Metabolomics is the study of metabolomes, the sets of metabolites observed in living systems. Metabolism interconverts these metabolites to provide the molecules and energy necessary for life processes. Many disease processes, including cancer, have a significant metabolic component that manifests as differences in what metabolites are present and in what quantities they are produced and utilized. Thus, using metabolomics, differences between metabolomes in disease and non-disease states can be detected and these differences improve our understanding of disease processes at the molecular level. Despite the potential benefits of metabolomics, the comprehensive investigation of metabolomes remains difficult. A popular analytical technique for metabolomics is mass spectrometry. Advances in Fourier transform mass spectrometry (FT-MS) instrumentation have yielded simultaneous improvements in mass resolution, mass accuracy, and detection sensitivity. In the metabolomics field, these advantages permit more complicated, but more informative experimental designs such as the use of multiple isotope-labeled precursors in stable isotope-resolved metabolomics (SIRM) experiments. However, despite these potential applications, several outstanding problems hamper the use of FT-MS for metabolomics studies. First, artifacts and data quality problems in FT-MS spectra can confound downstream data analyses, confuse machine learning models, and complicate the robust detection and assignment of metabolite features. Second, the assignment of observed spectral features to metabolites remains difficult. Existing targeted approaches for assignment often employ databases of known metabolites; however, metabolite databases are incomplete, thus limiting or biasing assignment results. Additionally, FT-MS provides limited structural information for observed metabolites, which complicates the determination of metabolite class (e.g. lipid, sugar, etc. ) for observed metabolite spectral features, a necessary step for many metabolomics experiments. To address these problems, a set of tools were developed. The first tool identifies artifacts with high peak density observed in many FT-MS spectra and removes them safely. Using this tool, two previously unreported types of high peak density artifact were identified in FT-MS spectra: fuzzy sites and partial ringing. Fuzzy sites were particularly problematic as they confused and reduced the accuracy of machine learning models trained on datasets containing these artifacts. Second, a tool called SMIRFE was developed to assign isotope-resolved molecular formulas to observed spectral features in an untargeted manner without a database of expected metabolites. This new untargeted method was validated on a gold-standard dataset containing both unlabeled and 15N-labeled compounds and was able to identify 18 of 18 expected spectral features. Third, a collection of machine learning models was constructed to predict if a molecular formula corresponds to one or more lipid categories. These models accurately predict the correct one of eight lipid categories on our training dataset of known lipid and non-lipid molecular formulas with precisions and accuracies over 90% for most categories. These models were used to predict lipid categories for untargeted SMIRFE-derived assignments in a non-small cell lung cancer dataset. Subsequent differential abundance analysis revealed a sub-population of non-small cell lung cancer samples with a significantly increased abundance in sterol lipids. This finding implies a possible therapeutic role of statins in the treatment and/or prevention of non-small cell lung cancer. Collectively these tools represent a pipeline for FT-MS metabolomics datasets that is compatible with isotope labeling experiments. With these tools, more robust and untargeted metabolic analyses of disease will be possible.

<p><strong>Background: </strong>Lung cancer is the leading cause of cancer-related death and one of the most common cancer types worldwide. Epidermal growth factor receptor (EGFR) has been shown to be an important therapeutic target in non-small cell lung cancer. Kirsten rat sarcoma viral oncogene homologue (KRAS) is a downstream signalling molecule in the EGFR pathway. Lung cancer patients with <em>EGFR </em>mutations respond to tyrosine EGFR inhibitor therapy, in contrast, patients with <em>KRAS </em>mutations do not benefit of such treatment.</p><p><strong>Methods: </strong>This study investigates the frequency of <em>EGFR </em>and <em>KRAS </em>mutations in non-small cell lung cancer patients. Fifty-one lung cancer patients with primary non-small cell lung cancer diagnosed between 1995 and 2005 in the Uppsala-Örebro region were analysed by Sanger sequencing and Pyrosequencing to determine the mutation status of these genes.</p><p><strong>Results: </strong>Five <em>EGFR </em>mutations were found in four patients (8%), two deletions in exon 19, one point mutation in exon 20 and two point mutations in exon 21. <em>KRAS </em>mutations were found in 12 patients (24%), ten codon 12 mutations and two codon 61 mutations.</p><p><strong>Conclusions: </strong>This study confirms previous observations regarding the frequency of <em>EGFR </em>and <em>KRAS </em>mutations in non-small cell lung cancer. Mutations in <em>EGFR </em>and <em>KRAS </em>were mutually exclusive, indicating that both mutations present relevant tumorigenic genomic aberrations.</p>

