made with all the patients who attended to the consultation. Results Conclusions: 1- More patients in 2012 than in 2008, but there were no differences in success. 2- Patients in 2008 smoked more and had more dependence. 3- There were fewer relapses in 2012. 4- We got more abstinence days in 2008.

Background: The efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in EGFR-mutant non-small cell lung cancer (NSCLC) is limited by adaptive activation of cell survival signals. We hypothesized that both signal transducer and activator of transcription 3 (STAT3) and Src-YES-associated protein 1 (YAP1) signaling are dually activated during EGFR TKI treatment to limit therapeutic response.Methods: We used MTT and clonogenic assays, immunoblotting, and quantitative polymerase chain reaction to evaluate the efficacy of EGFR TKI alone and in combination with STAT3 and Src inhibition in three EGFR-mutant NSCLC cell lines. The Chou-Talalay method was used for the quantitative determination of drug interaction. We examined tumor growth inhibition in one EGFR-mutant NSCLC xenograft model (n = 4 mice per group). STAT3 and YAP1 expression was evaluated in tumors from 119 EGFR-mutant NSCLC patients (64 in an initial cohort and 55 in a validation cohort) by quantitative polymerase chain reaction. Kaplan-Meier and Cox regression analyses were used to assess the correlation between survival and gene expression. All statistical tests were two-sided.Results: We discovered that lung cancer cells survive initial EGFR inhibitor treatment through activation of not only STAT3 but also Src-YAP1 signaling. Cotargeting EGFR, STAT3, and Src was synergistic in two EGFR-mutant NSCLC cell lines with a combination index of 0.59 (95% confidence interval [CI] = 0.54 to 0.63) for the PC-9 and 0.59 (95% CI = 0.54 to 0.63) for the H1975 cell line. High expression of STAT3 or YAP1 predicted worse progression-free survival (hazard ratio [HR] = 3.02, 95% CI = 1.54 to 5.93, P = .001, and HR = 2.57, 95% CI = 1.30 to 5.09, P = .007, respectively) in an initial cohort of 64 EGFR-mutant NSCLC patients treated with firstline EGFR TKIs. Similar results were observed in a validation cohort.Conclusions: Our study uncovers a coordinated signaling network centered on both STAT3 and Src-YAP signaling that limits targeted therapy response in lung cancer and identifies an unforeseen rational upfront polytherapy strategy to minimize residual disease and enhance clinical outcomes.

This paper presents a study of industry equipment electrical responses under voltage dips with different magnitude, duration and point-on-wave initiation. The analysis provides electrical sensitive curves for safe and reliable operation based on experimental tests performed over real AC-contactors, ground fault relays, variable speed drives, industrial computers and lighting loads. The presence of a relevant scatter-band where the sensitivity of some equipments in the presence voltage dips responds in a stochastic manner is analysed. The effect of the working temperature of the device is studied as well. A detailed description of the testing procedure is provided, proposing additional voltage dip parameters to characterize the equipment sensitivity in detail. In this way, existing standards and previously published contributions are reviewed and compared with the obtained results. The obtained results are useful for OEMs, Utilities as well as Uninterruptible Power Supply Manufacturers; where a more precise counting of equipment failures due to voltage dips is essential for an economic and technical assessment of power quality.

Lung cancer is a lethal disease, and most cases have already disseminated at the time of diagnosis. Driver mutations in the EGFR tyrosine kinase domain (mainly deletions in exon 19 and L858R mutation in exon 21) have been identified in lung adenocarcinomas, mostly in never smokers, at frequencies of 20–60%. The EGFR tyrosine kinase inhibitors (TKIs) gefitinib or erlotinib attain a response rate of 70% and progression-free survival of 9–13 months, although there are subgroups of patients with long-lasting remissions. No significant correlation between EGFR overexpression and response to treatment has been found, while controversial results have been reported regarding EGFR gene amplification. The pretreatment presence of the T790M mutation, initially identified as an acquired resistance mutation to treatment with EGFR TKIs, has also been reported and may indicate a genetically distinct disease. Finally, other genetic factors, such as mRNA expression of BRCA1 and components of the NF-κB pathway, can modulate response to EGFR TKIs in EGFR-mutated patients.

The available wind speed potential strongly depends on the existing wind profiles at different locations. In this way, the exploitation of this resource can be determined by the wind turbine power curve, relating both wind speed at the rotor hub and electric power from the wind turbine. According to the current international Standard for power performance, the wind speed at hub height is the main input parameter for the characterization of the power curve, being considered the air density as a secondary factor. Under this framework, the wind speed is considered as constant over the whole turbine swept rotor area. This assumption can achieve high level of accuracy for small wind turbines. However, current wind turbines are characterized by a large rotor diameter, being exposed to remarkable variations in wind speed with height. Moreover, this Standard will be modified in a short-term, with the aim of taking into account several wind measurements within the swept rotor area This paper reviews recent solutions to measure wind speed and direction based on remote sensing systems: SODAR and LIDAR technology. These systems are able to collect vertical wind profiles over a wide range of heights, providing a significant advantage in comparison with conventional approaches.

The majority of non-small-cell lung cancer (NSCLC) patients present with locally advanced (35%) or metastatic disease (40%); in this setting, it is of the utmost importance to balance efficacy with toxicity. However, with platinum combinations, survival has reached a " plateau" , with median overall survival times of a mere 10-12 months, making it mandatory to search for new strategies and to identify more effective treatment. Molecular characteristics can be more informative than clinical features in predicting clinical benefit, and the identification of molecular markers can help define subgroups of patients who are likely to respond to different treatments, thus avoiding unnecessary toxicities and costs and providing the maximum benefit to each patient. Here we review research on biomarker assessment that was presented during the Molecular Biology Workshop held in Palma de Mallorca on 25 November 2010, during the Fifth Educational Symposium of the Spanish Lung Cancer Group. © 2011.

Purpose: Advanced non–small-cell lung cancer (NSCLC) patients harboring epidermal growth factor receptor (EGFR) mutations (deletion in exon 19 or L858R) show an impressive progression-free survival of 14 months when treated with erlotinib. However, the presence of EGFR mutations can only imperfectly predict outcome. We hypothesized that progression-free survival could be influenced both by the pretreatment EGFR T790M mutation and by components of DNA repair pathways. Experimental Design: We assessed the T790M mutation in pretreatment diagnostic specimens from 129 erlotinib-treated advanced NSCLC patients with EGFR mutations. The expression of eight genes and two proteins involved in DNA repair and four receptor tyrosine kinases was also examined. Results: The EGFR T790M mutation was observed in 45 of 129 patients (35%). Progression-free survival was 12 months in patients with and 18 months in patients without the T790M mutation (P = 0.05). Progression-free survival was 27 months in patients with low BRCA1 mRNA levels, 18 months in those with intermediate levels, and 10 months in those with high levels (P = 0.02). In the multivariate analysis, the presence of the T790M mutation (HR, 4.35; P = 0.001), intermediate BRCA1 levels (HR, 8.19; P < 0.0001), and high BRCA1 levels (HR, 8.46; P < 0.0001) emerged as markers of shorter progression-free survival. Conclusions: Low BRCA1 levels neutralized the negative effect of the T790M mutation and were associated with longer progression-free survival to erlotinib. We advocate baseline assessment of the T790M mutation and BRCA1 expression to predict outcome and provide alternative individualized treatment to patients based on T790M mutations and BRCA1 expression. Clin Cancer Res; 17(5); 1–9. ©2011 AACR.

The survival of advanced non-small-cell lung cancer patients is short in spite of advances in new combination chemotherapy regimens. The benefit of adding antiangiogenic drugs and/or EGFR inhibitors is unclear. For the vast majority of patients without EGFR mutations, treatment approaches based on customization should be pursued. BRCA1 is central to the repair of DNA damage and is an important modulator of the differential effect of chemotherapy. Retrospective and prospective data indicate that low BRCA1 mRNA levels predict better response and survival when patients are treated with cisplatin, non-taxane combinations. For an important subgroup of patients with EGFR mutations, selective treatment with EGFR tyrosine kinase inhibitors is a major advance, with a dramatic impact on clinical outcomes. In a prospective study of customized erlotinib [1] , overall response rate was 70% (including 12% complete responses), median progression free survival was 14 months (even longer in women and in patients with del 19), 20% of patients were disease-free at three years, and median survival was 27 months. Nonetheless, these clinical outcomes fall short of curability and continuous treatment with erlotinib or gefitinib is required. It is plausible that several genetically defined subclasses of EGFR mutations could help to improve current clinical outcomes by combining erlotinib or gefitinib with other targeted drugs.

Activating mutations in the form of deletions in exon 19 (del 19) or the missense mutation L858R in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) predict outcome to use of EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib. Pooled data from several phase II studies show that gefitinib and erlotinib induce responses in over 70% of NSCLC patients harboring EGFR mutations, with progression-free survival (PFS) ranging from 9 to 13 months. Two studies in Caucasian and Asian patients have confirmed that these subgroups of patients attain PFS up to 14 months. These landmark outcomes have been accompanied by new challenges, primarily the additional role of chemotherapy and the management of tumors with the secondary T790M mutation that confers resistance to EGFR TKIs. Mechanisms of resistance to reversible EGFR TKIs should be further clarified and could be related to modifications in DNA repair.

Background: Surgical resection remains the best treatment option for early-stage endometrial (CE), colorectal (CRC) and lung cancer (LC) patients. However, a proportion of patients (p) develop tumor recurrence, even after curative resection. Peritoneal or pleural lavages may be performed during surgery but little data is available about their potential as liquid biopsies with predictive value for recurrence. This proof-of concept study aimed to determine if somatic mutations can be detected in these lavages. Methods: We analyzed mutations in paired surgical biopsies, blood and peritoneal or pleural lavages supernatants in an early-stage cohort of 25 EC, 12 CRC and 19 LC p. Molecular characterization of surgical biopsies was performed using the GeneReader NGS platform (Qiagen) and the Actionable Insight Tumor Panel, which includes 12 genes (KRAS, NRAS, KIT, BRAF, PDGFRA, ALK, EGFR, ERBB2, PIK3CA, ERBB3, ESR1, and RAF1). cfDNA was purified from blood and lavages using an automatic extractor and the hotspot mutations identified in tumor tissue were tested using a sensitive Taqman assay for codons 12, 13, and 61 in KRAS, codons 542, 545 and 1047 in PIK3CA and codon 600 in BRAF. Results: NGS of tumor biopsies revealed hotspot KRAS, PIK3CA or BRAF mutations in a total of 11/25 (44%) early-stage EC p, 7/12 (58%) early-stage CRC p and 6/19 (32%) early-stage LC p. In peritoneal lavages, hotspot mutations were found in 6/11 (55%) EC p and 2/7 (29%) CRC p and, in pleural lavages, hotspot mutations appeared in 2/6 (33%) LC p. Furthermore, 2/11 (18%) blood samples from EC p, 1/7 (14%) blood samples from CRC p and 1/6 (17%) blood samples from LC p were positive. Conclusions: Our study reveals that mutational analysis in peritoneal or pleural lavages from early stage tumors is feasible and that mutations are more frequently detected in lavages than in blood. Further research will determine if positivity in blood or lavages is associated with early relapse. Citation Format: Núria Jordana Ariza, Mónica Garzón Ibañez, Alejandro Martinez-Bueno, Sonia Gatius Caldero, Clara Mayo de las casas, Ana Velasco, Maria Ruiz, Berta Roman Canal, Jordi Bertran Alamillo, Sonia Rodríguez Muñoz, Raquel Campos Fuentes, Beatriz García, Ariadna Balada Bel, Sergi Villatoro, Miguel Ángel Molina-Vila, Xavier Matias-Guiu. Utility of peritoneal and pleural lavages for detection of somatic alterations: A proof-of-concept study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3643.