<p>This thesis investigated the predictive and the prognostic powers of angiogenesis related markers in both operable and inoperable non-small cell lung cancer (NSCLC) patients.</p><p>In the first and second study, we investigated the serological fractions of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in 2 cohorts of patients with either operable or inoperable NSCLC. </p><p>Regarding operable NSCLC, we demonstrated significant correlations between VEGF and tumour volume and overall survival. Regarding bFGF, significant correlations with recurrent disease and survival were demonstrated. VEGF and bFGF correlated to each other and with platelet counts. In multivariate analysis, bFGF proved to be a significantly independent prognostic factor.</p><p>Regarding inoperable NSCLC, we demonstrated that patients with elevated bFGF levels before any treatment and during chemotherapy had a significantly poorer survival. During chemotherapy, each rise of one unit of bFGF (ng/L) corresponded to a 4 times increased risk of death. Regarding VEGF, elevated levels after radiotherapy corresponded with better survival. All prognostic information demonstrated in this study concerned patients with a, co-sampled, normal platelet count.</p><p>In the third study, three putative markers, HER-2, EGFR and COX-2, suitable for targeted therapies in resected NSCLC were investigated in a panel of 53 tumours and further investigated for a possible correlation with microvessel density. We demonstrated that HER-2 and COX-2 were mainly expressed in adenocarcinomas, whereas EGFR was only expressed in squamous cell carcinomas. COX-2 showed a trend towards a correlation with microvesssel density. The expression profile, HER-2+/EGFR-, was significantly correlated to poorer survival. </p><p>In the fourth study, a predictive model for recurrences consisting of p53, CD34 and CD105, and circulating serum fractions of VEGF and bFGF, was investigated. The two endothelial markers correlated with each other. CD105 expression correlated with p53 expression. No other significant correlations between markers could be demonstrated. A significant correlation between p53 overexpression and recurrent disease was demonstrated. The mutational status could not confirm the immunohistochemical correlation between p53 and recurrences. </p><p> In conclusion, the present thesis demonstrates that the angiogenic factors VEGF and bFGF analysed in sera have both predictive and prognostic information when measured in operable and inoperable NSCLC. Since HER-2 is overexpressed in NSCLC and linked with prognostic information, this marker might be a suitable target for therapy in NSCLC. Furthermore, in patients with operable NSCLC, p53 expression status was linked with recurrent disease and mean MVD. </p>