Background: Non-small cell lung cancer (NSCLC) tumors harboring mutations in the EGF receptor (EGFR) ultimately relapse to therapy with EGFR tyrosine kinase inhibitors (TKIs). S49076 is a potent ATP-competitive tyrosine kinase inhibitor of MET, AXL/MER and FGFR1/2/3 currently in a phase I clinical trial in combination with Gefinitib in NSCLC resistant to EGFR inhibitors. Methods: We generated 6 resistant lines by treating EGFR-mutated, TKI sensitive PC9 cells with increasing concentrations of gefitinib (GR1-5) or erlotinib (ER). The six lines conserved the exon 19 deletion but the T790M resistance mutation emerged in two of them (GR1, GR4), which remained sensitive to osimertinib. Six additional cell lines resistant to osimertinib were derived from GR1 and GR4 by exposure to increasing concentrations of the inhibitor. The 12 resistant cell lines were genotyped and characterized for AXL, GAS6, MET, FGFR1 and FGFR2 expression by qRT-PCR, immunohistochemistry and Western blotting. The effects of S49076 and the aurora kinase B (AURKB) inhibitor barasertib were analyzed by MTT, flow cytometry and immunocytochemistry. Western blotting and ELISA of key signal transduction proteins and gene silencing was used to gain insight in the mechanism of action of the drug. Results: Several mechanisms associated with resistance to EGFR TKIs were represented in our panel of resistant cell lines and they frequently co-occurred, including AXL/GAS6 and FGFR1 overexpression, MET activation or emergence and loss of the p.T790M. When using in vitro models, we discovered that the cell lines of our panel were relatively insensitive to single-agent treatment with inhibitors of AXL, MET or FGFR1. In contrast, resistant cells not harboring the p.T790M were sensitive in vitro and also in xenografts to S49076, a drug targeting AXL, MET and FGFR1/2/3. At higher doses (above 300 nM in cultured cells), S49076 blocks AURKB, which could therefore be considered as a further target of the compound. Since partial silencing of AURKB rendered cells resistant to S49076, we investigated the role of this protein. We found that the levels of phospho-histone H3 (pH3), but not Ki67, were increased in all the EGFR TKI resistant cells of our panel, revealing a widespread AURKB activation not related to increased cell proliferation. Treatment with S49076 or barasertib down-regulated pH3 and induced G1/S arrest and polyploidy. In cells harboring the p.T790M, polyploid cells underwent senescence. However, in absence of the resistance mutation, polyploidy was followed by cell death. Conclusions: AURKB is a novel target in non-T790M NSCLC with acquired resistance to first and third line EGFR TKIs. Multitargeted agents such as S49076, which inhibit not only AXL, MET or FGFR1 but also AURKB, might be more effective in this setting than agents targeting the receptor tyrosine kinases alone. Citation Format: Jordi Bertran-Alamillo, Valérie Cattan, Marie Schoumacher, Jordi Codony-Servat, Frederique Cantero, Ruth Roman, Sonia Rodriguez, Ana Gimenez-Capitan, Josep Castellvi, Cristina Teixido, Rafael Rosell, Miguel A. Molina-Vila. Aurora B, a potential new target in non-T790M lung cancer cells with acquired resistance to anti-EGFR therapy, is effectively blocked by the MET/AXL/FGFR inhibitor S49076 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3924.

Background: Immunization against Epidermal Growth Factor (EGF) has demonstrated clinical efficacy in a phase III trial including unselected NSCLC patients. We are currently analyzing if anti-EGF antibodies generated by vaccination (anti-EGF VacAbs) showed antitumor activity in EGFR-mutant, Kras-mutant (mut) and Anaplastic Lymphoma Kinase (ALK) translocated non-small cell lung cancer cells (NSCLC), alone or in combination with tyrosine kinase inhibitors (TKI). In an EGFR-mut and in an ALK translocated cell lines we are also studying if the anti-EGF VacABs can delay the emergence of resistance to TKIs. Methods: Anti-EGF VacAbs were obtained by immunizing rabbits with recombinant EGF. Cell lines were treated with anti-EGF VacABs alone and in combination with TKIs in EGFR (PC9, H1975) and ALK translocated (H3122, H2228) cell lines. In KRAS-mut cells (A549, H23) the combination was with chemotherapy agents. Cell viability was analyzed by MTT and apoptosis and cell cycle by fluorescence-activates cell sorting analysis (FACS). Changes of total and phosphorylated proteins were determined by Western blot. Sera from advanced NSCLC patients immunized with anti-EGF vaccine were also tested Results: Anti-EGF VacAbs suppressed EGF-induced cell proliferation and inhibited downstream EGFR signaling in all cell lines tested. In combination, the anti-EGF VacAbs significantly enhanced the antitumor activity of gefitinib, erlotinib, osimertinib and afatinib in EGFR-mut cells, potentiated Erk ½ phosphorylation, arrested cell cycle progression and increased apoptosis. In addition, anti-EGF VacAbs blocked the activation of STAT3, downregulated the expression of proteins related to EGFR resistance, such as AXL, and significantly delayed the emergence in vitro of clones resistant to EGFR TKI. Sera from patients vaccinated against EGF also suppressed Erk ½ phosphorylation. Results for the combination of ALK TKIs and chemotherapy agents in ALK translocated and KRAS-mut cell lines will be presented at the meeting. Conclusions: Anti-EGF VacAbs decreased cell proliferation and inhibited the activation of EGFR-pathway downstream proteins in EGFR-mut, KRAS-mut and ALK translocated cell lines. Also, they potentiate the effects of TKIs and prevent the emergence of resistance in EGFR-mut NSCLC cells. Two clinical trials are currently testing anti-EGF vaccination in EGFR-wt and EGFR-mut advanced NSCLC patients. Citation Format: Silvia Garcia-Roman, Jordi Codony-Servat, Miguel Angel Molina-Vila, Jordi Bertran-Alamillo, Ana Gimenez-Capitán, Niki Karachaliou, Erik d'Hondt, Rafael Rosell. Antitumor effects of anti-EGF antibodies generated by vaccination in NSCLC tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 837.

3076Background: In advanced cancer, the overall response rate (ORR) following anti-PD1 monotherapy is variable (0% to 50%). Here, we hypothesized that this observation is partly explained by differ...

Purpose of review Classic activating mutations in the form of deletions in exon 19 or a missense mutation L858R in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) predict dramatic responses to EGFR tyrosine kinase inhibitors such as gefitinib and erlotinib. We review here the clinical benefits of targeted therapy with erlotinib and gefitinib in white and Asian nonsmall-cell lung cancer patients. Recent findings Two separate analyses of pooled data from small phase II prospective studies show that therapy with gefitinib and erlotinib induces responses in over 70% of nonsmall-cell lung cancer patients harboring classic EGFR mutations, with progression-free survival ranging from 9 to 13 months and median survival of around 23 months. Two separate studies in white and Asian patients have recently confirmed that these subgroups of patients attain response rates of 70% with erlotinib and gefitinib, including complete responses, progression-free survival of up to 14 months, and median survival of up to 27 months. The serial monitoring of EGFR mutations in the blood will permit the assessment of molecular responses and be an important tool for the surveillance of clinical progression. Summary Nonsmall-cell lung cancer with EGFR mutations constitute a new entity with a unique opportunity for further refinement of different genetic subgroups among patients with EGFR mutations, requiring different personalized treatment strategies. Despite the impressive outcomes attained with EGFR tyrosine kinase inhibitors, patients with EGFR mutations at present require continuous treatment, and only a fraction of these patients will reach sustainable long-term survival.

Key "driver" mutations have been discovered in specific subgroups of non-small-cell lung cancer (NSCLC) patients. Activating mutations in the form of deletions in exon 19 (del 19) or the missense mutation L858R in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) predict outcome to EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib. Pooled data from several phase II studies show that gefitinib and erlotinib induce responses in over 70% of NSCLC patients harbouring EGFR mutations, with progression-free survival (PFS) ranging from 9 to 13 months and median survival of around 23 months. Two studies in Caucasian and Asian patients have confirmed that these subgroups of patients attain response rates of 70% with erlotinib and ge- fitinib, including complete responses, PFS up to 14 months and median survival up to 27 months. These landmark outcomes have been accompanied by new challenges: the additional role of chemotherapy and the management of tumours with the secondary T790M mutation that confers resistance to EGFR TKIs. Mechanisms of resistance to reversible EGFR TKIs should be further clarified and could be related to modifications in DNA repair. The presence of double mutations (T790M plus either L858R or del 19) at the time of diagnosis could be much more frequent than originally thought. The sensitivity to EGFR TKIs could be greatly influenced by the expression of genes involved in the repair of DNA double-strand breaks by homologous recombination and non-homologous end joining.

The presence of somatic mutations of the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene in patients with advanced nonsmall cell lung cancer (NSCLC) correlates with a good response to tyrosine kinase inhibitors. The usefulness of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) for the detection of EGFR mutations in cells recovered from malignant mediastinal nodes in patients with NSCLC was assessed. All patients with lung adenocarcinoma or unspecified NSCLC referred for staging with EBUS-TBNA were included. Nodes with a short-axis diameter of >5 mm were sampled, and genomic DNA from metastatic tumour cells was obtained for analysis of exons 19 and 21. The impact of sampling on management was assessed. EGFR gene analysis of the EBUS-TBNA sample was feasible in 26 (72.2%) out of the 36 patients with lymph node metastasis. Somatic mutations of the EGFR gene were detected in tissue obtained through EBUS-TBNA in two (10%) out of 20 patients with metastasic lung adenocarcinoma. Malignant tissue samples obtained by EBUS-TBNA from patients with nodal metastasis of NSCLC are suitable for the detection of EGFR mutations in most cases, and this technique demonstrates mutated neoplastic cells in a tenth of patients with adenocarcinoma.

Evaluation of: Rebouissou S, Amessou M, Couchy G et al.: Frequent in-frame somatic deletions activate gp130 in inflammatory hepatocellular tumours. Nature 457, 200–204 (2009). Recently, in-frame somatic deletions activating gp130, the receptor of IL-6, have been identified in inflammatory hepatocellular adenomas. This is a novel class of mutation in a receptor that is central to the IL-6/gp130/STAT3 pathway, which is relevant in a variety of cancers. STAT3 is a cancer transcriptional node activating several prosurvival and proliferation genes. The activation of IL-6/gp130 also promotes the activation of the RAS and PI3K pathways. Intriguingly, the overexpression of IL-6 and STAT3 has been observed in lung adenocarcinomas, including those with EGF-receptor mutations. Further clinical research should be performed on the IL-6/gp130/STAT3 signaling axis, since it constitutes an autocrine and paracrine amplification loop in several tumors, including those with RAS mutations.

Background Detection of epidermal growth factor receptor (EGFR) mutations in advanced non-small cell lung cancer (NSCLC) patients has relied on DNA purification from biopsies, amplification, and sequencing. However, the number of tumor cells in a sample is often insufficient for EGFR assessment. Methods We prospectively screened 1380 NSCLC patients for EGFR mutations but found that 268 were not evaluable because of insufficient tumor tissue. We therefore developed and validated a method of detecting EGFR mutations in these samples. Tumor cells were microdissected into polymerase chain reaction buffer and amplified. EGFR mutations were detected by length analysis of fluorescently labeled polymerase chain reaction products and TaqMan assay. Results We determined EGFR status in 217 (81%) of the 268 primary NSCLC samples not evaluable in our original study—fresh and paraffin-embedded with less than 150 cells. Exon 19 deletions were detected in 11.5% of patients and exon 21 L858R mutations in 5.5%. In addition, the exon 20 T790M mutation was detected in 6 of 15 (40%) patients at the time of progression to erlotinib. The primary, sensitive mutation was present in all tumor cells, whereas the T790M mutation was absent in some groups. Conclusions The method presented here eliminates the need for DNA purification and allows for detection of EGFR mutations in samples containing as few as eight cancer cells.

Objective: Cost-effectiveness analysis of the major tobacco control tools medium long term. Method: This is a retrospective, descriptive study of the results obtained after analyzing the sample of all patients who attended the Unit Specialized Treatment Smoking during two years. Without drug treatment: the costs and effectiveness of the four options used in our Unit for smoking cessation compared: 1) Bupropion+ NRT, 2) Varenicline, 3) Varenicline and NRT and 4) No pharmacology treatment. Costs and efficiency: Efficiencies rates for comparison are the result of our study for the 4 options. All costs are expressed in euros, both costs and an effect beyond the first year rate of 3.5% per annum is deducted. Results: Of the 559 patients, 32.7% received no treatment. 34.5% (n=194) of patients was treated with NRT (gum, patches or in combination with varenicline). Varenicline is the second most frequently used drug with 22.9% and, finally, bupropion (9.8%). The analysis results show that the greater efficacy of varenicline is a better cost/effectiveness (C/E) in the treatment of smoking medium to long term that comes to fully offset the higher cost of treatment compared to the other options. So both varenicline monotherapy as associated with TSN have average costs of € 2.491,21 and € 2.432,22 per patient who quit smoking compared to the other options with Bupropion and TSN 4.136,55 € or drug treatment to 5.783,88 € in a perspective of two years. Conclusion: The option of giving Varenicline is a better cost/effectiveness (C/E) in the treatment of smoking-half longer than the other options.