This thesis investigated the predictive and the prognostic powers of angiogenesis related markers in both operable and inoperable non-small cell lung cancer (NSCLC) patients. In the first and second study, we investigated the serological fractions of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in 2 cohorts of patients with either operable or inoperable NSCLC. Regarding operable NSCLC, we demonstrated significant correlations between VEGF and tumour volume and overall survival. Regarding bFGF, significant correlations with recurrent disease and survival were demonstrated. VEGF and bFGF correlated to each other and with platelet counts. In multivariate analysis, bFGF proved to be a significantly independent prognostic factor. Regarding inoperable NSCLC, we demonstrated that patients with elevated bFGF levels before any treatment and during chemotherapy had a significantly poorer survival. During chemotherapy, each rise of one unit of bFGF (ng/L) corresponded to a 4 times increased risk of death. Regarding VEGF, elevated levels after radiotherapy corresponded with better survival. All prognostic information demonstrated in this study concerned patients with a, co-sampled, normal platelet count. In the third study, three putative markers, HER-2, EGFR and COX-2, suitable for targeted therapies in resected NSCLC were investigated in a panel of 53 tumours and further investigated for a possible correlation with microvessel density. We demonstrated that HER-2 and COX-2 were mainly expressed in adenocarcinomas, whereas EGFR was only expressed in squamous cell carcinomas. COX-2 showed a trend towards a correlation with microvesssel density. The expression profile, HER-2+/EGFR-, was significantly correlated to poorer survival. In the fourth study, a predictive model for recurrences consisting of p53, CD34 and CD105, and circulating serum fractions of VEGF and bFGF, was investigated. The two endothelial markers correlated with each other. CD105 expression correlated with p53 expression. No other significant correlations between markers could be demonstrated. A significant correlation between p53 overexpression and recurrent disease was demonstrated. The mutational status could not confirm the immunohistochemical correlation between p53 and recurrences. In conclusion, the present thesis demonstrates that the angiogenic factors VEGF and bFGF analysed in sera have both predictive and prognostic information when measured in operable and inoperable NSCLC. Since HER-2 is overexpressed in NSCLC and linked with prognostic information, this marker might be a suitable target for therapy in NSCLC. Furthermore, in patients with operable NSCLC, p53 expression status was linked with recurrent disease and mean MVD.

Introduction: New treatments for stage IV (adv) NSCLC have emerged this past decade. Recent pharmacoeconomic research has focused on cost of treatment, comparative costs of therapies, and cost/cost effectiveness of adding a biologic to traditional therapy. Drug cost is thought to be a primary driver of cost change in NSCLC, yet to our knowledge, characterization of the direct cost of NSCLC has not been published since the new treatments have emerged in the guidelines. Our primary objective was to characterize the direct and segmented cost of adv NSCLC from 2000-9. We also want to determine cost impact of new therapies, and cost trend from 2000-9. Methods: This PharMetrics claims database study includes diagnosed NSCLC patients [greater than or equal to] 20 yo. Small cell lung cancer was excluded. Claims were divided into disease segments and time periods representative of changes in therapy ("pre" (2000-2), "transition" (2003-5), and "current" (2006-9) periods). Descriptive statistics (median, interquartile range (IQR)), chi-square test (nominal data), and Wilcoxan rank sum tests were performed on the data. To adjust for baseline confounders, multivariate least squares regression models were created. Results: Costs are reported as medians in terms of per patient per month (pppm). Overall monthly cost (n=969) was $10,281 pppm. Diagnosis cost $6,601 pppm, active treatment cost $9,287 pppm, and end-of life cost $12,215 pppm. There was no difference in cost between the “transition” (n=439) and “current” (n=503) periods overall or for any segment of disease. Comorbidities had no effect on cost. For patients receiving at least 5 months of active treatment medication (n=316) total median cost was $144,147 per patient ($9,371 pppm). Discussion: There was no difference in cost between the transition and current periods, in regards to either overall cost or segmented cost. The most expensive segment was end-of-life, with a median cost exceeding $12,000 pppm. Surprisingly, comorbidities had no effect on cost. Newer agents (biologics, TKIs, and pemetrexed) represent only a modest portion of cost, with a majority of cost for stage IV NSCLC comprised of non-drug costs. / text

EGFR TKIs are standard therapy for advanced NSCLC. In order to define their role in early disease, we implemented a phase II trial of neoadjuvant gefitinib in clinical stage I NSCLC. Tumour shrinkage was seen in 43% of patients, with 11% achieving RECIST partial response (PR). Analysis of molecular markers showed EGFR TKD mutations in 17% of cases, being the only associated with PR. For the first time we defined the histopathological response of NSCLC to these agents, characterized by reduction in tumour cellularity and proliferative index as well as presence of non-mucinous BAC histology. Clinical PR tumours also presented large areas of stromal fibrosis with presence of focal residual tumour. In a characterization of intracellular signalling response, EGFR dephosphorylation in the residues Y1068 and Y1173 was not concordant and only the former was significantly reduced. pAkt Ser473/Akt and Thr308/Akt ratios were significantly reduced but observed among both, clinical responders and resistant patients. Interestingly, reduction in pEGFR Y1068 was significantly associated with increase in tumour cellularity (p=0.047), Ki-67 index (p=0.018) and tumour growth (p=0.019) with a residual perinuclear localization been detected, suggesting a novel mechanism of resistance involving receptor internalization. Finally, we determined that the EGFR protein remains stable up to one hour of post resection ischemia but two to three tumour samples are necessary for an adequate tumour representation. Furthermore, EGFR cytoplasmic compartment presented the best association with clinical response in our cohort. Taking all together, we were able to generate the first clinical trial exploring the use of an EGFR TKI in early NSCLC, characterizing for the first time the histopathological and signalling responses to these agents with an evidence of a potential novel mechanism of resistance. Finally, we observed that multiple samples collection for an adequate tumour representation, and assessment of the cytoplasmic compartment, are warrant.

EGFR TKIs are standard therapy for advanced NSCLC. In order to define their role in early disease, we implemented a phase II trial of neoadjuvant gefitinib in clinical stage I NSCLC. Tumour shrinkage was seen in 43% of patients, with 11% achieving RECIST partial response (PR). Analysis of molecular markers showed EGFR TKD mutations in 17% of cases, being the only associated with PR. For the first time we defined the histopathological response of NSCLC to these agents, characterized by reduction in tumour cellularity and proliferative index as well as presence of non-mucinous BAC histology. Clinical PR tumours also presented large areas of stromal fibrosis with presence of focal residual tumour. In a characterization of intracellular signalling response, EGFR dephosphorylation in the residues Y1068 and Y1173 was not concordant and only the former was significantly reduced. pAkt Ser473/Akt and Thr308/Akt ratios were significantly reduced but observed among both, clinical responders and resistant patients. Interestingly, reduction in pEGFR Y1068 was significantly associated with increase in tumour cellularity (p=0.047), Ki-67 index (p=0.018) and tumour growth (p=0.019) with a residual perinuclear localization been detected, suggesting a novel mechanism of resistance involving receptor internalization. Finally, we determined that the EGFR protein remains stable up to one hour of post resection ischemia but two to three tumour samples are necessary for an adequate tumour representation. Furthermore, EGFR cytoplasmic compartment presented the best association with clinical response in our cohort. Taking all together, we were able to generate the first clinical trial exploring the use of an EGFR TKI in early NSCLC, characterizing for the first time the histopathological and signalling responses to these agents with an evidence of a potential novel mechanism of resistance. Finally, we observed that multiple samples collection for an adequate tumour representation, and assessment of the cytoplasmic compartment, are warrant.

No description available.

Lung cancer is the leading cause of cancer mortality worldwide. Understanding biological processes of specific antitumor immune response remains of an eminent interest and represents necessity for designing successful antitumor immunotherapeutic strategies. The theoretical part of the thesis describes components of the immune system that are involved in antitumor response and discusses their role in the hitherto known and used lung cancer immunotherapy. In the practical part of the thesis, two studies studying different aspects of anticancer immune response are described. Both studies were conducted in cooperation with 3rd Surgical Department 1st Faculty of Medicine, Charles University and University Hospital Motol and with the biotechnology company Sotio a.s. The first study is focused on the humoral component of the specific antitumor response and prospectively analyses serum frequency of antitumor antibodies against NY-ESO-1, Her2/neu and MAGE-A4 antigens in 121 patients with NSCLC. Here it was shown for the first time that tobacco smoking significantly increases the frequency of NY- ESO-1 antibodies in sera of smokers in comparison to ex-smokers and non-smokers. The second study is focused on the cellular component of the specific antitumor response investigating the activity of the dendritic...