Objective: To assess the impact of a telemedicine project that uses the Medtep platform in patients diagnosed with asthma. Method: An asthma consultation in the Integrated Care Management center of Albacete, in collaboration with the Pneumology department, which treat patients with possible asthma and/or acute asthma. In March of 2015 a group of 25 patients diagnosed with asthma, previously invited to participate in the study, were selected. Several training meetings were scheduled to assist them in using a web portal to control their asthma. They were also delivered a Peak-Flow and taught how it works. For 6 months clinical evolution and web portal entrances to follow. The data which was collected: smoking, how long they were diagnosed with asthma, asthma attacks, hospitalizations. Treatments- Evolution, According to patient level. Results: Out of the total 25 patients diagnosed, 17 (67%) were men and 8 (32%) women. The patient’s average age is of 34 years old. The total amount of smokers were 5 patients (20%), 3 of the remaining patients had never smoked. The average cigarette consumption of the smokers was of 2 packs a day. 42% had been diagnosed with asthma for less than 1 year; 20% between 1 and 5 years and the rest of patients had been diagnosed for over 5 years. Before entering the study, 50% had to attend emergency visits at least once (a year?), and some patients as much as 4 times in the last year. After entering the study, none of the patients had to attend ER visits. 80% of the total patients entered their health related data on a daily basis, as well as medication registers, which was above 95% in those who assiduously used the system. The total Peak flow of the patients saw an increase of 7% over baseline. More than 50% of patients increased their treatment before entering the study, and none and even 25% decreased their dose of treatment. 32% of patients have improved their level depending on the base treatment. Conclusions: Improvements in asthma control were achieved in patients who received Medtep Asthma support for 6 months, decreasing assistance in emergency services and, as a result, requiring less treatment.

Submit Manuscript | http://medcraveonline.com The trial, which is being led by Dr. Ele-na Rodriguez has involving various services such as ophthalmology, hematology and neurophysiology. Seven patients are the first who are benefiting from this experiment anesthesia and it is being held in the unit cell production of the Arrixaca. Patient recruitment for this first phase Zo eat it in October 2014 and were 74 patients on requesting information. Finally, after conducting the necessary studies are seven patients who met all the criteria for inclusion in this trial. Those responsible for the Ministry of Health subrayan reconcile the need for research as well as assistance measures to make progress in the possible origin of the disease and seek treatment alternati -vos and effective. These experiments are seGuidos with great interest by the Association of Retina Affected Region of Murcia (Retimur).

Previous studies have indicated that EGFR exon 19 deletions in non-small cell lung cancer (NSCLC) are associated with better outcomes to tyrosine kinase inhibitors (TKIs) than the L858R mutation. This study aimed to evaluate whether T790M, a resistant mutation, is more likely to coexist with L858R mutation than with exon 19 deletions in pretreatment NSCLC patients.We searched MEDLINE and EMBASE up to Nov 30th, 2015 to identify randomized controlled trials (RCTs) and observational studies that reported pretreatment T790M and EGFR-activating mutation. A meta-analysis was performed using a random-effects model. The primary outcome was odds ratio (OR) of pretreatment T790M mutation in NSCLC co-existing with L858R mutation and exon 19 deletions. Stratified analysis was performed based on sensitivity of mutation detection methods for T790M.We identified 15 observational studies and 3 RCTs for analysis. Pretreatment T790M was more frequent in L858R than in exon 19 mutated patients. The association of T790M and L858R was statistically significant in observational studies (OR, 1.65, 95% CI, 1.17-2.32), with less precision in RCTs (OR, 1.84, 95% CI, 0.96-3.52). In the stratified analysis based on the sensitivity of the mutation detection methods, the association was observed in the studies using intermediately (detection limit5% and ≥ 0.1%; OR, 2.23, 95% CI, 1.19-4.17) and highly sensitive methods (detection limit0.1%; OR, 1.74, 95% CI, 1.10-2.73), but not in those using low sensitivity methods (detection limit5%; OR, 1.28, 95% CI, 0.74-2.23).Pretreatment EGFR T790M mutation is more likely to coexist with L858R mutation than with exon 19 deletions in NSCLC. This association was observed only in studies using sensitive mutation detection methods (5%).

Importance The EURTAC trial demonstrated the greater efficacy of erlotinib compared with chemotherapy for the first-line treatment of European patients with advanced non–small-cell lung cancer (NSCLC) harboring oncogenic epidermal growth factor receptor ( EGFR ) mutations (exon 19 deletion or L858R mutation in exon 21) in tumor tissue. Objective To assess the feasibility of using circulating free DNA (cfDNA) from blood samples as a surrogate for tumor biopsy for determining EGFR mutation status and to correlate EGFR mutations in cfDNA with outcome. Design, Setting, and Participants This prespecified analysis was a secondary objective of the EURTAC trial using patients included in the EURTAC trial from 2007 to 2011 with available baseline serum or plasma samples. Patients had advanced NSCLC, oncogenic EGFR mutations in the tumor, and no prior chemotherapy for metastatic disease and were treated with erlotinib or chemotherapy. EGFR mutations were examined in cfDNA isolated from 97 baseline blood samples by our novel peptide nucleic acid–mediated 5´ nuclease real-time polymerase chain reaction (TaqMan) assay. Main Outcomes and Measures Overall survival (OS), progression-free survival (PFS), and response to therapy were correlated with type of EGFR mutations in cfDNA. Results In samples from 76 of 97 (78%) patients with usable blood samples, EGFR mutations in cfDNA were detected. Median OS was shorter in patients with the L858R mutation in cfDNA than in those with the exon 19 deletion (13.7 [95% CI, 7.1-17.7] vs 30.0 [95% CI, 19.3-37.7] months; P EGFR mutations in cfDNA identified the L858R mutation in tumor tissue or in cfDNA as a marker of shorter OS (hazard ratio [HR], 2.70 [95% CI, 1.60-4.56]; P P = .008). For patients with the L858R mutation in tissue, median OS was 13.7 (95% CI, 7.1-17.7) months for patients with the L858R mutation in cfDNA and 27.7 (95% CI, 16.1-46.2) months for those in whom the mutation was not detected in cfDNA (HR, 2.22 [95% CI, 1.09-4.52]; P = .03). In the multivariate analysis of the 76 patients with EGFR mutations in cfDNA, only erlotinib treatment remained an independent predictor of longer PFS (HR, 0.41 [95% CI, 0.23-0.74]; P = .003). Conclusions and Relevance The peptide nucleic acid–mediated 5´ nuclease real-time polymerase chain reaction (TaqMan) assay used in this study can be used to efficiently assess EGFR mutations in cfDNA. The L858R mutation in cfDNA may be a novel surrogate prognostic marker. Trial Registration clinicaltrials.gov Identifier:NCT00446225

This paper describes an alternative approach based on symbolic computations to simulate wind turbines equipped with Doubly–Fed Induction Generator (DFIG). The actuator disk theory is used to represent the aerodynamic part, and the one-mass model simulates the mechanical part. The 5th–order induction generator is selected to model the electric machine, being this approach suitable to estimate the DFIG performance under transient conditions. The corresponding non–linear integro-differential equation system has been reduced to a linear state-space system by using an ad-hoc local linearization This novel Symbolic Computation (SYMB) method has been implemented by using two different software-packages, with the purpose of solving simultaneously a remarkable number of individual wind turbines models submitted to different wind speed profiles and/or grid voltage waveforms.The obtained results are compared with traditional Finite Difference Discretization (FDD) methods, widely proposed for this type of studies. The results offer a good agreement between the proposed SYMB method and the FDD solutions, considering variations of both wind speed profiles and electrical transient events.

Background: The HER-2 receptor undergoes a proteolytic cleavage generating an NH2-terminally truncated fragment, p95HER-2, that is membrane-associated and tyrosine-phosphorylated. We have reported that p95HER-2, but not the full-length receptor, p185HER-2, correlated with the extent of lymph node involvement in patients with breast cancer and its expression was significantly enhanced in nodal metastatic tissue. These facts suggested an important role for p95HER-2 either as a marker or cause of metastasis and poor outcome in breast cancer. In this work, we have studied the prognostic value of p95HER-2 in breast cancer.Methods: Primary breast tumor tissues (n = 483) were from surgical resections conducted in hospitals in two different countries: the U.S. (n = 334) and Spain (n = 149). HER-2 protein forms, including p185HER-2 and p95HER-2, were examined in extracts of primary breast tumors by Western blot analysis. The levels of the two forms (high or low) were tested for association with other clinicopathologic factors and for correlation with disease-free survival.Results: The median follow-up was 46 months. A high level of p95HER-2 in primary tumor tissue correlated with reduced 5-year disease-free survival (hazard ratio, 2.55; 95% confidence interval, 2.13-8.01; P < 0.0001). The median time for disease-free survival was 32 versus 139 months in patients with low levels of p95HER-2. In comparison, high levels of the full-length p185HER-2 did not significantly correlate with poor outcome (P > 0.1). Multivariate analysis revealed that high p95HER-2 was an independent predictor of disease-free survival (hazard ratio, 1.59; 95% confidence interval, 1.246-1.990; P = 0.0004).Conclusions: p95HER-2 expression is an independent prognostic factor in breast cancer and defines a group of patients with HER-2-positive breast cancer with significantly worse outcome.

Background: Programmed death-ligand 1 (PD-L1) may be induced by oncogenic signals or can be upregulated via interferon gamma (IFN-γ). We have explored whether the expression of IFNG, the gene encoding IFN-γ, is associated with clinical response to the immune checkpoint blockade in non-small cell lung cancer (NSCLC) and melanoma patients. The role of inflammation-associated transcription factors STAT3, IKBKE, STAT1 and other associated genes has also been examined. Methods: Total RNA from 17 NSCLC and 21 melanoma patients was analyzed by quantitative reverse transcription PCR. STAT3 and Rantes, YAP1 and CXCL5, DNMT1, RIG1 and TET1, EOMES, IFNG, PD-L1 and CTLA4, IKBKE and NFATC1 mRNA were examined. PD-L1 protein expression in tumor and immune cells and stromal infiltration of CD8+ T-cells were also evaluated. Progression-free survival and overall survival were estimated. Results: A total of 17 NSCLC patients received nivolumab and 21 melanoma patients received pembrolizumab. Progression-free survival with nivolumab was significantly longer in NSCLC patients with high versus low IFNG expression (5.1 months versus 2 months, p = 0.0124). Progression-free survival with pembrolizumab was significantly longer in melanoma patients with high versus low IFNG expression (5.0 months versus 1.9 months, p = 0.0099). Significantly longer overall survival was observed for melanoma patients with high versus low IFNG expression (not reached versus 10.2 months p = 0.0183). There was a trend for longer overall survival for NSCLC patients with high versus low IFNG expression. Conclusions: IFN-γ is an important marker for prediction of response to immune checkpoint blockade. Further research is warranted in order to validate whether IFNG is more accurate than PD-L1.

EEG Morphology and Spectral Analysis in Attention Deficit/ Hyperactivity Disorder. Effect of Methylphenidate Treatment Introduction: Attention deficit/hyperactivity disorder (ADHD) is the most commonly diagnosed disorder in childhood. It has been described structural and functional abnormalities in the brain of these patients. However, the etiology of this disorder is still unknown. The aim of this study is to investigate cortical activity alterations in patients with attention Deficit Hyperactivity disorder (ADHD), that could help diagnose this pathology and monitor electrophysiological prognosis after medical treatment in order to improve the management of this disorder.

BIM is a proapoptotic protein that initiates apoptosis triggered by EGFR tyrosine kinase inhibitors (TKI). mTOR negatively regulates apoptosis and may influence response to EGFR TKI. We examined mRNA expression of BIM and MTOR in 57 patients with EGFR-mutant NSCLC from the EURTAC trial. Risk of mortality and disease progression was lower in patients with high BIM compared with low/intermediate BIM mRNA levels. Analysis of MTOR further divided patients with high BIM expression into two groups, with those having both high BIM and MTOR experiencing shorter overall and progression-free survival to erlotinib. Validation of our results was performed in an independent cohort of 19 patients with EGFR-mutant NSCLC treated with EGFR TKIs. In EGFR-mutant lung adenocarcinoma cell lines with high BIM expression, concomitant high mTOR expression increased IC50 of gefitinib for cell proliferation. We next sought to analyse the signalling pattern in cell lines with strong activation of mTOR and its substrate P-S6. We showed that mTOR and phosphodiesterase 4D (PDE4D) strongly correlate in resistant EGFR-mutant cancer cell lines. These data suggest that the combination of EGFR TKI with mTOR or PDE4 inhibitors could be adequate therapy for EGFR-mutant NSCLC patients with high pretreatment levels of BIM and mTOR.