L'EGFR est un récepteur transmembranaire à activité tyrosine kinase (TK) qui transduit des signaux de prolifération et de survie cellulaire. Dans les cancers du poumon, son activité est fréquemment dérégulée par surexpression et/ou par mutation au niveau de son domaine TK. Ces mutations sont principalement de deux types (EGFR-L858R et EGFR-Del19) et sont dites activatrices car elles induisent une activation constitutive des signalisations oncogéniques de l'EGFR. Elles sont aussi un facteur prédictif de réponse aux EGFR-TKIs qui inhibent spécifiquement ce récepteur. P14ARF est un suppresseur de tumeur qui restreint la prolifération cellulaire et maintient la stabilité génomique. Nous avons décrit son inactivation dans les cancers du poumon et démontré que son expression freine leur développement. Nos résultats récents montrent que l'expression de p14ARF est inhibée dans une très grande majorité d'adénocarcinomes pulmonaires présentant une mutation activatrice de l'EGFR. Sur la base de ces résultats nous avons émis l'hypothèse que l'inhibition de l'expression de p14ARF contribuerait à l'expansion clonale des tumeurs porteuses d'un EGFR muté. P14ARF pourrait ainsi être un frein à l'activité oncogénique de l'EGFR.Dans différents modèles d'adénocarcinomes pulmonaires exprimant un mutant EGFR-L858R nous montrons que l'expression transitoire de p14ARF active une signalisation pro-apoptotique dépendante de STAT3 et de Bcl2. En retour, l'EGFR inhibe l'expression de p14ARF et bloque ses fonctions pro-apoptotiques. Nous montrons aussi que l'activation de l'EGFR (sauvage ou muté) inhibe l'expression de p14ARF à un niveau transcriptionnel. Ceci implique une translocation nucléaire de l'EGFR contrôlée par les PI3Ks de classe III (Vps34) et la fixation de l'EGFR sur le promoteur de ARF. L'ensemble de ces travaux identifie pour la première fois un lien fonctionnel entre les voies de signalisation de l'EGFR et de p14ARF. Ils mettent en évidence un nouveau mécanisme de progression tumorale par lequel une signalisation nucléaire de l'EGFR inactive le suppresseur de tumeur p14ARF afin de permettre la croissance tumorale. / EGFR is a transmembrane tyrosine kinase (TK) receptor which activates proliferative and survival signals. In lung cancer, its activity is frequently deregulated by overexpression and/or mutation in its TK domain. These mutations are mainly of two types (L858R and Del19) and are called « driver mutations » because they induce constitutive activation of EGFR oncogenic signaling. They also represent a predictive responsive factor to EGFR TKIs that specifically inhibit this receptor. P14ARF is a tumor suppressor that restricts cellular proliferation and maintains genomic stability. We described its inactivation in lung cancer and demonstrated that its expression inhibits their development. Our recent results show that the expression of p14ARF is inhibited in a majority of lung adenocarcinomas expressing an activating EGFR mutation. Based on these results we hypothesized that inhibition of p14ARF expression contributes to clonal expansion of mutated EGFR-bearing tumor. P14ARF could be a break to EGFR oncogenic activity.In different models of lung adenocarcinoma expressing a L858R EGFR mutant we show that transient expression of p14ARF activates a pro-apoptotic STAT3/Bcl-2-dependent signaling pathway. In turn, EGFR inhibits the expression of p14ARF and blocks its pro-apoptotic function. We also show that EGFR (wild type or mutated) activation inhibits the expression of p14ARF at the transcriptional level. This implies a nuclear translocation of EGFR controlled by Class III PI3K (Vps34) and its fixation to the ARF promoter. This work identifies for the first time a functional link between EGFR and p14ARF signaling pathways. They highlight a new mechanism of tumor progression by which a nuclear EGFR signaling inactivates the tumor suppressor p14ARF to allow tumor growth.