Background: Rearrangements in anaplastic lymphoma kinase (ALK) gene can be detected in 5-7% of EGFR and KRAS wild-type advanced NSCLC patients (p). Fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC) are currently used for screening but are unable to identify the specific fusion partner and are unpractical to monitor clinical responses due to difficulty of obtaining rebiopsies. The RT-PCR technique has the potential to overcome this pitfall and allow patient monitorization in blood. Methods: A total of 405 formalin-fixed paraffin-embedded (FFPE) samples from advanced NSCLC were analyzed by ALK IHC (Ventana D5F3) and FISH (Vysis). Positive patients were confirmed by RT-PCR and submitted to Sanger in order to identify the variant. In a subset of 36 patients with EML4-ALK-rearranged tumors who were treated with crizotinib, fusion transcripts were analyzed by RT-PCR in mRNA purified from plasma and platelets and correlated with clinical response. Results: ALK IHC was analyzed in 405 NSCLC patients and 37 tested positive (9.1%) whereas 25 (7.7%) were identify as translocated by FISH (n=323). ALK fusion transcripts were analyzed by RT-PCR and a new fusion variant of ALK was identified. A total of 36 p benefited from crizotinib treatment, including the p with the new variant. Monitoring of EML4-ALK fusion transcripts in the plasma ad platelets of 35 ALK positive patients revealed a good correlation with clinical outcome to crizotinib treatment, with the fusion transcripts becoming undetectable in p with good clinical responses. Conclusions: Analysis of ALK fusion transcripts in mRNA purified from plasma and platelets can have a value in patients with no biopsy available and to monitor the course of the disease. Citation Format: Cristina Aguado, Cristina Teixido, Ana Gimenez-Capitan, Maria de los Llanos Gil, Sonia Rodriguez, Santiago Viteri, Niki Karachaliou, Erika Aldeguer, Vicente Peg, Lidia Alonso, Miguel Angel Molina-Vila, Rafael Rosell. Analysis of EML4-ALK fusion transcripts in plasma and platelets to monitor response to crizotinib in EML4-ALK positive non-small cell lung cancer patients (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1739. doi:10.1158/1538-7445.AM2017-1739

Background: Aberrant activity of the MET, FGFR1 and AXL receptors has been associated with the development of resistance to first, second and third generation EGFR tyrosine kinase inhibitors (TKI) in EGFR-mutated non-small cell lung cancer (NSCLC) patients. Methods: We obtained 6 resistant lines by treating EGFR-mutated (exon 19), TKI sensitive PC9 cells with increasing concentrations of gefitinib or erlotinib. The p.T790M resistance mutation emerged in two cell lines (GR1, GR4), which remained sensitive to osimertinib, a third generation EGFR TKI. Six new cell lines to resistant to “second line” osimertinib were generated from GR1 and GR4 by exposure to increasing concentrations of the inhibitor. Finally, six more cell lines resistant to “first line” osimertinib were derived from the PC9 parental cells. All resistant cell lines were genotyped for selected genes (including EGFR) and characterized for AXL, MET and FGFR1 expression and activation by Q-RT-PCR, immunohistochemistry and Western blotting. The effects of AXL (BGB324), MET (crizotinib, capmatinib) and FGFR1 (nindetanib) inhibitors on the parental and the 18 resistant cell lines were analyzed by MTT and, in some cases, by colony formation. AXL was stably silenced in some of the resistant cell lines. Results: All cell lines resistant to “first line” gefitinib, erlotinib and osimertinib maintained the exon 19 EGFR sensitizing mutation. In contrast, three of the resistant cell lines to “second line” osimertinib lost the exon 19 and the p.T790M mutations. In two more, the p.T790M dropped to low allelic fractions (1% and 0.03%). Regardless of the EGFR status, AXL overexpression was the most common event related to EGFR TKI resistance in our panel of 18 cell lines, with FGFR1 and MET overexpression or activation as less frequent events. In proliferation assays, the IC50 of the EGFR TKI resistant cell lines for BGB324 (AXL inhibitor) was indistinguishable from the IC50 of the parental, EGFR TKI sensitive cell line. Similar results were obtained in the case of capmatinib, crizotinib (MET inhibitors) and nintedanib (FGFR inhibitor). Stable silencing of AXL on some of the AXL-overexpressing resistant cell lines had no effects in terms of doubling times, morphology of cells or sensitivity to EGFR TKIs. In combination experiments, the effect of BGB and MET inhibitors was found to be additive. Conclusions: In tumor cell line models of acquired resistance to EGFR TKIs, overexpression or activation of AXL, MET and FGFR1 was not associated to sensitivity to single-agent treatment with AXL, MET or FGFR inhibitors. Multitargeted approaches might be more effective in this setting. Citation Format: Jordi Bertran-Alamillo, Miguel Angel Molina-VIla, Cristina Teixidó, Jordi Codony-Servat, Ana Giménez-Capitán, Carles Codony-Servat, Silvia García-Román, Erika Aldeguer, Sonia Rodríguez, Rafael Rosell. Tumor cells with acquired resistance to EGFR inhibitors and overexpression or activation of AXL, MET and FGFR1 are insensitive to single-agent treatment targeting AXL, MET or FGFR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3077. doi:10.1158/1538-7445.AM2017-3077

Background: Mutations in the KRAS proto-oncogene, GTPase (KRAS) are driver alterations in several tumors, such as non-small-cell lung (NSCLC) and colorectal cancer (CRC). However, their role as a prognostic marker in liquid biopsies is unclear. We studied the status of KRAS mutations in the peripheral blood of advanced NSCLC and colorectal cancer patients visited in our hospital and evaluated its potential value as a monitoring marker in the clinical practice. Methods: We developed a sensitive TaqMan assay, in the presence of a PNA clamp, for the determination of KRAS mutations (codons 12, 13 and 61) in circulating-free DNA (cfDNA). The assay detected 2.5 pg mutated DNA/µL and a ratio of KRAS mutated versus wild type allele of 1:20000 and was validated in 80 cancer patients previously genotyped in tumor tissue showing a clinical sensitivity of 72.5% and specificity of 100%. cfDNA was isolated from serum and plasma specimens, using an automatic extractor and mutational analyses were performed in quadruplicates samples. Serum and plasma samples were drawn at diagnosis, during follow-up and progression. Results: 239 NSCLC and 11 colorectal cancer patients were screened for KRAS mutations in cfDNA at presentation in the period of 2013 to 2016. In addition, 160 cfDNA samples collected during disease monitoring of 61 patients were analyzed. Paired biopsies were available from 110/250 p at diagnosis (60 positive for KRAS in FFPE and 50 WT). The KRAS mutation was detected in the cfDNA of 76.6% (46/60) p positive in tumor tissue vs. only in 1/50 WT patients where a G12C was detected in cfDNA in a consistent manner, probably associated with a heterogeneity of the tumor. In patients with no biopsy available, we detected KRAS mutations in cfDNA in 14.3% (20/140) cases at diagnosis. The most prevalent mutations observed in cfDNA were G12C (44.7 %), G12V (22.4%) and G12D (10.4%) for NSCLC patients and G13D (5.9%) for colorectal patients. The Q61H mutation was detected in the cfDNA of one NSCLC and one CRC p. Of the 67 patient’s positive in cfDNA at presentation, KRAS mutations were detected in serum and plasma in 38 patients, only in plasma in 24 and only in serum in 5. Regarding the 61 patients with serial blood samples, we observed a correlation between the KRAS mutation load in cfDNA and the course of the disease, with a decrease in patients responding to chemotherapy and an increase during disease progression. Conclusions: Our results demonstrate the feasibility of KRAS mutation analysis in the cfDNA of advanced NSCLC and colorectal patients. Analysis of KRAS mutations in the blood of mutated patients can have a value in patients with no biopsy available and to monitor the course of the disease. Citation Format: Mónica Garzón Ibañez, Clara Mayo de las casas, Nùria Jordana Ariza, Ariadna Balada Bel, Beatriz Garcia, Sergio Villatoro, Jordi Bertrán-Alamillo, Alejandro Martinez-Bueno, Santiago Viteri Ramirez, Ana Pérez Rosado, Daniela Morales Espinosa, Maria José Catalán, Niki Karachaliou, Miguel Ángel Molina-Vila, Rafael Rosell. Routine testing for KRAS mutations in cfDNA from blood of advanced cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5688. doi:10.1158/1538-7445.AM2017-5688

Background: Stand alone tests such as PCR-derived techniques, FISH or IHC are usually employed to determine clinically relevant alterations in non-small cell lung cancer (NSCLC). However, they target single genes and proteins. Mutiplex techniques can reduce the turnaround time and quantity of sample in this setting, but require a careful validation. Methods: A total of 41 cfDNA samples from serum and plasma from advanced NSCLC p were analyzed with the Actionable Insights Tumor Panel, which covers mutations in 15 clinically relevant genes, using the Gene Reader platform (Qiagen). The samples had been previously genotyped for EGFR, KRAS and BRAF mutations by stand alone, PNA-Taqman assays. Paired biopsies were available in 37 cases. The remaining 4 corresponded to p.T790M-positive blood samples of p progressing to EGFR TKIs. Results: Of the 41 samples taken into the GeneReader workflow, some had a DNA input concentration below specifications, in spite of this limitation, good results were obtained. 14 mutations were fully concordant between tissue, Taqman and GeneReader and the four p.T790M mutations were concordant between Taqman and GeneReader. Five mutations present in tissue were detected by GeneReader and not by Taqman and 11 mutations detected by Taqman were below the 1% detection threshold of GeneReader. Finally, 12 mutations present in tissue were not detected in cfDNA by any of the assays. Concordance between the stand alone tests and the Gene Reader in cfDNA was 64%, raising to 84% if mutations Conclusions: Application of NGS to liquid biopsies is challenging and requires a careful validation. However, once fully validated, NGS will probably become the methodology of choice for cfDNA analysis in NSCLC patients at presentation and at progression. Citation Format: Clara Mayo de las Casas, Mónica Garzón, Nuria Jordana Ariza, Ariadna Balada, Jordi Bertran-Alamillo, Beatriz García, Sergio Villatoro, Erika Aldeguer, Sonia Rodriguez, Raquel Campos, Santiago Viteri Ramirez, Maria Gonzalez-Cao, Niki Karachaliou, Rafael Rosell Costa, Miguel Angel Molina-Vila. Next generation sequencing of circulating-free DNA from advanced non small cell lung cancer patients using Gene Reader® [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5698. doi:10.1158/1538-7445.AM2017-5698

11504 Background: PD-L1 can be induced by oncogenic signals or up-regulated via INFG in a STAT1- and NFκB-dependent manner. STAT3 opposes STAT1-mediated anti-tumor immune responses. I kappa B kinase epsilon (IKBKE) is an interferon signaling inducer. We explored whether INFG expression in pre-treatment tumors is associated with to the efficacy of ICB in NSCLC and melanoma patients. The role of inflammation-associated transcription factors STAT3, IKBKE and STAT1 was also examined. Methods: Total RNA from 17 NSCLC and 21 melanoma patients, was analyzed by qRT-PCR. INFG, STAT3, IKBKE, STAT1 and PD-L1 mRNA were examined. PD-L1 protein expression in tumor and immune cells was evaluated (Ventana SP142 assay). Progression free survival (PFS) and overall survival (OS) were estimated. Results: 17 previously treated NSCLC patients received nivolumab; 71% lung adenocarcinoma, 71% male, 53% smokers, 35% KRAS mutant, 88% EGFR wild-type (wt). 21 previously treated melanoma patients received pembrolizumab; 67% male, 67% BRAF wt. PFS to nivolumab was significantly longer in NSCLC patients with high vs. low INFG expression (5.12 vs. 2mo, p = 0.0124). PFS to pembrolizumab was significantly longer in melanoma patients with high vs. low INFG expression (4.99 vs. 1.86mo, p = 0.0099). Significantly longer OS was observed for melanoma patients with high vs. low INFG expression (not reached vs. 3.10mo p = 0.0183). There was a trend for longer OS for NSCLC patients with high vs. low INFG expression (10.15 vs. 4.86mo, p = 0.0687). The other gene levels and PD-L1 protein levels in tumor and immune cells did not affect the outcome to ICB. IKBKE was positively correlated with INFG and PD-L1 expression (NSCLC Spearman’s ρ = 0.58 and 0.65; melanoma Spearman’s ρ = 0.61 and 0.59), and STAT3 expression was loosely anticorrelated with PD-L1 expression (NSCLC Spearman’s ρ = −0.21; melanoma Pearson’s ρ = −0.01). Conclusions: INFG is an important marker for qRT-PCR mediated prediction of response to ICB in NSCLC and melanoma patients. Further research is warranted in order to validate that INFG is more accurate than PD-L1.